The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

BACKGROUND The role of fatty acid ethyl esters (FAEEs) during human alcoholic pancreatitis is unknown. We compared FAEE levels to their non-esterified fatty acids (NEFA) precursors during alcohol intoxication and clinical alcoholic pancreatitis. The pathophysiology underlying FAEE increase and their role as diagnostic biomarkers for alcoholic pancreatitis was investigated METHODS: A prospective blinded study compared FAEE, NEFA and ethanol blood levels on hospitalization for alcoholic pancreatitis (n=31), alcohol intoxication (n=25) and in normal controls (n=43). Serum FAEEs were measured at admission for non-alcoholic pancreatitis (n=75). Mechanistic cell and animal studies were done. RESULTS Median FAEEs were similarly elevated during alcohol intoxication (205nM; CI 71.8-515nM, P<0.001) and alcoholic pancreatitis (103.1nM; CI 53-689nM, P<0.001), vs. controls (1.7nM; CI 0.02-4.3nM) or non-alcoholic pancreatitis (8nM; CI 1.1-11.5nM). Alcoholic pancreatitis increased serum NEFA (1024± 710 μM vs. 307±185 μM in controls, p<0.05). FAEEs comprised 0.1-2% of the parent NEFA concentrations. FAEES correlated strongly with NEFAs independent of ethanol levels in alcoholic pancreatitis but not during alcohol intoxication. On receiver operating characteristic (ROC) analysis for diagnosing alcoholic pancreatitis, serum FAEE's area under the curve (AUC) was 0.87 (CI 0.78-0.95, P<0.001). In mice and cells, alcohol administration transiently increased all FAEEs. OAEE, was the only FAEE with a sustained increase up to 24 hours by intraperitoneal OA+ ethanol. CONCLUSIONS The sustained, alcohol independent, large (20-50-fold) increase in circulating FAEEs during alcoholic pancreatitis results from their visceral release and mirrors the 2-4-fold increase in parent NEFA. The large AUC of FAEEs on ROC analysis supports their role as alcoholic pancreatitis biomarkers.

