Podcasts about IC50

Welcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: A primer on why computational predictive toxicology is hard, published by Abhishaike Mahajan on August 19, 2024 on LessWrong. Introduction There are now (claimed) foundation models for protein sequences, DNA sequences, RNA sequences, molecules, scRNA-seq, chromatin accessibility, pathology slides, medical images, electronic health records, and clinical free-text. It's a dizzying rate of progress. But there's a few problems in biology that, interestingly enough, have evaded a similar level of ML progress, despite there seemingly being all the necessary conditions to achieve it. Toxicology is one of those problems. This isn't a new insight, it was called out in one of Derek Lowe's posts, where he said: There are no existing AI/ML systems that mitigate clinical failure risks due to target choice or toxicology. He also repeats it in a more recent post: '…the most badly needed improvements in drug discovery are in the exact areas that are most resistant to AI and machine learning techniques. By which I mean target selection and predictive toxicology.' Pat Walters also goes into the subject with much more depth, emphasizing how difficult the whole field is. As someone who isn't familiar at all with the area of predictive toxicology, that immediately felt strange. Why such little progress? It can't be that hard, right? Unlike drug development, where you're trying to precisely hit some key molecular mechanism, assessing toxicity almost feels…brutish in nature. Something that's as clear as day, easy to spot out with eyes, easier still to do with a computer trained to look for it. Of course, there will be some stragglers that leak through this filtering, but it should be minimal. Obviously a hard problem in its own right, but why isn't it close to being solved? What's up with this field? Some background One may naturally assume that there is a well-established definition of toxicity, a standard blanket definition to delineate between things that are and aren't toxic. While there are terms such as LD50, LC50, EC50, and IC50, used to explain the degree by which something is toxic, they are an immense oversimplification. When we say a substance is "toxic," there's usually a lot of follow-up questions. Is it toxic at any dose? Only above a certain threshold? Is it toxic for everyone, or just for certain susceptible individuals (as we'll discuss later)? The relationship between dose and toxicity is not always linear, and can vary depending on the route of exposure, the duration of exposure, and individual susceptibility factors. A dose that causes no adverse effects when consumed orally might be highly toxic if inhaled or injected. And a dose that is well-tolerated with acute exposure might cause serious harm over longer periods of chronic exposure. The very definition of an "adverse effect" resulting from toxicity is not always clear-cut either. Some drug side effects, like mild nausea or headache, might be considered acceptable trade-offs for therapeutic benefit. But others, like liver failure or birth defects, would be considered unacceptable at any dose. This is particularly true when it comes to environmental chemicals, where the effects may be subtler and the exposure levels more variable. Is a chemical that causes a small decrease in IQ scores toxic? What about one that slightly increases the risk of cancer over a lifetime (20+ years)? And this is one of the major problems with applying predicting toxicology at all - defining what is and isn't toxic is hard! One may assume the FDA has clear stances on all these, but even they approach it on a 'vibe-based' perspective. They simply collate the data from in-vitro studies, animal studies, and human clinical trials, and arrive to an approval/no-approval conclusion that is, very often, at odds with some portion of the medical comm...

Link to original articleWelcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: A primer on why computational predictive toxicology is hard, published by Abhishaike Mahajan on August 19, 2024 on LessWrong. Introduction There are now (claimed) foundation models for protein sequences, DNA sequences, RNA sequences, molecules, scRNA-seq, chromatin accessibility, pathology slides, medical images, electronic health records, and clinical free-text. It's a dizzying rate of progress. But there's a few problems in biology that, interestingly enough, have evaded a similar level of ML progress, despite there seemingly being all the necessary conditions to achieve it. Toxicology is one of those problems. This isn't a new insight, it was called out in one of Derek Lowe's posts, where he said: There are no existing AI/ML systems that mitigate clinical failure risks due to target choice or toxicology. He also repeats it in a more recent post: '…the most badly needed improvements in drug discovery are in the exact areas that are most resistant to AI and machine learning techniques. By which I mean target selection and predictive toxicology.' Pat Walters also goes into the subject with much more depth, emphasizing how difficult the whole field is. As someone who isn't familiar at all with the area of predictive toxicology, that immediately felt strange. Why such little progress? It can't be that hard, right? Unlike drug development, where you're trying to precisely hit some key molecular mechanism, assessing toxicity almost feels…brutish in nature. Something that's as clear as day, easy to spot out with eyes, easier still to do with a computer trained to look for it. Of course, there will be some stragglers that leak through this filtering, but it should be minimal. Obviously a hard problem in its own right, but why isn't it close to being solved? What's up with this field? Some background One may naturally assume that there is a well-established definition of toxicity, a standard blanket definition to delineate between things that are and aren't toxic. While there are terms such as LD50, LC50, EC50, and IC50, used to explain the degree by which something is toxic, they are an immense oversimplification. When we say a substance is "toxic," there's usually a lot of follow-up questions. Is it toxic at any dose? Only above a certain threshold? Is it toxic for everyone, or just for certain susceptible individuals (as we'll discuss later)? The relationship between dose and toxicity is not always linear, and can vary depending on the route of exposure, the duration of exposure, and individual susceptibility factors. A dose that causes no adverse effects when consumed orally might be highly toxic if inhaled or injected. And a dose that is well-tolerated with acute exposure might cause serious harm over longer periods of chronic exposure. The very definition of an "adverse effect" resulting from toxicity is not always clear-cut either. Some drug side effects, like mild nausea or headache, might be considered acceptable trade-offs for therapeutic benefit. But others, like liver failure or birth defects, would be considered unacceptable at any dose. This is particularly true when it comes to environmental chemicals, where the effects may be subtler and the exposure levels more variable. Is a chemical that causes a small decrease in IQ scores toxic? What about one that slightly increases the risk of cancer over a lifetime (20+ years)? And this is one of the major problems with applying predicting toxicology at all - defining what is and isn't toxic is hard! One may assume the FDA has clear stances on all these, but even they approach it on a 'vibe-based' perspective. They simply collate the data from in-vitro studies, animal studies, and human clinical trials, and arrive to an approval/no-approval conclusion that is, very often, at odds with some portion of the medical comm...

BUFFALO, NY- December 27, 2023 – A new #research paper was #published in Oncotarget's Volume 14 on December 20, 2023, entitled, “The pharmacodynamic and mechanistic foundation for the antineoplastic effects of GFH009, a potent and highly selective CDK9 inhibitor for the treatment of hematologic malignancies.” To evade cell cycle controls, malignant cells rely upon rapid expression of select proteins to mitigate pro-apoptotic signals resulting from damage caused by both cancer treatments and unchecked over-proliferation. Cyclin-dependent kinase 9 (CDK9)-dependent signaling induces transcription of downstream oncogenes promoting tumor growth, especially in hyperproliferative ‘oncogene-addicted' cancers, such as human hematological malignancies (HHMs). In this new study, researchers Fusheng Zhou, Lili Tang, Siyuan Le, Mei Ge, Dragan Cicic, Fubo Xie, Jinmin Ren, Jiong Lan, and Qiang Lu from GenFleet Therapeutics Inc. and Sellas Life Sciences Group aimed to summarize current knowledge underlying the mechanism of action (MOA) of GFH009 and explain its robust anti-cancer activity. “Understanding GFH009's MOA allows for a more optimal clinical development path, given the potential for meaningful benefits in patients with hematological malignancies.” GFH009, a potent, highly selective CDK9 small molecule inhibitor, demonstrated antiproliferative activity in assorted HHM-derived cell lines, inducing apoptosis at IC50 values below 0.2 μM in 7/10 lines tested. GFH009 inhibited tumor growth at all doses compared to controls and induced apoptosis in a dose-dependent manner. Twice-weekly injections of GFH009 maleate at 10 mg/kg significantly prolonged the survival of MV-4-11 xenograft-bearing rodents, while their body weight remained stable. There was marked reduction of MCL-1 and c-MYC protein expression post-drug exposure both in vitro and in vivo. Through rapid ‘on-off' CDK9 inhibition, GFH009 exerts a proapoptotic effect on HHM preclinical models triggered by dynamic deprivation of crucial cell survival signals. “Our results mechanistically establish CDK9 as a targetable vulnerability in assorted HHMs and, along with the previously shown superior class kinome selectivity of GFH009 vs other CDK9 inhibitors, strongly support the rationale for currently ongoing clinical studies with this agent in acute myeloid leukemia and other HHMs.” DOI - https://doi.org/10.18632/oncotarget.28543 Correspondence to - Jiong Lan - jlan@genfleet.com Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28543 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, GFH009, CDK9, leukemia, cell cycle About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551225v1?rss=1 Authors: Baert, L., Rudy, S., Pellisson, M., Doll, T., Rocchetti, R., Kaiser, M., Mäser, P., Müller, M. Abstract: The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimise the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new research paper was published in Oncotarget's Volume 14 on June 21, 2023, entitled, “Diphenyl ditelluride anticancer activity and DNA topoisomerase I poisoning in human colon cancer HCT116 cells.” Diphenyl ditelluride (DPDT) is an organotellurium (OT) compound with pharmacological properties, including antioxidant, antigenotoxic and antimutagenic activities when applied at low concentrations. However, DPDT as well as other OT compounds also show cytotoxicity against mammalian cells when treatments occur at higher drug concentrations. The underlying mechanisms of toxicity of DPDT against tumor cells have previously been poorly explored. In this new study, researchers André Luiz Mendes Juchem, Cristiano Trindade, Juliana Bondan da Silva, Miriana da Silva Machado, Temenouga Nikolova Guecheva, Jaqueline Cesar Rocha, Jenifer Saffi, Iuri Marques de Oliveira, João Antonio Pêgas Henriques, and Alexandre Escargueil from the Federal University of Rio Grande do Sul, Sorbonne Université, Federal University of Health Sciences of Porto Alegre, Lutheran University of Brazil, Bulgarian Academy of Sciences, and University of Vale do Taquari aimed to investigate the effects of DPDT against both human cancer and non-tumorigenic cells. “Together, our results will help to better define DPDT as a potential drug candidate for treating CRC [colorectal cancer].” The researchers used the colonic HCT116 cancer cells and the MRC5 fibroblasts as models. Their results showed that DPDT preferentially targets HCT116 cancer cells when compared to MRC5 cells with IC50 values of 2.4 and 10.1 μM, respectively. This effect was accompanied by the induction of apoptosis and a pronounced G2/M cell cycle arrest in HCT116 cells. Furthermore, DPDT induces DNA strand breaks at concentrations below 5 μM in HCT116 cells and promotes the occurrence of DNA double strand breaks mostly during S-phase as measured by γ-H2AX/EdU double staining. Finally, DPDT forms covalent complexes with DNA topoisomerase I, as observed by the TARDIS assay, with a more prominent effect observed in HCT116 than in MRC5 cells. Taken together, the results of this study show that DPDT preferentially targets HCT116 colon cancer cells likely through DNA topoisomerase I poisoning. “This makes DPDT an interesting molecule for further development as an anti-proliferative compound in the context of cancer.” Read the full paper: DOI: https://doi.org/10.18632/oncotarget.28465 Correspondence to: João Antonio Pêgas Henriques, Alexandre Escargueil Email: pegas.henriques@ufrgs.br, alexandre.escargueil@gmail.com Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28465 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - colorectal cancer, HCT116, diphenyl ditelluride, organotellurium, topoisomerase I About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534814v1?rss=1 Authors: Chang, W.-J., Harpel, Z., Circelli, J., Chen, R., Chang, I., Rivera, J., Wu, S., Wei, Z. Abstract: Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37{degrees}C) where NP1 does not form the oral apparatus. We found that cytarabine was the only tested analog that did not inhibit growth, and phagocytosis was not required for pyrimidine analog entry. IC50 values did not significantly differ between strains for the same analog at either temperature. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, 5-methyluridine) to help determine whether the inhibitory effects from analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534108v1?rss=1 Authors: Hoffstadt, J. G., Wotring, J. W., Porter, S., Halligan, B. S., O'Meara, M. J., Tai, A. W., Sexton, J. Z. Abstract: Dengue Virus (DENV) causes dengue fever, a pandemic-potential disease with currently no FDA-approved antivirals. Additionally, the available vaccine for DENV can increase the risk of severe dengue fever for those who have never had a DENV infection due to antibody-dependent enhancements. Thus, there is an urgent need to identify dengue virus antivirals. Antivirals that target NS4B, the replication compartment forming protein of DENV and the flavivirus family, are a promising new drug class that minimize cytotoxic effects to host cells. Drug-repurposing and high-content screening were leveraged to efficiently identify antivirals likely to inhibit NS4B. Using high-content screening, we quantified the morphological patterns of NS4B and envelope (E) protein expression versus time and developed a viral pseudotime model that was able to predict the infection progression to enable drug screening. We then developed a single-cell infection classifier for antiviral efficacy and performed high-throughput drug screening of 960 compounds. We identified four concentration-dependent inhibitors of DENV with nanomolar potencies including: Nexium, Pralatrexate, GW4064, and LY411575. LY411575, a gamma secretase inhibitor, exhibited an IC50 of 72nM and reduced percent infection to levels indistinguishable from the mock infection control. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

 This antidepressant is the #1 cause of major (life threatening) effects in overdose reported to U.S. Poison CentersIt is difficult to manage due toPotential for delays seizuresUnique cardiogenic shock in overdosePotential wide complex arrhythmia refractory to Sodium Bicarbonate Potential interference with brain death testingToxicityIt increases dopamine and norepinephrine, it also blocks the gap junction in the cardiac myocyteRohr 2004- Gap junction blockade can cause a wide QRVink 2004 Connexin 43 is the most important protein for connexon formation and cardiac signal transmissionCallier 2012- Bupropion does not block sodium channels, and does exhibit similar effects on the cardiac action potential as known gap junctionBurnham 2014 Bupropion has an IC50 for connexin 43 >50 uMol, larger than other drugs such as fluoextine and lamotrigineShaikh Quereshi 2014 Bupropion interferes with connexin43 production and localization in chicken cardiac myoctes at concentration >50 uMolEffectsSympathetic toxidromeSeizuresTL;DRYour patient can seize 8-24 hours in, usually they have neurologic symptoms and tachycardia before handTachycardia may be masked by coingestions and symptoms may be very delayedDo not discharge a patient without discussing observation time with a toxicologist or poison centerDo not dismiss tachycardia and anxiety as situational in a bupropion overdoseShepherd 2004- Seizures in primarily sustained release productsMost seizures had prodromal neuropsychiatric symptomsStarr 2009- Seizure in XL products. Tachycardia, tremor, agitation most associated with seizuresSeizure occured as late as 24 hours and 25% occurred after 8 hoursOfferman 2020- Primarily sustained/extended release productsTachycardia duration, and extent (>120) predicted seizure. (Hypotnesion and neuropsych symptoms also predict)Late seizure occurred only in those with symptoms on presentationThose who had cardiac arrest had prehospital seizure= bad signRianprakaisang 2021- ToxIC review of risk factors for seizuresQTc and HR>140 predict seizuresUnique cardiogenic shock in overdosePotential wide complex arrhythmia refractory to Sodium Bicarbonate Potential interference with brain death testingTreatment DecontaminationAggressive whole bowel irrigation or charcoal may be indicated if large ingestionSupportive careIntubation if airway compromisedBenzodiazepine for agitationBenzodiazepines and GABA-ergic AED's for status epilepticsTachycardia, tremor, and agitation are risk factor for seizuresTachycardia may be masked by alpha 2 agonist co ingestionsSeizures may occur 24 hour outSodium bicarbonate for wide QRS (it may be refractory)Inodilators and vasopressors for cardiogenic shockECMO for refractory shock or arrhythmiaAwareness that severe bupropion toxicity can mimic brain deathsend analytical confirmation of bupropion if possible to rule out confoundingEnhanced eliminationlimited options due to protein binding, not routineFocused antidoteConsider IV fat emulsion if the patient is peri arrestObservation timesTalk to a toxicolleague about observation times, decontamination, and use of invasive therapies to avoid falling into a trapNot all ingestions are made the same   

Rohr 2004- Gap junction blockade can cause a wide QRS Vink 2004 Connexin 43 is the most important protein for connexon formation and cardiac signal transmissionCallier 2012- Bupropion does not block sodium channels, and does exhibit similar effects on the cardiac action potential as known gap junction  Burnham 2014 Bupropion has an IC50 for connexin 43 >50 uMol, larger than other drugs such as fluoextine and lamotrigineShaikh Quereshi 2014 Bupropion interferes with connexin43 production and localization in chicken cardiac myoctes at concentration >50 uMol

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.25.525351v1?rss=1 Authors: Baraibar, A. M., de Pascual, R., Jimenez, V., Hernandez, N., Aguirregabiria, I. E., Hernandez-Guijo, J. M. Abstract: Aluminium (Al3+) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release. Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1 uM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 uM. This blockade was irreversible, since it did not recover after wash-out. Moreover, Al3+ produced a bigger blockade on N-, P- and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (419 uM). This inhibition was reversible. Voltage-dependent potassium currents were non-significantly affected by Al3+. Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 uM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductances altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminium has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

What is the secret to connecting with young people? Have you ever felt frustrated because you find your teenager shuts down and is uncommunicative? Marie Arymar works with children, young people and adults, sharing simple ideas on connection, resilience and wellbeing. In this conversation she explains the four common barriers to listening - this includes: Criticism and judgments Chatter in the Head 'Yes but'   Being Solutions focussed Marie explains that young people don't want to be fixed, they actually want to be heard. We also explore the 'mental health crisis' and explore whether Social Media is really as bad as it is often made out.  We see how the pandemic has (for some young people) been a blessing and we talk about Marie's simple, easy book 'Do you Want to Know a Secret?" which is perfect for young and old alike. You can find out more about Marie and buy her book at realisationworks.com