This letter is in reference to the study by Freedberg et al1Freedberg D.E. et al.Gastroenterology. 2020; 159: 1129-1131Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar recently published in Gastroenterology. This retrospective analysis of an inpatient cohort admitted to 2 hospitals in New York found that patients with coronavirus disease 2019 (COVID-19) who were treated with famotidine exhibited a lower risk of death or mechanical ventilation as composite outcomes over a 30-day period. The study was based on computational modelling, which proposed famotidine might inhibit viral replication through direct interaction with the 3-chymotrypsin-like protease,2Wu C. et al.Acta Pharm Sin B. 2020; 10: 766-788Crossref PubMed Scopus (1581) Google Scholar and was also preceded by a small, short-term follow-up, outpatient study suggesting that famotidine use was associated with symptomatic improvement.3Janowitz T. et al.Gut. 2020; 69: 1592-1597Crossref PubMed Scopus (87) Google Scholar However, the current study published in Gastroenterology provides additional value because the previous cohort was much smaller in number, included patients without a proven COVID-19 diagnosis, and lacked a control group. The current study by Freedberg et al1Freedberg D.E. et al.Gastroenterology. 2020; 159: 1129-1131Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar brings a few questions to mind, which we hope the authors can answer. In the conclusions, the authors stated, "The study was premised on the assumption that use of famotidine represented a continuation of home use." In the Results section, they also say, "Home use of famotidine was documented on admission medication reconciliation in 15% of those who used famotidine while hospitalized." These points bring up a few questions.1.Does this indicate that the remaining 85% patients given famotidine during hospitalization were using over the counter formulations at home which were not prescribed by a physician?2.Were these formulations continued in-patient?3.What were the formulations of famotidine used by the hospital pharmacies?4.Did some included patients receive concomitant treatment with antacids, particularly calcium-containing compounds?5.It is also mentioned that 28% hospitalized COVID-19 patients received famotidine intravenously. Was there a difference in outcomes between the intravenously treated group and the orally treated group? We ask since popular over the counter famotidine formulations such as Pepcid Complete commonly contain 800 mg calcium carbonate per 10 mg famotidine. The answers to these questions are pathophysiologically relevant as we have recently published a report in your journal,4El-Kurdi B. et al.Gastroenterology. 2020; 159: 1015-1018Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar suggesting that the hypocalcemia commonly seen in severe COVID-19 disease is prognostically and mechanistically relevant to disease outcomes. We proposed that calcium supplementation early in the disease can, by interacting with fatty acids, decrease the lipotoxicity, which may exacerbate the disease and result in organ failure. Using in silico molecular docking screens, famotidine has been characterized as potentially being able to bind papain-like protease (PLpro) and 3 chymotrypsin-like protease (Mpro) of SARS-CoV-2.2Wu C. et al.Acta Pharm Sin B. 2020; 10: 766-788Crossref PubMed Scopus (1581) Google Scholar,5Sen Gupta P.S. et al.J Biomol Struct Dyn. 2020; 24: 1-7Google Scholar To explore this notion further, we downloaded crystal structures of PLpro and Mpro from RCSB.org (PDB IDs 6WX4 and 6LU7, respectively) and imported these to Schrodinger Maestro. The structures were prepared for docking, and famotidine was docked to both proteases using the XP docking protocol. Famotidine was found to dock to PLpro with a GlideScore of –6.86 kcal/mol and to Mpro with a GlideScore of –4.05 kcal/mol. This finding represents a weak, nonspecific binding of famotidine to both PLpro and Mpro, and is in contradiction to previous molecular docking studies. Recently, in vitro experiments have shown that famotidine does not inhibit PLpro or Mpro, and it does not directly inhibit SARS-CoV-2 infection,6Malone R.W. et al.Res Sq. 2020 Jun 23; (rs.3.rs-30934)Google Scholar,7Anson B.J. et al.Res Sq. 2020; (rs.3.rs-26344)Google Scholar supporting our molecular docking data that famotidine does not bind to either protease. It has been hypothesized that famotidine could indirectly treat COVID-19 through antagonism or inverse agonism of histamine signaling as a result of binding to the H2 receptor,6Malone R.W. et al.Res Sq. 2020 Jun 23; (rs.3.rs-30934)Google Scholar but this hypothesis has yet to be rigorously tested. Although the results of the randomized clinical trial on the benefits of intravenous famotidine in treating COVID-19 (NCT04370262) are excitedly awaited; the clues gained by the studies published in both Gastroenterology1Freedberg D.E. et al.Gastroenterology. 2020; 159: 1129-1131Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar,4El-Kurdi B. et al.Gastroenterology. 2020; 159: 1015-1018Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar and Gut,3Janowitz T. et al.Gut. 2020; 69: 1592-1597Crossref PubMed Scopus (87) Google Scholar give hope that COVID-19 could be combated by delving deeper into, and understanding the mechanistic basis of what was observed. Famotidine Use Is Associated With Improved Clinical Outcomes in Hospitalized COVID-19 Patients: A Propensity Score Matched Retrospective Cohort StudyGastroenterologyVol. 159Issue 3PreviewCoronavirus Disease 2019 (COVID-19) caused 2 million cases and more than 150,000 deaths worldwide as of mid-April 2020.1 Clinical trials are under way to assess the efficacy of a variety of antiviral drugs; however, many of these drugs have toxicities and thus far no drug has been proven to improve outcomes in patients with COVID-19. Full-Text PDF ReplyGastroenterologyVol. 160Issue 5PreviewSingh et al1 are interested in the formulation of famotidine received by patients in our study and whether there was concurrent antacid use. In our retrospective study,2 15% of patients who received famotidine during hospitalization for Coronavirus Disease 2019 (COVID-19) had home use of famotidine documented on the electronic medication reconciliation that must be performed at the time of hospital admission (compared with 1% of patients who did not receive famotidine during hospitalization for COVID-19, P < .01). Full-Text PDF

Histone deacetylase 3 (HDAC3) plays an important role in signal-dependent transcription and is dysregulated in diseases such as cancer. Previous studies have shown that the function of HDAC3 requires an activation step, which is mediated by the interactions of HDAC3 with the deacetylase-activation domain (DAD) of nuclear receptor corepressors and inositol tetra phosphate (IP4). However, the role of the unique HDAC3 C terminal region in HDAC3 activation is elusive. Here multiple biochemical, structural, and functional studies show that HDAC3 activation requires a priming step mediated by the C terminus to remodel HDAC3 conformation. We show that multiple C-terminal mutations prevent HDAC3 activation by preventing this C terminus-dependent conformational change. Mechanistically, we demonstrate that the C terminus- mediated function in altering HDAC3 conformation is required for proper complex formation of HDAC3 with DAD and IP4 by allowing HDAC3 to undergo IP4-dependent interaction with DAD. Remarkably, we found that this C terminus function is conformation dependent, being necessary for HDAC3 activation prior to but not after the conformational change. Together, our study defines two functional states of free HDAC3, reveals the complete HDAC3 activation pathway, and links the C terminus function to the specific interaction between HDAC3 and DAD. These results also have implications in how signaling pathways may converge on the C terminus to regulate HDAC3 and suggest that the C terminus- mediated conformational change could represent a new target for inhibiting HDAC3 in diseases such as cancer.

Although most coronavirus disease 2019 (COVID-19) infections are self-limited, some develop into sepsis and multisystem organ failure (MSOF),1 resembling lipotoxic acute pancreatitis.2,3 Understanding underlying mechanisms may guide supportive care while clinical trials are ongoing. Unsaturated fatty acids (UFAs) generated by adipose lipolysis2,3 cause MSOF, including acute lung injury.2 Severe acute pancreatitis and severe COVID-19 share obesity as a risk factor,4 along with lipase elevation,5 hypoalbuminemia,1 and hypocalcemia.