Here’s why eating garlic and onions can prevent hypertension and diabetes Federal University of Technology (Nigeria), April 16, 2021 n a recent study, researchers at the Federal University of Technology in Nigeria investigated the benefits of eating garlic, white onion and purple onion against serious conditions like diabetes and hypertension. They confirmed these by looking at how extracts from the three alliums affect the activity of diabetes-related enzymes, such as a-amylase and a-glucosidase, and the hypertension-related enzyme, angiotensin-converting enzyme (ACE). The researchers reported their findings in an article published in the Journal of Dietary Supplements. Garlic, white onion and purple onion show antioxidant, antidiabetic and antihypertensive properties Garlic and onions are spices commonly used in cooking. They also serve as ingredients in several traditional delicacies in Nigeria that are known to contain plenty of polyphenols. To assess the beneficial properties of garlic, white onion and purple onion, the researchers first obtained extracts from each and assessed their inhibitory effects on certain enzymes. They also conducted assays to determine the antioxidant capacities of the extracts. ACE is the enzyme responsible for converting angiotensin I into angiotensin II, the hormone that increases blood pressure, as well as body water and sodium content. Angiotensin II elevates blood pressure by constricting the blood vessels; hence, chemicals that can inhibit the activity of ACE, which is responsible for the production of angiotensin II, are used for the treatment of hypertension. (Related: Meet the “two-day cure” plant: An African medicinal plant that can naturally lower blood pressure.) a-Amylase is the enzyme that breaks down starch and glycogen into glucose and maltose (two glucose molecules bound together). In humans, this enzyme is produced by the salivary glands and the pancreas. a-Glucosidase, on the other hand, is responsible for breaking down carbohydrates in the small intestine and facilitating the absorption of glucose. Inhibiting the activity of this enzyme is one of the strategies currently used to prevent the rise of blood sugar levels following a carbohydrate-filled meal. The researchers reported that the garlic, purple onion and white onion extracts inhibited the activities of ACE, a-amylase and a-glucosidase in vitro in a concentration-dependent manner. At a half maximal inhibitory concentration (IC50) of 0.59 mg/mL, the purple onion extract exhibited a higher inhibitory effect on ACE than the white onion extract (IC50 = 0.66 mg/mL) and the garlic (IC50 = 0.96 mg/mL) extract. Meanwhile, the white onion extract showed a significantly stronger inhibitory effect on a-amylase at an IC50 of 3.93 mg/mL than the garlic extract (IC50 = 8.19 mg/mL) and the purple onion (IC50 = 8.27 mg/mL) extract. The garlic extract, on the other hand, showed a similar inhibitory effect (IC50 = 4.50 mg/mL) on a-glucosidase as the white and purple onion extracts. All three extracts also showed dose-dependent free radical scavenging activity and reducing power in the antioxidant assays. Based on these findings, the researchers concluded that garlic, white onion and purple onion can be used to treat or prevent diabetes and hypertension, thanks to their ability to inhibit ACE, a-amylase and a-glucosidase activity, as well as lipid peroxidation in the pancreas and the heart.   Adolescents with lack of empathy show early signs of psychopathy   University of Coimbra (Portugal), April 14, 2021 A pioneering study with the Portuguese population shows that adolescents with high levels of callous-unemotional traits demonstrate lower levels of anticipated guilt towards the possibility of committing an immoral act and struggle to judge an immoral act as a wrong one. Researchers have evaluated the callous traits, that is, the lack of empathy and disregard for the wellbeing and feelings of others, of 47 adolescents between 15 and 18 years old. The teenagers watched video animations portraying examples of moral transgressions, such as incriminating someone or keeping money that fell from someone else's pocket. "This approach allowed us to create more realistic scenarios that happen in daily life," explains Oscar Gonçalves, a neuroscientist at Proaction Lab and co-author of the study. The adolescents were asked how guilty they would feel if they were the ones to commit the moral transgressions and how wrong they think the actions were.  Although the callous-unemotional traits in adolescents are known to be precursors of psychopathy in adulthood, the results of the study differ from what is known about psychopaths. "Adults with psychopathic traits show low levels of anticipated guilt but consider immoral actions as wrong. However, in our study, adolescents with high CU levels show levels of guilt and judge immoral actions as less wrong," explains Margarida Vasconcelos, first author.  However, researchers have found evidence of a dissociation between moral emotions and moral judgment, that is, between the feelings of guilt and the judgment of immoral actions. "Even in adolescents with sub-clinical levels of callous-unemotional traits, this dissociation typical in psychopathy in adulthood is already happening during development," explains the study coordinator Ana Seara Cardoso.  The results of the study will "contribute to the development of a severe anti-social behavior model" and allow the "development of intervention targets, rehabilitation and early prevention of anti-social behavior," says Ana Seara Cardoso.   Omega-3 supplements do double duty in protecting against stress   Ohio State University, April 20, 2021 A high daily dose of an omega-3 supplement may help slow the effects of aging by suppressing damage and boosting protection at the cellular level during and after a stressful event, new research suggests. Researchers at The Ohio State University found that daily supplements that contained 2.5 grams of omega-3 polyunsaturated fatty acids, the highest dose tested, were the best at helping the body resist the damaging effects of stress. Compared to the placebo group, participants taking omega-3 supplements produced less of the stress hormone cortisol and lower levels of a pro-inflammatory protein during a stressful event in the lab. And while levels of protective compounds sharply declined in the placebo group after the stressor, there were no such decreases detected in people taking omega-3s. The supplements contributed to what the researchers call stress resilience: reduction of harm during stress and, after acute stress, sustained anti-inflammatory activity and protection of cell components that shrink as a consequence of aging. The potential anti-aging effects were considered particularly striking because they occurred in people who were healthy but also sedentary, overweight and middle-aged—all characteristics that could lead to a higher risk for accelerated aging. "The findings suggest that omega-3 supplementation is one relatively simple change people could make that could have a positive effect at breaking the chain between stress and negative health effects," said Annelise Madison, lead author of the paper and a graduate student in clinical psychology at Ohio State. The research is published today (Monday, April 19, 2021) in the journal Molecular Psychiatry. Madison works in the lab of Janice Kiecolt-Glaser, professor of psychiatry and psychology and director of the Institute for Behavioral Medicine Research at Ohio State. This paper is a secondary analysis of one of Kiecolt-Glaser's earlier studies showing that omega-3 supplements altered a ratio of fatty acid consumption in a way that helped preserve tiny segments of DNA in white blood cells. Those short fragments of DNA are called telomeres, which function as protective caps at the end of chromosomes. Telomeres' tendency to shorten in many types of cells is associated with age-related diseases, especially heart disease, and early mortality. In the initial study, researchers were monitoring changes to telomere length in white blood cells known as lymphocytes. For this new study, the researchers looked at how sudden stress affected a group of biological markers that included telomerase, an enzyme that rebuilds telomeres, because levels of the enzyme would react more quickly to stress than the length of telomeres themselves. Specifically, they compared how moderate and high doses of omega-3s and a placebo influenced those markers during and after an experimental stressor. Study participants took either 2.5 grams or 1.25 grams of omega-3s each day, or a placebo containing a mix of oils representing a typical American's daily intake. After four months on the supplements, the 138 research participants, age 40-85, took a 20-minute test combining a speech and a math subtraction task that is known to reliably produce an inflammatory stress response. Only the highest dose of omega-3s helped suppress damage during the stressful event when compared to the placebo group, lowering cortisol and a pro-inflammatory protein by an average of 19% and 33%, respectively. Results from blood samples showed that both doses of omega-3s prevented any changes in telomerase levels or a protein that reduces inflammation in the two hours after participants experienced the acute stress, meaning any needed stress-related cell repair—including telomere restoration—could be performed as usual. In the placebo group, those repair mechanisms lost ground: Telomerase dropped by an average of 24% and the anti-inflammatory protein decreased by an average of at least 20%. "You could consider an increase in cortisol and inflammation potential factors that would erode telomere length," Madison said. "The assumption based on past work is that telomerase can help rebuild telomere length, and you want to have enough telomerase present to compensate for any stress-related damage. "The fact that our results were dose-dependent, and we're seeing more impact with the higher omega-3 dose, would suggest that this supports a causal relationship." The researchers also suggested that by lowering stress-related inflammation, omega-3s may help disrupt the connection between repeated stress and depressive symptoms. Previous research has suggested that people with a higher inflammatory reaction to a stressor in the lab may develop more depressive symptoms over time. "Not everyone who is depressed has heightened inflammation—about a third do. This helps explain why omega-3 supplementation doesn't always result in reduced depressive symptoms," Kiecolt-Glaser said. "If you don't have heightened inflammation, then omega-3s may not be particularly helpful. But for people with depression who do, our results suggest omega-3s would be more useful." The 2.5-gram dose of omega-3s is much higher than what most Americans consume on a daily basis, but study participants showed no signs of having problems with the supplements, Madison said.           Want to be robust at 40-plus? Meeting minimum exercise guidelines won't cut it 5 hours of moderate activity a week may be required to avoid midlife hypertension, UCSF-led study shows University of California at San Francisco, April 15, 2021 Young adults must step up their exercise routines to reduce their chances of developing high blood pressure or hypertension - a condition that may lead to heart attack and stroke, as well as dementia in later life. Current guidelines indicate that adults should have a minimum of two-and-a-half hours of moderate intensity exercise each week, but a new study led by UCSF Benioff Children's Hospitals reveals that boosting exercise to as much as five hours a week may protect against hypertension in midlife - particularly if it is sustained in one's thirties, forties and fifties.  In the study publishing in American Journal of Preventive Medicine on April 15, researchers followed approximately 5,000 adults ages 18 to 30 for 30 years. The participants were asked about their exercise habits, medical history, smoking status and alcohol use. Blood pressure and weight were monitored, together with cholesterol and triglycerides.  Hypertension was noted if blood pressure was 130 over 80 mmHg, the threshold established in 2017 by the American College of Cardiology/American Heart Association.  The 5,115 participants had been enrolled by the Coronary Artery Risk Development in Young Adults (CARDIA) study and came from urban sites in Birmingham, Ala., Chicago, Minneapolis and Oakland, Calif. Approximately half the participants were Black (51.6 percent) and the remainder were White. Just under half (45.5 percent) were men.  Fitness Levels Fall Fast for Black Men Leading to More Hypertension Among the four groups, who were categorized by race and gender, Black men were found to be the most active in early adulthood, exercising slightly more than White men and significantly more than Black women and White women. But by the time Black men reached age 60, exercise intake had slumped from a peak of approximately 560 exercise units to around 300 units, the equivalent to the minimum of two-and-a-half hours a week of moderate intensity exercise recommended by the U.S. Department of Health and Human Services. This was substantially less exercise than White men (approximately 430 units) and slightly more than White women (approximately 320 units). Of the four groups, Black women had the least exercise throughout the study period and saw declines over time to approximately 200 units. "Although Black male youth may have high engagement in sports, socio-economic factors, neighborhood environments, and work or family responsibilities may prevent continued engagement in physical activity through adulthood," said first author Jason Nagata, MD, of the UCSF Division of Adolescent and Young Adult Medicine. Additionally, Black men reported the highest rates of smoking, which may preclude physical activity over time, he noted. Physical activity for White men declined in their twenties and thirties and stabilized at around age 40. For White women, physical activity hovered around 380 exercise units, dipping in their thirties and remaining constant to age 60. Rates of hypertension mirrored this declining physical activity. Approximately 80-to-90 percent of Black men and women had hypertension by age 60, compared with just below 70 percent for White men and 50 percent for White women.  "Results from randomized controlled trials and observational studies have shown that exercise lowers blood pressure, suggesting that it may be important to focus on exercise as a way to lower blood pressure in all adults as they approach middle age," said senior author Kirsten Bibbins-Domingo, MD, PhD, of the UCSF Department of Epidemiology and Biostatistics.  "Teenagers and those in their early twenties may be physically active but these patterns change with age. Our study suggests that maintaining physical activity during young adulthood - at higher levels than previously recommended - may be particularly important." More Exercise from Youth to Midlife Offers Best Protection Against Hypertension When researchers looked at the 17.9 percent of participants who had moderate exercise for at least five hours a week during early adulthood - double the recommended minimum - they found that the likelihood of developing hypertension was 18 percent lower than for those who exercised less than five hours a week. The likelihood was even lower for the 11.7 percent of participants who maintained their exercise habits until age 60.  Patients should be asked about physical activity in the same way as they are routinely checked for blood pressure, glucose and lipid profiles, obesity and smoking, Nagata said, and intervention programs should be held at schools, colleges, churches, workplaces and community organizations. Black women have high rates of obesity and smoking, and low rates of physical activity, he said, and should be an important group for targeted intervention.  "Nearly half of our participants in young adulthood had suboptimal levels of physical activity, which was significantly associated with the onset of hypertension, indicating that we need to raise the minimum standard for physical activity," Nagata said. "This might be especially the case after high school when opportunities for physical activity diminish as young adults transition to college, the workforce and parenthood, and leisure time is eroded."     Study finds association between periodontal disease and low intake of minerals, vitamins and dietary fiber in young adult women Tokyo Medical and Dental University, April 12, 2021 According to news reporting out of Tokyo, Japan, research stated, “Dietary habits of middle-aged and elderly individuals affected by periodontal disease (PD) differ from those who are unaffected by it, according to previous reports. However, in young adults, there are only a few reports that show a correlation between nutrient/food intake and PD.” Our news journalists obtained a quote from the research from Tokyo Medical and Dental University (TMDU), “Moreover, no report till date has assessed the correlation between dietary habits and PD using a self-administered diet history questionnaire (DHQ). Therefore, we assessed this correlation using a DHQ in young adult women who are likely to develop PD. The participants were enrolled from 2 universities and included 120 female college students a mean age of 20.4 y. The participants were assessed for the presence of PD according to the community periodontal index and were divided into two groups, the PD group and the non-PD group. Their dietary habits were investigated using a DHQ and the level of difficulty in chewing food was assessed. The PD group had a significantly lower nutrient intake of minerals, fat-soluble vitamins, water-soluble vitamins, and dietary fiber than the non-PD group. In terms of food groups, the PD group consumed significantly lesser amounts of green and yellow vegetables (GYV) than the non-PD group. Multivariate analysis revealed that the PD group had significantly lower intakes of vitamin E and GYV than the non-PD group. The PD group consumed significantly lesser amounts of hard foods than the non-PD group.” According to the news editors, the research concluded: “Young adult women who were evaluated for PD by a screening test had a significantly lower nutrient/food intake than those without a PD.” This research has been peer-reviewed.       Just 2 days of increased sugar intake can harm your gut health, warn researchers University of Alberta, April 16, 2021 Researchers from the University of Alberta in Canada found that short-term increases in sugar intake can increase the risk of inflammatory bowel disease. Their finding, which was published in Scientific Reports, is a reminder that eating healthy must be sustained in order to keep your gut in good shape. “Surprisingly, our study shows that short-term sugar consumption can really have a detrimental impact, and so this idea that it’s OK to eat well all week and indulge in junk food on the weekend is flawed,” said Karen Madsen, one of the study researchers. Increased sugar intake is bad for the gut Previous studies have shown that diets can affect your susceptibility to disease. Western diets, for example, have been implicated in the development of inflammatory bowel disease. But it’s still unclear when a poor diet begins to take a toll on your health, much less how it does so. To investigate, the researchers placed adult mice on a chow diet or a high-sugar diet and treated them with dextran sodium sulfate to induce ulcerative colitis, one of the major forms of inflammatory bowel disease. Disease severity was assessed daily. After two days, the mice on the high-sugar diet were at great risk of developing colitis. Their immune response also weakened while their gut permeability increased, allowing more bacteria and toxins to enter their bloodstream. “We wanted to know how long it takes before a change in diet translates into an impact on health. In the case of sugar and colitis, it only took two days, which was really surprising to us. We didn’t think it would happen so quickly,” said Madsen. The researchers attributed these effects to sugar’s impact on the gut bacteria. Eating sugary foods decreases the amount of “good” gut bacteria that produce short-chain fatty acids, which are critical for a strong immune response. Meanwhile, sugar feeds “bad” bacteria that promote inflammation and weaken your immunity.  Fortunately, the researchers found that supplementing with short-chain fatty acids helped reduce the negative effects of a high-sugar diet. Having these supplements as an option will be great for people struggling to change their bad eating habits. “People want to eat what they want to eat, so short-chain fatty acids could possibly be used as supplements to help protect people against the detrimental effects of sugar on inflammatory bowel disease,” said Madsen.     Rose water is an antimicrobial and anti-inflammatory remedy for skin infections Teikyo University (Japan), April 15, 2021 Rosa damascena, commonly known as Damask rose, is one of the most important and medicinally useful members of the Rosaceae (rose) family. It is an ornamental plant widely used to make perfumes and is reported to have plenty of beneficial properties. According to multiple studies, Damask rose has anti-HIV, antibacterial, antioxidant, antitussive, hypnotic and antidiabetic properties. It has also shown relaxant effects on the tracheal chains of guinea pigs. In a recent study, researchers at Teikyo University in Japan investigated two biological properties of Damask rose, specifically it’s antimicrobial and anti-inflammatory properties. They tested rose water made from high-quality Damask rose petals on two microbial pathogens, namely, Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), which commonly cause skin infections. The researchers reported their findings in an article published in Biological and Pharmaceutical Bulletin. Damask rose water is a natural antibiotic and anti-inflammatory agent Damask rose is a multipurpose plant widely known for its culinary and medicinal applications, among other things. Edible parts of Damask rose are used in various cuisines, including its young shoots, petals, fruits, leaves and seeds. Damask rose petals are used to make jams and add flavor to beverages, baked goods and desserts. They are also used for cooking dishes. Rosewater, which can be sweetened to produce rose syrup, is a byproduct of rose oil production. It is usually obtained by steam distilling Damask rose petals and taking the hydrosol portion of the rose petal distillate.  In different parts of the world, rose water, rose oil and a decoction made of Damask rose roots are used in traditional medicine for the treatment of various ailments, such as abdominal and chest pain, digestive problems and inflammation, especially of the neck. In North America, Indian tribes use the decoction as a cough remedy for children. Rose oil is used to treat depression and reduce stress and tension. Inhaling the vapor produced by heating rose oil is also believed to be an effective remedy for allergies, headaches and migraine. Damask rose water, on the other hand, is traditionally used to treat skin conditions, such as erythema (skin redness), itchiness and swelling. To evaluate its antimicrobial and anti-inflammatory properties, the researchers tested Damask rose water against C. albicans and MRSA and assessed its effects on the function of neutrophils, which are white blood cells that serve as key regulators of inflammatory reactions. The researchers reported that Damask rose water (2.2. percent solution) inhibited the mycelial growth of C. albicans and reduced the viability of MRSA within an hour of treatment. Damask rose water (five to 15 percent) also suppressed the activation of neutrophils induced by treatment with lipopolysaccharide (LPS), a bacterial toxin; tumor necrosis factor-alpha (TNF-a), a cell-signaling protein produced by immune cells; and N-formyl-Met-Leu-Phe (fMLP), a macrophage activator. Additionally, Damask rose water reduced LPS- and TNF-a-induced cell surface expression of the adhesion-related molecule, cluster of differentiation 11b (CD11b), which is rapidly elevated by the activation of neutrophils. The amount of CD11b in neutrophils is said to correlate with their activation and inflammation. However, Damask rose water did not affect the migratory capacity of neutrophils (with or without a chemoattractant). Based on these findings, the researchers concluded that Damask rose water can reduce the pathogenicity of microbes and attenuate neutrophil stimulation, thus inhibiting skin inflammation caused by microbial infections.     Study shows how chronic stress may inhibit the body's cancer-fighting ability University of Western Ontario, April 15, 2021 New research from Western University has shown how psychological stress hinders the immune system's defenses against cancer. By investigating the effects of chronic stresson the immune system's "emergency responders," researchers at the Schulich School of Medicine & Dentistry found that a stress-induced hormone impairs the ability of these immune cells to carry out their cancer-fighting function. Led by Mansour Haeryfar, Ph.D., the research looked specifically at innate-like T cells, which when functioning properly enable the immune system to look for potentially cancerous cells in the body and destroy them. The study was published today in Cell Reports. Innate-like T cells include invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, which were the subjects of this investigation. iNKT cells are present in small numbers in many tissues but are especially enriched in the human omentum, an apron-like layer of fatty tissue. MAIT cells are present in relatively high numbers in the human peripheral blood, gut, lungs and liver among other organs. "These innate-like T cells are our immune system's emergency responders," said Haeryfar. "They react quickly to pathogens and cancer cells and are in a pre-activated mode, so they are like loaded guns, ready to respond." Previous studies have shown that when a person experiences chronic psychological and emotional stress, the body's immune system is suppressed, dampening its ability to fight cancer and opportunistic infections. This happens in large part because stress hormones kill off some of the body's immune cells. However, Haeryfar and his team showed that innate-like T cells actually don't die as a result of chronic stress but their cancer-fighting abilities are drastically impaired by stress-induced hormones called glucocorticoids. This impairment led to a striking increase in cancer metastasis in a mouse model. "We found that innate-like T cells survive when the host is under stress, but their functions are compromised," Haeryfar said. "The cells cannot make enough of their beneficial mediators to help fight cancer, so the metastatic burden is increased because of the stress." The team also looked at the effects of natural and synthetic glucocorticoids on innate-like T cells in human blood and liver tissue, where they are abundant. This was important to providing initial evidence that some of the discoveries made in the mouse models were valid for human cells as well, said Patrick Rudak, Ph.D. Candidate in Haeryfar's lab. One of the important implications of this work is that innate-like T cells are currently being investigated for cancer immunotherapy treatment. This study demonstrates that their therapeutic potential can be dampened by psychological stress, said Haeryfar, and this finding needs to be considered when designing or administering those therapies. Rudak added: "Our study demonstrates that, despite being capable of instigating robust anti-tumor immune responses under normal conditions, innate-like T cells completely fail to protect against tumors during psychological stress." Because the study also uncovered the mechanisms by which stress diminishes T cell function, the researchers hope they can use the information to help design immunotherapies involving these cells that will still be effective in psychologically stressed patients.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.344424v1?rss=1 Authors: An, H., Park, J. Abstract: Currently, more than 33 million peoples have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and more than a million people died from coronavirus disease 2019 (COVID-19), a disease caused by the virus. There have been multiple reports of autoimmune and inflammatory diseases following SARS-CoV-2 infections. There are several suggested mechanisms involved in the development of autoimmune diseases, including cross-reactivity (molecular mimicry). A typical workflow for discovering cross-reactive epitopes (mimotopes) starts with a sequence similarity search between protein sequences of human and a pathogen. However, sequence similarity information alone is not enough to predict cross-reactivity between proteins since proteins can share highly similar conformational epitopes whose amino acid residues are situated far apart in the linear protein sequences. Therefore, we used a hidden Markov model-based tool to identify distant viral homologs of human proteins. Also, we utilized experimentally determined and modeled protein structures of SARS-CoV-2 and human proteins to find homologous protein structures between them. Next, we predicted binding affinity (IC50) of potentially cross-reactive T-cell epitopes to 34 MHC allelic variants that have been associated with autoimmune diseases using multiple prediction algorithms. Overall, from 8,138 SARS-CoV-2 genomes, we identified 3,238 potentially cross-reactive B-cell epitopes covering six human proteins and 1,224 potentially cross-reactive T-cell epitopes covering 285 human proteins. To visualize the predicted cross-reactive T-cell and B-cell epitopes, we developed a web-based application "Molecular Mimicry Map (3M) of SARS-CoV-2" (available at https://ahs2202.github.io/3M/). The web application enables researchers to explore potential cross-reactive SARS-CoV-2 epitopes alongside custom peptide vaccines, allowing researchers to identify potentially suboptimal peptide vaccine candidates or less ideal part of a whole virus vaccine to design a safer vaccine for people with genetic and environmental predispositions to autoimmune diseases. Together, the computational resources and the interactive web application provide a foundation for the investigation of molecular mimicry in the pathogenesis of autoimmune disease following COVID-19. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378018v1?rss=1 Authors: Eberle, R. J., Olivier, D. S., Amaral, M. S., Willbold, D., Arni, R. K., Coronado, M. A. Abstract: Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) up to now WHO reported more than 50 million confirmed cases and more than one million losses, globally. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide resulting in a pandemic of unprecedented magnitude. To date, no clinically safe drug or vaccine is available and the development of molecules to combat SARS-CoV-2 infections is imminent. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is the repurposing of clinically developed drugs, e.g., anti-parasitic drugs. The results described in this study demonstrate the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CLpro). Quinacrine and suramin molecules present a competitive and non-competitive mode of inhibition, respectively, with IC50 and KD values in low M range. Using docking and molecular dynamics simulations we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin in combination with quinacrine showed promising synergistic efficacy to inhibit SARS-CoV-2 3CLpro. The identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease. Drug repositioning offers hope to the SARS-CoV-2 control. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.06.371534v1?rss=1 Authors: Ghosh, D., Bansode, S., Joshi, R. S., Kolte, B., Gacche, R. Abstract: The serine protease, elastase exists in various forms and plays diverse roles in the human body. Pharmacological inhibition of elastase has been investigated for its therapeutic role in managing conditions such as diabetes, pneumonia and arthritis. Sivelestat, a synthetic molecule, is the only elastase inhibitor to have been approved by any major drug regulatory authority (PMDA, in this case), but still has failed to attain widespread clinical usage owing to its high price, cumbersome administration and obscure long-term safety profile. In order to find a relatively better-suited alternative, screening was conducted using plant flavonoids, which yielded Baicalein, a molecule that showed robust inhibition against Pancreatic Elastase inhibition (IC50: 3.53 micromolar). Other than having an IC50 almost 1/5th of that of sivelestat, baicalein is also cheaper, safer and easier to administer. While Microscale thermophoresis validated baicalein-elastase interaction, enzyme-kinetic studies, molecular docking and molecular dynamic simulation revealed the mode of inhibition to be non-competitive. Baicalein exhibited binding to a distinct allosteric site on the enzyme. The current study demonstrates the elastase inhibition properties of baicalein in an in-vitro and in-silico environment. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.31.363549v1?rss=1 Authors: L, L., V, V., Srihari, R., CN, P. Abstract: Introduction: Type 2 Diabetes Mellitus (T2DM) is a long-term metabolic disorder that primarily characterized by impaired insulin resistance to become hyperglycemia. People suffering from T2DM have a higher risk of developing various diseases but, on top of that, some diabetic drugs are also suspected of increasing the risk in some cases. Aldose reductase is a key target enzyme to catalyze the reduction of glucose to sorbitol and does not readily diffuse across cell membranes and cause retinopathy and neuropathy. The aldolase reductase inhibitors prevent the conversion of glucose to sorbitol and may have the capacity of preventing and / or treating several diabetic complications. It will be expected to be twofold in the subsequent decade due to intensification in the senile population with the number of people affected, thus adding to the liability on medical providers in poor developed countries using herbal medicine to control the diabetes. In recent investigation, the antidiabetic property of phytochemicals extracted from leafs of Abutilon indicum (L.) is elucidated using animal models. Materials and Methods: In the current study using aldose reductase enzyme assay inhibitor of Rat lens Aldose reductase were treated with A. indicum methanolic leaf extract at different concentrations (6.25, 12.5, 25, 50, 100, and 200microgram/mL). Copper sulphate was used as reference drug and docking studies to predict the screen the best aldose reductase inhibitor. Results and Discussion: The crude extract exhibited cytotoxicity against rat lens aldose reductase (IC50 = 135.8 {+/-} 956;g/L vs ref 13.60 {+/-} 956;g/L) using In Vitro. The docking is performed with 11 compounds shows Ertugliflozin, 9H-Cycloisolongifolene, 8-oxo and 7-hydroxycadalene showed a good binding interaction with aldose reductase. Conclusion: We are concluding that the invitro and in silico analysis helps researchers to utilize these compound for aldose reductase inhibitors and further can be used for clinical applications. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.29.339317v1?rss=1 Authors: The COVID Moonshot Consortium,, Achdout, H., Aimon, A., Bar-David, E., Barr, H., Ben-Shmuel, A., Bennett, J., Bobby, M. L., Brun, J., BVNBS, S., Calmiano, M., Carbery, A., Cattermole, E., Chodera, J. D., Clyde, A., Coffland, J. E., Cohen, G., Cole, J., Contini, A., Cox, L., Cvitkovic, M., Dias, A., Douangamath, A., Duberstein, S., Dudgeon, T., Dunnett, L., Eastman, P. K., Erez, N., Fairhead, M., Fearon, D., Fedorov, O., Ferla, M., Foster, H., Foster, R., Gabizon, R., Gehrtz, P., Gileadi, C., Giroud, C., Glass, W. G., Glen, R., Glinert, I., Gorichko, M., Gorrie-Stone, T., Griffen, E. J., Heer Abstract: Herein we provide a living summary of the data generated during the COVID Moonshot project focused on the development of SARS-CoV-2 main protease (Mpro) inhibitors. Our approach uniquely combines crowdsourced medicinal chemistry insights with high throughput crystallography, exascale computational chemistry infrastructure for simulations, and machine learning in triaging designs and predicting synthetic routes. This manuscript describes our methodologies leading to both covalent and non-covalent inhibitors displaying protease IC50 values under 150 nM and viral inhibition under 5 uM in multiple different viral replication assays. Furthermore, we provide over 200 crystal structures of fragment-like and lead-like molecules in complex with the main protease. Over 1000 synthesized and ordered compounds are also reported with the corresponding activity in Mpro enzymatic assays using two different experimental setups. The data referenced in this document will be continually updated to reflect the current experimental progress of the COVID Moonshot project, and serves as a citable reference for ensuing publications. All of the generated data is open to other researchers who may find it of use. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.12.336412v1?rss=1 Authors: Zhen, Y., Ewert, K. K., Fisher, W. S., Steffes, V. M., Li, Y., Safinya, C. R. Abstract: Lipid-based carriers of the hydrophobic drug paclitaxel (PTX) are used in clinical trials as next-generation agents for cancer chemotherapy. Improving the loading capacity of these carriers requires enhanced PTX solubilization. We compared the solubility of PTX in cationic liposomes (CLs) with lipid tails containing one (oleoyl; C18:1 {Delta}9 DOTAP/DOPC) or two (linoleoyl; C18:2 {Delta}9 DLinTAP/DLinPC) cis double bonds with newly synthesized cationic DLinTAP (2,3-dilinoleoyloxy-propyl-trimethyl-ammonium methylsufate). We used differential-interference-contrast microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams representing the time-dependence of PTX solubility as a function of PTX content in the membrane. Replacing tails bearing one cis double bond (DO lipids) with those bearing two (DLin lipids) significantly increased PTX membrane solubility in CLs. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the membrane solubility limit which has been the focus of most previous fundamental studies and clinical trials) in DOTAP/DOPC CLs. The large increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX upon replacement of oleoyl by linoleoyl tails, rather than by the transition in membrane structure from lipid bilayers to inverse cylindrical micelles observed in small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against human prostate cancer (PC3) cells from measurements of the IC50 of PTX cytotoxicity was unaffected by changing the lipid tails, and toxicity of the CL carrier alone was negligible. Moreover, efficacy was approximately doubled against human melanoma (M21) cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. The findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading well over the typically used 3 mol% while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.323741v1?rss=1 Authors: Panter, F., Bader, C. D., Mueller, R. Abstract: Soil dwelling bacteria such as myxobacteria defend themselves by using secondary metabolites to inhibit growth of competing microorganisms. In this work we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous recombination mediated exchange of the native promoter sequence against a vanillate inducible system led to production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A-G. The sandarazol structure contains intriguing features such as an -chlorinated ketone, an epoxyketone and a (2R)-2-amino-3-(N,N-dimethylamino)-propionic acid building block. In depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthesis steps. The chlorinated congener sandarazol C shows an IC50 value of 0.5 M against HCT 116 cells and a MIC of 14 M against Mycobacterium smegmatis, which points at the sandarazol's potential function as defensive secondary metabolites or toxins. The sandarazols' BGC location on pSa001 is one of the very few example of large multimodular BGCs on a replicative plasmid, whose existence points at the mechanism of horizontal gene transfer events of entire multimodular BGCs to exchange chemical warfare capabilities between bacterial species. Copy rights belong to original authors. Visit the link for more info

The cover for issue 39 of Oncotarget features Figure 4, "Apoptosis assay of NRXN1-targeted ADC at IC50 dose calculated by growth inhibition curves," by Yotsumoto, et al. which reported that the authors identified transmembrane proteins overexpressed specifically in SCLC with little or no expression in normal tissues and decided to focus on the cell adhesion molecule neurexin-1. The cell surface overexpression of NRXN1 was confirmed using flow cytometry in SCLC cell lines. The combination of a primary anti-NRXN1 monoclonal antibody and a secondary ADC exhibited anti-tumor activity in SCLC cell lines. Moreover, the knockout of NRXN1 in SHP77 cells resulted in a loss of the anti-tumor activity of NRXN1-mediated ADC therapy. Thus, NRXN1 could be a novel target for ADC therapy for the treatment of SCLC that is worth further research. Dr. Daiya Takai from The University of Tokyo Hospital and Dr. Takuma Yotsumoto from The University of Tokyo Graduate School of Medicine said, "Small cell lung cancer (SCLC) accounts for 10–15% of lung cancer, and its prognosis has remained relatively dismal for years." Considering the high sensitivity of SCLC to chemotherapy, the selective delivery of a cytotoxic agent using ADC could be a novel treatment strategy for SCLC. Five ADCs have been approved by The Food and Drug Administration: Brentuximab vedotin for Hodgkin lymphoma Ado-trastuzumab emtansine for HER2-positive metastatic breast cancer Inotuzumab ozogamicin for acute lymphoblastic leukemia Gemtuzumab ozogamicin for CD33-positive acute myeloid leukemia, and Trastuzumab deruxtecan for unresectable or metastatic HER2-positive breast cancer patients who have received two or more prior anti-HER2-based regimens in a metastatic setting. In SCLC, DLL3, a cell surface Notch ligand that appears to be a direct downstream target of ASCL1, has been identified as a novel target for ADCs. In this study, the Oncotarget authors aimed to identify novel molecular targets for ADCs in SCLC. They herein report that NRXN1-mediated ADC exhibited anti-tumor activity in vitro, and thus NRXN1 could be a novel target of ADCs for SCLC. The Takai/Yotsumoto Research Team concluded in their Oncotarget Research Paper "we identified NRXN1 as a new target for ADCs by screening membrane proteins using a computational-biological approach. The combination of the primary anti-NRXN1 monoclonal antibody and the secondary ADC exhibited anti-tumor activity in an NRXN1-expression dependent manner. NRXN1 could be a novel potential target of ADCs for SCLC that is worth further research." Sign up for free Altmetric alerts about this article DOI - https://doi.org/10.18632/oncotarget.27718 Full text - https://www.oncotarget.com/article/27718/text/ Correspondence to - Daiya Takai - dtakai-ind@umin.ac.jps and Takuma Yotsumoto - tyotsumoto-ths@umin.ac.jp Keywords - antibody-drug conjugates, small cell lung cancer, novel molecular targets, NRXN1, cell adhesion molecule About Oncotarget Oncotarget is a weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit http://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957x105

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.21.299776v1?rss=1 Authors: Zaidman, D., Gehrtz, P., Filep, M., Fearon, D., Prilusky, J., Duberstein, S., Cohen, G., Owen, D., Resnick, E., Strain-Damerell, C., Lukacik, P., Covid-Moonshot Consortium,, Barr, H., A. Walsh, M., von Delft, F., London, N. Abstract: Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 M. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.15.299164v1?rss=1 Authors: Ma, C., Hu, Y., Townsend, J. A., Lagarias, P., Marty, M. T., Kolocouris, A., Wang, J. Abstract: There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (Mpro) is one of the most extensively studied drug targets. Mpro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, Mpro has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that Mpro inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as Mpro inhibitors, with several of which also showed antiviral activity in cell culture. In this study, we investigated the mechanism of action of six previously reported Mpro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of Mpro by these six compounds is non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit Mpro, but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 2Apro and 3Cpro from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC50 values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 Mpro inhibitors, and urge the scientific community to be stringent with hit validation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.05.283978v1?rss=1 Authors: Al-Hamashi, A. A., Chen, D., Deng, Y., Dong, G., Huang, R. Abstract: Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC50) of 2.8 nM and

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.28.271569v1?rss=1 Authors: Wang, S., Sun, Q., Xu, Y., Pei, J., Lai, L. Abstract: The COVID-19 pandemic calls for rapid development of effective treatments. Although various drug repurpose approaches have been used to screen the FDA-approved drugs and drug candidates in clinical phases against SARS-CoV-2, the coronavirus that causes this disease, no magic bullets have been found until now. We used directed message passing neural network to first build a broad-spectrum anti-beta-coronavirus compound prediction model, which gave satisfactory predictions on newly reported active compounds against SARS-CoV-2. Then we applied transfer learning to fine-tune the model with the recently reported anti-SARS-CoV-2 compounds. The fine-tuned model was applied to screen a large compound library with 4.9 million drug-like molecules from ZINC15 database and recommended a list of potential anti-SARS-CoV-2 compounds for further experimental testing. As a proof-of-concept, we experimentally tested 7 high-scored compounds that also demonstrated good binding strength in docking study against the 3C-like protease of SARS-CoV-2 and found one novel compound that inhibited the enzyme with an IC50 of 37.0 M. Our model is highly efficient and can be used to screen large compound databases with billions or more compounds to accelerate the drug discovery process for the treatment of COVID-19. Copy rights belong to original authors. Visit the link for more info

Buying as much loperamide as you possibly can Loperamide history1969- Synthesized (1)1976 FDA Approved as schedule V (2)Jaffe trial of "abuse potential"- https://pubmed.ncbi.nlm.nih.gov/7438696/1982- Descheduled (3)2010-Annually Increasing in # of poison center calls, cases of arrhythmia and hospitalization (4,5,6)2016- Submission to DEA for rescheduling of loperamide denied (7)2019- FDA works with manufactures to reduce package size to 48 tablets (8)Pharmacist knowledge of abuse remains low https://pubmed.ncbi.nlm.nih.gov/32641253/Toxic MechanismFun theories about co evolution of PGP and CYP https://pubmed.ncbi.nlm.nih.gov/10837556/Inhibition of sodium channels, and to a higher affinity, Human Ether a Go-Go Related (HERG) channel leads to prolonged repolarization (9)IC50 for HERG Ikr ~ 40 nm/l (1908 ng/dl), inhibits as low as 10 nm/l (10)Case reports of conduction disturbance with level of 22 ng/ml (14)Levels in fatalities vary but  reported as high as 270 ng/ml in some studies  (15)Prolonged re polarization leads to torsadesEarly after depolarizations may trigger, which are then propagated torsades via re entrant rhythms (11)TreatmentACMT loperamide guidelines (12)Supportive careArrhythmia managementTorsades (13)Electrical cardioversion (terminates re entrant rhythm)Magnesium (prevents early after depolarization)Target Mg >2 and K >4Lidocaine-> Recommended in 2006 Sudden cardiac death guidlines, not mentioned in 2017, however one of the only VT recommended antiarryhtmics that do not prolong QTc (others, sotalol, amiodarone, and procainamide, do)If preceded by bradycardia, Overdrive pacing with isoproterenol to target HR~ 100Beta blockers are recommended in patients with LQTSSodium channel blockade induced wide QRS complex tachycardia (12)Hypertonic sodium to over whelm sodium channel blockade (1-2 amps of 8.4% Sodium Bicarbonate given IV)Where do we go in the future?More research will help us understand the true incidence of how often this occurs and what impact the FDA decisions will haveAny concerned citizen can submit for rescheduling of loperamide. Interested? Reach out at toxtalk1@gmail.comDrug Enforcement Agency. The Controlled Substances Act. Available at: https://www.dea.gov/controlled-substances-act.Florey, Klaus (1991). Profiles of Drug Substances, Excipients and Related Methodology, Volume 19. Academic Press. p. 342. ISBN9780080861142."IMODIUM FDA Application No.(NDA) 017694". U.S. Food and Drug Administration (FDA). 1976.https://www.deadiversion.usdoj.gov/schedules/orangebook/orangebook.pdf.Miller H, Panahi L, Tapia D, Tran A, Bowman JD. Loperamide misuse and abuse. J Am Pharm Assoc (2003). 2017;57(2S):S45eS50.Feldman R, Everton E. National assessment of pharmacist awareness of loperamide abuse and ability to restrict sale if abuse is suspected [published online ahead of print, 2020 Jul 5]. J Am Pharm Assoc (2003). 2020;S1544-3191(20)30264-8. doi:10.1016/j.japh.2020.05.021Eggleston W, Marraffa JM, Stork CM, et al. Notes from the Field: Cardiac Dysrhythmias After Loperamide Abuse — New York, 2008–2016. MMWR Morb Mortal Wkly Rep 2016;65:1276–1277. DOI: http://dx.doi.org/10.15585/mmwr.mm6545a7https://www.chpa.org/PDF/09_05_17_CommentsCitizenPetitionLoperamide.aspxhttps://www.fda.gov/drugs/drug-safety-and-availability/fda-limits-packaging-anti-diarrhea-medicine-loperamide-imodium-encourage-safe-useKang J, Compton DR, Vaz RJ, Rampe D. Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic. Naunyn Schmiedebergs Arch Pharmacol. 2016;389(10):1133-1137. doi:10.1007/s00210-016-1286-7Klein MG, Haigney MCP, Mehler PS, Fatima N, Flagg TP, Krantz MJ. Potent Inhibition of hERG Channels by the Over-the-Counter Antidiarrheal Agent Loperamide. JACC Clin Electrophysiol. 2016;2(7):784-789. doi:10.1016/j.jacep.2016.07.008https://www.sciencedirect.com/science/article/pii/S1880427611800050Eggleston W, Palmer R, Dubé PA, et al. Loperamide toxicity: recommendations for patient monitoring and management. Clin Toxicol (Phila). 2020;58(5):355-359. doi:10.1080/15563650.2019.1681443Al-Khatib SM, Stevenson WG, Ackerman MJ, et al. 2017 AHA/ACC/HRS Guideline for Management of Patients With Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Rhythm Society [published correction appears in J Am Coll Cardiol. 2018 Oct 2;72(14):1760]. J Am Coll Cardiol. 2018;72(14):e91-e220. doi:10.1016/j.jacc.2017.10.054Marraffa JM, Holland MG, Sullivan RW, et al. Cardiac conduction disturbance after loperamide abuse. Clin Toxicol (Phila). 2014;52(9):952-957. doi:10.3109/15563650.2014.969371Miller H, Panahi L, Tapia D, Tran A, Bowman JD. Loperamide misuse and abuse. J Am Pharm Assoc (2003). 2017;57(2S):S45-S50. doi:10.1016/j.japh.2016.12.079

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.25.266361v1?rss=1 Authors: De Leo, F., Giacomo, Q., De Marchis, F., Malisa, M. V., Bianchi, M. E., Musco, G. Abstract: HMGB1 is a key molecule that both triggers and sustains inflammation following infection or injury, and is involved in a large number of pathologies, including cancer. HMGB1 participates to the recruitment of inflammatory cells forming a heterocomplex with the chemokine CXCL12 (HMGB1/CXCL12), herewith activating the G-protein coupled receptor CXCR4. Thus, identification of molecules that disrupt this heterocomplex can offer novel pharmacological opportunities to treat inflammation related diseases. To identify new HMGB1/CXCL12 inhibitors we have performed a study on the ligandability of the single HMG boxes of HMGB1 followed by a virtual screening campaign on both HMG boxes using Zbc Drugs and three different docking programs (Glide, AutoDock Vina, AutoDock 4.2.6). The best poses in terms of scoring functions, visual inspection and predicted ADME properties were further filtered according to a pharmacophore model based on known HMGB1 binders and clustered according to their structures. Eight compounds representative of the clusters were tested for HMGB1 binding by NMR. We identified 5,5' methylenedi-2,3-cresotic acid (2a) as binder of both HMGB1 and CXCL12; 2a also targets the HMGB1/CXCL12 heterocomplex. In cell migration assays 2a inhibited the chemotactic activity of HMGB1/CXCL12 with IC50 in the subnanomolar range, the best documented up to now. These results pave the way for future structure activity relationship studies to optimize the pharmacological targeting of HMGB1/CXCL12 for anti-inflammatory purposes. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.28.223784v1?rss=1 Authors: Liu, W. R., Yang, K. S., Ma, X. R., Ma, Y., Alugubelli, Y. R., Scott, D. A., Vatansever, E. C., Drelich, A. K., Geng, Z. Z., Blankenship, L. R., Sankaran, B., Ward, H. E., Sheng, Y. J., Hsu, J. C., Kratch, K. K., Zhao, B., Liu, J., Li, P., Fierke, C. A., Tseng, C.-T. K., Xu, S., Hayatshahi, H. S. Abstract: The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain beta-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 uM and A549 cells at 0.16-0.31 uM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.24.219998v1?rss=1 Authors: Marahatha, R., Basnet, S., Bhattarai, B. R., Budhathoki, P., Aryal, B., Adhikari, B., Lamichhane, G., Poudel, D. K., Parajuli, N. Abstract: Hypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generates atherosclerosis and cholesterol crystals. {beta}-Hydroxy {beta}-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some commercially available enzyme inhibiting drugs have effectively reduced the cholesterol level, most of them have failed to meet the requirements of being apt drug candidates. Here, we have carried out an in-silico analysis of secondary metabolites that have already shown good inhibitory activity against XO and HMGR. Out of 118 secondary metabolites reviewed, sixteen molecules inhibiting XO and HMGR were taken based on IC50 values reported in vitro assays. Further, receptor-based virtual screening was carried out against secondary metabolites using GOLD Protein-Ligand Docking Software, combined with subsequent post-docking, to study the binding affinities of ligands to the enzymes. In-Silico ADMET analysis was carried out to study their pharmacokinetic properties, followed by toxicity prediction through ProTox-II. The molecular docking of amentoflavone (1) (GOLD score 70.54), and ganomycin I (9) (GOLD score 59.61) evinced that the drug has effectively bind at the competitive site of XO and HMGR, respectively. Besides, 6-paradol (3) and selgin (4) could be potential drug candidates to inhibit XO. Likewise, n-octadecanyl-O--D-glucopyranosyl(6[->]1")-O--D-glucopyranoside (10) could be potential drug candidates to maintain serum cholesterol. In-silico ADMET analysis showed that the sixteen metabolites were optimal within the categorical range in comparison to commercially available XO and HMGR inhibitors, respectively. Toxicity analysis through Protox-II revealed that 6-gingerol (2), ganoleucoin K (11), and ganoleucoin Z (12) are toxic for human use. This computational analysis supports earlier experimental evidence towards the inhibition of XO and HMGR by natural products. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of hypercholesterolemia. Copy rights belong to original authors. Visit the link for more info

06.27.19 )) . . (( lack lust for nurses drifting person is not by the bank of some

Optimizing the receptor binding kinetics of new drugs can have significant benefits, ranging from improved duration of action to enhanced efficacy through the insurmountable antagonism of dynamic physiological systems. Despite this, the kinetics of new receptor ligands are rarely measured early in the drug discovery process, largely because current assays are technically difficult and relatively low-throughput. This webinar episode will review the current methods for measuring receptor kinetics and then describe the development of a novel approach using time-resolved FRET in continuous read mode that is capable of simultaneously measuring the kinetics of hundreds of compounds. This offers the potential for placing a kinetics assay at the top of a screening cascade, negating the need to first run “IC50 curves” to assess affinity at the receptor. It also presents the opportunity to screen fragment libraries at receptors in a kinetic mode. Sponsored by BMGLabtech & CISBio If you'd like to view the original webinar recording then follow the link below: https://www.ddw-online.com/webinars/p321001-the-kinetics-of-drug-receptor-binding:-why-it-is-important-and-how-we-can-measure-it.html To register for our NEW upcoming webinar: ‘Multiplexing Specificity and Species Cross Reactivity Assays in Biologics Discovery’, please follow the link below. https://www.ddw-online.com/webinars/p322511-multiplexing-specificity-and-species-cross-reactivity-assays-in-biologics-discovery.html For more information on Drug Discovery World, head to: https://www.ddw-online.com

Background To overcome the limitations of animal-based experiments, 3D culture models mimicking the tumor microenvironment in vivo are gaining attention. Herein, we investigated an alginate-based 3D scaffold for screening of 5-fluorouracil (5-FU) or/and curcumin on malignancy of colorectal cancer cells (CRC). Methods The potentiation effects of curcumin on 5-FU against proliferation and metastasis of HCT116 cell and its corresponding isogenic 5-FU-chemoresistant cells (HCT116R) were examined in a 3D-alginate tumor model. Results CRC cells encapsulated in alginate were able to proliferate in 3D-colonospheres in a vivo-like phenotype and invaded from alginate. During cultivation of cells in alginate, we could isolate 3 stages of cells, (1) alginate proliferating (2) invasive and (3) adherent cells. Tumor-promoting factors (CXCR4, MMP-9, NF-κB) were significantly increased in the proliferating and invasive compared to the adherent cells, however HCT116R cells overexpressed factors in comparison to the parental HCT116, suggesting an increase in malignancy behavior. In alginate, curcumin potentiated 5-FU-induced decreased capacity for proliferation, invasion and increased more sensitivity to 5-FU of HCT116R compared to the HCT116 cells. IC50 for HCT116 to 5-FU was 8nM, but co-treatment with 5 μM curcumin significantly reduced 5-FU concentrations in HCT116 and HCT116R cells (0.8nM, 0.1nM, respectively) and these effects were accompanied by down-regulation of NF-κB activation and NF-κB-regulated gene products. Conclusions Our results demonstrate that the alginate provides an excellent tumor microenvironment and indicate that curcumin potentiates and chemosensitizes HCT116R cells to 5-FU-based chemotherapy that may be useful for the treatment of CRC and to overcome drug resistance.

Lung cancer is the leading cause of cancer deaths worldwide. Despite advances and progresses in surgery, chemotherapy, and radiotherapy over the last decades, the death rate from lung cancer has remained largely unchanged, which is mainly due to metastatic disease and multi drug resistance. Because of the overall poor prognosis, new treatment strategies for lung cancer patients are urgently needed. The aim of this study was to investigate the interactions between pemetrexed and vinorelbine for human adenocarcinoma via various chemotherapy schedules. Vinorelbine and pemetrexed caused a strong dose-dependent cytotoxic effect in both HCC and cisplatin resistant HCC (HCC-res) cells. The IC50 values of vinorelbine against HCC and HCC-res cells were 10.34±1.12 nM and 9.98±2.12 nM, respectively. The IC50 values of Pemetrexed against these cells were 110.77±17.28 nM and 118.89±18.77 nM respectively. The application of different therapy schedules induced a significant time dependent cell growth inhibition on HCC naïve and cisplatin resistant cells. The therapy scheme of cisplatin→pemetrexed→vinorelbine showed the strongest inhibitory effect on both HCC and HCC-res cells. The application of different therapy schedules on HCC and HCC-res cells increased the percentage of cells undergoing apoptosis, except the application of vinorelbine alone. In both HCC and HCC-res cells, cisplatin→pemetrexed→vinorelbine was found the most effective to induce apoptosis. The application of different therapy schedules on HCC and HCC-res cells increased cytoplasma calcium concentration. Only the application of vinorelbine alone failed to increase calcium concentration in HCC cells. The most elevated calcium concentration was found in the cells treated with cisplatin→pemetrexed→vinorelbine in both HCC and HCC-res cells As a conclusion, the sequential application of cisplatin, vinorelbine and pemetrexed has a synergistic effect in cell growth inhibition, apoptosis induction, and calcium concentration elevation in HCC and HCC-res cells. The calcium overload could lead to apoptosis, which was related to the cell growth inhibitory effect of chemotherapeutics in lung cancer cells. It might cast a light to develop chemotherapy schedules for patients, and to overcome cisplatin resistance in lung cancer.

Das Epstein-Barr Virus (EBV) ist mit einer Reihe von lebensbedrohlichen Krankheiten assoziiert. Dazu zählen unter anderem Nasopharynxkarzinome, Hodgkin-Lymphome und lymphoproliferative Erkrankungen nach Organtransplantationen. Dennoch gibt es bisher keinen wirksamen Therapieansatz, der sich spezifisch mit der Rolle von EBV in diesen malignen Erkrankungen auseinandersetzt. Das latente Membranprotein 1 (LMP1) ist das primäre Onkogen von EBV und essenziell für die Transformation von B-Zellen durch das Virus. Für eine effiziente Transformation von Zellen ist die Aktivierung verschiedener zellulärer Signalwege durch LMP1 notwendig. LMP1 besitzt jedoch keine enzymatische Aktivität und die Induktion der Signalwege ist somit abhängig von der Rekrutierung verschiedener zellulärer Adapterproteine. Die Ausbildung der notwendigen Signalkomplexe wird über zwei C-terminale Aktivierungs-Regionen (CTAR1 und CTAR2) vermittelt. Verschiedene Mitglieder der Tumornekrosefaktor (TNF)-Rezeptor-assoziierten Faktoren (TRAF)-Protein-Familie spielen bei der Induktion der Signalwege durch diese beiden CTAR-Domänen eine zentrale Rolle. Nach grundlegenden Protein-Protein-Interaktionsstudien zwischen LMP1 und rekombinanten TRAF-Proteinen wurde hier die Interaktion zwischen TRAF2 und LMP1 als Zielstruktur für Inhibitoren vorgestellt. TRAF2 ist essenziell für die Aktivierung des NF-κB-Signalweges durch die CTAR1-Domäne und somit für das Überleben EBV-transformierter Zellen. Die Bindung von TRAF2 an LMP1 wurde biochemisch näher charakterisiert und die gewonnen Erkenntnisse verwendet, um ein System zu etablieren, mit dem Inhibitoren gegen den Komplex aus LMP1 und TRAF2 identifiziert werden können. Dieses ELISA-basierte System erfüllt die Anforderungen, die allgemein an hochdurchsatzfähige Systeme gestellt werden. In einem Pilotscreen einer Bibliothek mit Naturstoffen wurden Substanzen identifiziert, die die Bindung von TRAF2 an LMP1 in vitro inhibierten. Die potenteste Substanz inhibierte die Interaktion von TRAF2 und LMP1 mit einem IC50 von 8 µM in diesen in vitro Studien. Weiterhin zeigte diese Substanz eine spezifische biologische Wirkung auf die Vitalität von EBV-transformierten B-Zellen. Zusätzlich konnte in den Protein-Protein-Interaktionsstudien zwischen den verschiedenen TRAF-Proteinen und LMP1 erstmals eine direkte Bindung von TRAF6 an LMP1 gezeigt werden. Entgegen der bisherigen Modellvorstellung, nach der TRAF6 indirekt über Adapterproteine an LMP1 gebunden wird, konnte hier gezeigt werden, dass TRAF6 direkt an die LMP1-Sequenz P379VQLSY innerhalb der CTAR2-Domäne bindet. Diese Sequenz ist essenziell für die Aktivierung verschiedener TRAF6-abhängiger Signalwege durch die CTAR2-Domäne. Auf der Oberfläche von TRAF6 wird die Bindung an LMP1 durch dieselbe Bindetasche vermittelt, über die auch die Interaktion mit zellulären Rezeptoren stattfindet. Diese direkte Interaktion zwischen LMP1 und TRAF6 ist wichtig für die Aktivierung des NF κB-Signalweges durch die CTAR2-Domäne. TRAF6-Mutanten, die nicht mehr in der Lage waren, mit LMP1 zu interagieren, waren ebenfalls nicht mehr dazu fähig, die Induktion von NF κB-Signalen durch die CTAR2-Domäne von LMP1 in embryonalen TRAF6-/- Mausfibroblasten wiederherzustellen. Ebenfalls konnte neben der direkten Bindung von TRAF6 an LMP1 hier eine weitere neue Protein-Protein-Interaktion für TRAF6 beschrieben werden. TRAF6 bindet direkt an das TNF-Rezeptor-assoziierte Todesdomänenprotein (TRADD). Die Interaktion zwischen TRAF6 und TRADD unterscheidet sich jedoch von der Bindung anderer TRAF-Proteine an TRADD. Die in vitro Studien zeigten, dass TRAF6 in der Lage ist, sowohl mit Teilen des N-Terminus, als auch mit Teilen des C-Terminus von TRADD zu interagieren. Diese bisher nicht beschriebene Art der direkten Interaktion von TRAF6 mit TRADD eröffnet neue Einblicke in den Aufbau des LMP1-Signalkomplexes.

Background: Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and alpha granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets. Objectives: To study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood. Methods: Human platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry. Results: NSC23766 (300 mu M) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC50 values for inhibition of TRAP-, collagen-, and atherosclerotic plaque-, were 50 +/- 18 mu M, 64 +/- 35 mu M, and 50 +/- 30 mu M NSC23766 (mean +/- SD, n = 3-7), respectively. In blood containing RGDS to block integrin alpha(IIb)beta(3)-mediated platelet aggregation, NSC23766 (300 mu M) completely inhibited P-selectin expression and reduced ATP-secretion after TRAP and collagen stimulation by 73% and 85%, respectively. In ADP-stimulated PRP, NSC23766 almost completely inhibited P-selectin expression, in contrast to aspirin, which was ineffective. Moreover, NSC23766 (300 mu M) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s(-1)) by 72%. Conclusions: Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in blood stimulated by a wide array of platelet agonists including atherosclerotic plaque. By specifically inhibiting platelet secretion, the pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

The pleiotropic cytokine interleukin-6 (IL-6) is one of the major growth factors for multiple myeloma cells. It was previously shown that IL-6 induces the activation of Src family kinases Hck, Lyn and Fyn and that Hck is associated with the IL-6-receptor beta chain (gp130) via an acidic domain in gp130. In the first part of this thesis the acidic domain is narrowed down from 41 to 18 amino acids by a peptide-based functional screening assay. A derivative of the acidic domain, an 18mer lipopeptide, peptide 18AD, is characterised on the cellular and molecular level. IL-6-dependent growth of human and murine myeloma cells is inhibited with an IC50 of 25-30 µM by the addition of peptide 18AD to the growth medium. These cells remain unaffected by treatment with a control peptide with scrambled sequence (18sc). Furthermore, growth of IL-6-independent myeloma cells is not inhibited by peptide 18AD. In IL-6-dependent cells, peptide 18AD causes the same degree of apoptosis induction as IL-6 deprivation. On the molecular level it is shown by peptide competition assays that the association of Hck and gp130 is inhibited by peptide 18AD in a concentration dependent way. Peptide 18AD blocked the IL-6-induced activity of the Src family kinases Hck, Lyn and Fyn. Results from different factor-dependent cell lines demonstrate a common mechanism of the molecular peptide effects on Src family kinase activity, but the involved pathways downstream of the kinases appear to differ among species and cell lines tested. Treatment of human IL-6-dependent myeloma cells with peptide 18AD reduced the activating tyrosine phosphorylation of the signal transducer and activator of transcription 3 (STAT3), while STAT3 activation remains unaffected in murine cells. The Co-immunoprecipitations from different overexpressed receptor-deletionmutants and Hck confirms that the association is mainly due to the interaction between the kinase and a 9 amino acids spanning region within the acidic domain which carries the highest accumulation of acidic residues. Apparently, a sequence of 8-9 amino acids within gp130 with the prevalence of acidic side chains resembles a potential pseudosubstrate domain of tyrosine kinases and is responsible for localisation and activation of Src family kinases in response to receptor stimulation. The identification of sequence motives similar to the acidic domain of gp130 in other cytokine receptors, receptor tyrosine kinases and adapter proteins by an in silicio motive scan might suggest a general role of such motives. This could be the efficient recruitment of cytoplasmic kinases to signalling complexes at the time of ligand stimulation. Here, the importance of the acidic domain-kinase interaction for the IL-6-signaling pathway is shown by the growth-inhibiting effect of peptide 18AD on myeloma cells. In the second part, a novel sequence and celltype-specific anti-myeloma agent, peptide 1A, is characterised. It was initially designed as the negative control for a "reverse alanine scan" to define the role of the acidic residues within the sequence of peptide 18AD. Unexpectedly it turned out to be at least a 25-fold more potent growth inhibitor of myeloma cells than peptide 18AD. Similar molecular bases of the observed effects of peptide 18AD and peptide 1A are very unlikely, because in contrast to peptide 18AD, peptide 1A efficiently kills IL-6-independent myeloma cells as well. Moreover, an excess of IL-6 fails to rescue the cells from peptide 1A-induced cell death. Peptide 1A shows, as tested so far, no effects on cells derived from non-tumor tissue or tumor cells of various origins other than multiple myeloma. Peptide 1A specifically kills myeloma cells by the induction of apoptosis and severely disturbs cell cycle progression. Apoptotic cell death induction by peptide 1A is shown by peptide 1A-induced caspase-3 activation and the cleavage of PARP, a substrate of effector caspases. Peptide-induced cell death can partly be inhibited by co-treatment with the pan-caspase inhibitor ZVAD-fmk. Major survival pathways like the STAT3, PI3K/Akt and the MAPK pathway are inhibited in peptide 1A treated cells. If a biotinylated version of the peptide is added to the growth medium, it is incorporated into human myeloma cells and localised in defined regions of the cytoplasm of myeloma cells. Here some of the molecular mechanisms of peptide 1A-induced cell death are elucidated. However, the direct molecular target(s) are still unknown. Despite the potential difference in the molecular mechanisms, both peptides 18AD and 1A are expected to prove useful for developing derivatives with possible applications for the treatment of multiple myeloma.

Aktivierende Mutationen in der juxtamembranösen Region (FLT3-Längenmutationen, FLT3-LM; FLT3- Interne Tandemduplikation, FLT3-ITD) und der Tyrosinkinase-Domäne (FLT3-TKD, FLT3-D835) von FLT3 stellen die häufigsten genetischen Alterationen in der AML dar und definieren eine klinisch-prognostische-Subgruppe in der AML. In der vorliegenden Arbeit wurden zwei neue Mutationen in FLT3 identifiziert und charakterisiert. Die erste Mutation (FLT3-840GS) wurde in zwei Patientenproben nachgewiesen. Moleculargenetisch stellte diese eine Längenmutation in Exon 20 dar, und war durch eine 6 bp-grosse Insertion bzw. Glycin+Serin Insertion in der Aktivierungsschleife der katalytischen Domäne zwischen den Kodons 840 und 841 nahe der Aktivierungsschleife verursacht. Die zweite Mutation, eine aktivierende Punktmutation in der JM-Region von FLT3 (FLT3-V592A) wurde in den AML-Zelllinien MonoMac 6 und MonoMac 1 identifiziert. Die funktionelle Analyse ergab, dass diese Mutanten eine konstitutive Tyrosinphosphorylierung aufweisen und zu einem IL-3 unabhängigem Wachstum in Ba/F3 Zellen führen, welches durch einen spezifischen Proteintyrosinekinase (PTK) Inhibitor gehemmt werden konnte. Diese Ergebnisse zeigen, dass neben den bisher beschriebenen noch weitere aktivierende Mutationen in der JM- und der katalytischen Domäne in FLT3 Gen existieren. Die weiteren Untersuchungen zeigten, dass verschiedene FLT3-TKD (D835) Mutationen eine deutlich unterschiedliche Empfindlichkeit gegenüber selektiven FLT3 PTK Inhibitoren aufwiesen. Weiter wurde gezeigt, dass durch kontinuierliche Exposition von FLT3-ITD transformierten Leukämiezellen mit dem FLT3 PTK Inhibitor SU5614 in vitro resistente Mutanten generiert werden können. Die molekulare und funktionelle Charakterisierung dieser SU5614-resistenten Zelllinien (Ba/F3 FLT3-ITDR1-4) ergab, dass spezifische Mutationen in der TKD-Domäne ursächlich für die Inhibitorresistenz verantwortlich waren. Die FLT3-ITD-R1-4 Zellen wiesen somit Doppelmutationen im FLT3-Gen (LM + TKD) auf und waren durch eine 7-26-fach höhere IC50 gegenüber dem Inhibitor gekennzeichnet. Solche Doppelmutanten von FLT3 wurden mittels in- vitro- Mutagenese generiert und rekapitulieren in Ba/F3 Zellen den SU5614-resistenten Phänotyp. Diese Ergebnisse zeigen, dass prä-existierende oder erworbene FLT3-TKD Mutationen Resistenzen gegenüber FLT3-PTK Inhibitoren in vitro induzieren können. Diese Befunde stellen die molekulare Basis für zelluläre Resistenzen gegenüber FLT3-PTK Inhibitoren bei Patienten mit AML dar.

GABA transporters GAT-1, GAT-2 and GAT-3 are new targets for drug design. The substitution of the nitrogen atoms in Nicopetic acid (11), Guvacine (12) and cis-4- Hydroxynicopetic acid (13) with appropriate bulky lipophilic groups resulted in very potent GABA uptake inhibitors for GAT-1 as well as for GAT-3. Pyrrolidine-2-acetic acid derivatives with the three N-substituents 24a-c (Scheme 54) also showed a highly potent inhibition at GAT-1 and GAT-3, respectively. My intention was to investigate how the potency of pyrrolindine-2-carboxylic acid derivatives and of pyrrolidine-2-acetic acid derivatives at GAT-1 and GAT-3 is influenced by the introduction of a hydroxy group or both of a hydroxy and a (4-methoxy)phenyl group at C-4. For this study, the N-substituents 24a-d were chosen. Thus, the four series of pyrrolidine derivatives 20-23 shown below were designed as potential GABA uptake inhibitors. L-trans-4-hydroxypyrrolidine [(2S,4R)-25] was chosen as a precursor, from which the four key intermediates (2S,4R)-38, (2R,4R)-38, (2S,4R)-86 and (2R,4R)-64 were synthesized. The known compounds (2S,4R)-38 and (2R,4R)-38 were prepared from (2S,4R)-25 according to literature procedures. N-protection of (2S,4R)-25 with Cbz group gave (2S,4R)-58 (90% yield). After a series of reactions  electrolysis (97% yield), O-silyl protection (85%), nucleophilic addition of 1- ethoxy-1-(trimethylsilyloxy)ethene (cis-71 79%; trans-71 9%) and finally O-deprotection (88%) and N-deprotection (89%)  (2R,4R)-64 was obtained in 46% overall yield [from (2S,4R)-25]. After the reaction conditions for the conversion of 70 into 71 have been optimized, the best stereoselectivity [a ratio of cis/trans 97:3; yield cis-71 74%, trans-(2S,4R)-71 1.7%] and a good yield of 88% (cis 79%, trans 9%) were achieved. BF3⋅Et2O appeared to be slightly better for a higher stereoselectivity than TiCl4. (2S,4R)-83 was obtained in 90% yield by protecting the hydroxy group of (2S,4R)-58. An Arndt-Eistert reaction (64% yield) starting from (2S,4R)-83 followed by a simultaneous N,Odeprotection (90% yield) of (2S,4R)-85 led to (2S,4R)-86 in 47% overall yield [from (2S,4R)- 25]. As illustrated in Scheme 58 and 59, (2S,4R)-38 and (2S,4R)-86, (2R,4R)-38 and (2R,4R)-64 were used as starting materials for the synthesis of the N-substituted target compounds (2S,4R)-40a-b, (2S,4S)-40a-b, (2R,4S)-40a-b, (2R,4R)-40a-b, (2S,4R)-89a-d, (2S,4S)-89a-d, (2R,4R)-89a-d and (2R,4S)-89a-d. N-alkylation of these four starting materials with the halides of 24a-d yielded the corresponding tertiary amines. Mitsunobu reactions gave access to the stereoisomers by inversion of the stereocenter at C-4 of the pyrrolidine ring. Finally upon hydrolysis, all the N-substituted pyrrolidine derivatives with a 2-carboxylic acid side chain or a 2-acetic acid side chain were obtained. In the same manners (scheme 58 and 59), the series 96 also were synthesized and finally their hydrogenolysis over Pd-C provided each of four stereoisomers 97. The N-substituted 4-oxopyrrolidine derivatives (2S)-52a-b and (2S)-100a-c (see Scheme 61) were prepared (81-92% yields) via Swern oxidation, and fortunately, the acid-sensitive Nsubstituent 24b was not affected. The N-Cbz-protected 4-oxopyrrolidine derivatives 60 and 108 (Scheme 61) were prepared in good yield (71-78%) via Jones’ oxidation, but in low yield (10-27%) via Swern oxidation. The addition reactions of the organometallic reagents to the N-substituted pyrrolidine derivatives 52a-b and (2S)-100b-c were carried out in two different ways. Depending on the starting material and the employed organometallic reagent, two different results were obtained: Under condition A [(4-MeOC6H4)MgBr at –78 °C in ether] the cis addition product (cis refers to the ester group) was formed as the major diastereomer and a good diastereoselectivity was achieved (cis/trans addition from 79:21 to 89:11; total yields 45- 65%); Under condition B [(4-MeOC6H4)MgBr/CeCl3 at –78 °C in THF], the trans addition product was obtained as a major diastereomer (cis/trans addition from 30:70 to 17:83; total yields 32-48%). In the case of the N-Cbz-protected pyrrolidine derivative (2S)-60, a single diastereomer (2S,4R)-61 was formed in 56% yield under condition B [(4-MeOC6H4)MgBr/CeCl3 in THF at – 60 °C for 4 h]. However, for the homologous (2S)-108 under the same conditions, (2S,4R)- 109 (25% yield) and a side product (5%) resulting from a simultaneous addition to the ester group were formed. The addition of (4-MeOC6H4)MgBr to (2S)-108 (at –78 °C in ether for 4 h), however, led to (2S,4R)-109 (38% yield) as a single diastereomer. Each of the N-substituted stereoisomers from the reaction above was subjected to a basic hydrolysis, which was followed by hydrogenolysis over Pd-C, where necessary, to afford the free amino acids (70-98% yields). Via the similar synthetic procedures as described above, the rest of the stereoisomers (2R,4R)- 57a-b and (2R,4S)-57a-b, (2R,4R)-104b-c and (2R,4S)-104b-c, (2R,4S)-63 and (2R,4S)-111 were obtained from (2R,4R)-39a-b, (2R,4R)-88b-c, (2R)-60 and (2R,4R)-73. The relative stereochemistry of the products obtained from the addition of the organometallic reagents to the 4-oxopyrrolidine derivatives was determined by chemical correlation and NOE measurements. NOE experiments performed with (2S,4R)-114 revealed that the phenyl group and H-2 are cis to each other. As important signals for the assignment overlapped in the 1H NMR spectrum of (2S,4R)-115, the NOE experiments were performed with (2S,4R)-111, which is the sodium salt of (2S,4R)-115. The NOE measurement revealed that the phenyl group is located cis to H- 2, thus, (2S,4R)-111 and (2S,4R)-115 being of the stereochemistry shown. The N-alkylation of (2S,4R)-114 with the bromide of 24a, and of (2S,4R)-115 with the bromide of 24c led to (2S,4R)-53a and (2S,4R)-102c, respectively. With these compounds as references, also the stereochemistry of all related compounds differing only in side chain on the amino nitrogen could be deduced. The target compounds obtained in this study were evaluated for their biological activities. Membrane fractions from frontal cortex of bovine brain (or porcine brain) were utilized to study the inhibitory potency of the test compounds regarding the GAT-1-mediated GABAuptake. For the determination of the potency as GAT-3 inhibitors, membrane fractions from brain stem of bovine brain (or porcine brain) were used. As compared to the corresponding 4-unsubstituted compounds with (2S) configuration (IC50 2.56 µM at GAT-1) and with (2R) configuration (IC50 18.5 µM), the 40a-b series containing a 4-hydroxy group showed a significant drop in the inhibitory potency at both GAT-1 and GAT-3, only one compound [(2R,4R)-40a] showed a reasonable potency at GAT-1 (IC50 9.4 µM) and no one of them for GAT-3 (IC50 > 100 µM). (2S,4S)-89a (IC50 3.29 µM at GAT-1) and (2S,4S)-89c (5.14 µM), (2S,4R)-89a (4.92 µM) and (2S,4R)-89c (3.15 µM) exhibiting a 4-hydroxypyrrolidine-2-acetic acid skeleton showed an inhibitory potency at GAT-1, which was only one order of magnitude lower than the potency of corresponding compounds with (2S) configuration without the 4-hydroxy group (with Nsubstituent 24a: IC50 0.40 µM and with N-substituent 24c: 0.34 µM). (2R,4S)-89b was the most potent inhibitor at GAT-3 (IC50 19.9 µM) of all the stereoisomers of series 89a-d and showed a much higher potency than its isomer (2R,4R)-89b (126 µM). According to these data a 4-hydroxy group is detrimental to the potency at both GAT-1 and GAT-3, and (2S)-configuration of the pyrrolidine-2-acetic acid unit is crucial for a reasonable potency at GAT-1. As compared to the 40b series, some stereoisomers of the 57b series, the latter exhibiting a (4-methoxy)phenyl group at C-4 of the pyrrolidine ring, showed an increased potency as inhibitors at GAT-1 and GAT-3 [e.g. (2S,4R)-57b: IC50 29.7 µM at GAT-3; (2R,4S)-57b: IC50 of 38.0 µM at GAT-3]. In contrast, the introduction of a (4-methoxy)phenyl group into C-4 of the 89b-c series, resulting in the compounds of 104b-c, gave rise to diverse biological results. As compared with (2R,4S)-89b, (2R,4R)-104b displayed a loss of inhibitory potency at GAT- 3 but some enhancement at GAT-1.

Background/Aim: Patients with idiopathic focal segmental glomerulosclerosis (FSGS) often develop a recurrence of the disease after kidney transplantation. In a number of FSGS patients, plasmapheresis and immunoadsorption procedures have been shown to transiently reduce proteinuria and are thought to do this by eliminating a circulating factor. Direct cellular effects of eluates from immunoadsorption procedures on podocytes, the primary target of injury in FSGS, have not yet been reported. Methods: Eluates were derived from antibody-based immunoadsorption of a patient suffering from primary FSGS, a patient with systemic lupus erythematosus, and a healthy volunteer. The cytosolic free Ca2+ concentration ({[}Ca2+](i)) of differentiated podocytes was measured by single-cell fura-2 microfluorescence measurements. Free and total immunoreactive kinin levels were measured by radioimmunoassay. Results: FSGS eluates increased the {[}Ca2+](i) levels concentration dependently (EC50 0.14 mg/ml; n = 3-19). 1 mg/ml eluate increased the {[}Ca2+](i) values reversibly from 82 +/- 12 to 1,462 +/- 370 nmol/l, and then they returned back to 100 16 nmol/l (n = 19). The eluate-induced increase of {[}Ca2+](i) consisted of an initial Ca2+ peak followed by a Ca2+ plateau which depended on the extracellular Ca2+ concentration. The eluate-induced increase of {[}Ca2+](i) was inhibited by the specific B-2 kinin receptor antagonist Hoe 140 in a concentration-dependent manner (IC50 2.47 nmol/l). In addition, prior repetitive application of bradykinin desensitized the effect of eluate on {[}Ca2+](i). A colonic epithelial cell line not reacting to bradykinin did not respond to eluate either (n = 6). Similar to FSGS eluates, the eluate preparations of both the systemic lupus patient and the healthy volunteer led to a biphasic, concentration-dependent {[}Ca2+](i) increase in poclocytes which again was inhibited by Hoe 140. Free kinins were detected in all eluate preparations. Conclusion: The procedure of antibody-based immunoadsorption leads to kinin in the eluate which elevates the {[}Ca2+](i) level of podocytes via B-2 kinin receptors. Copyright (C) 2002 S. Karger AG, Basel.