Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID‐19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses ...do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID‐19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell‐based phenotypic assays, the in vitro antiviral activity of itraconazole and 17‐OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Itraconazole demonstrated antiviral activity in human Caco‐2 cells (EC50 = 2.3 µM; 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17‐OH itraconazole, showed inhibition of SARS‐CoV‐2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17‐OH itraconazole resulted in a viral yield reduction in vitro of approximately 2‐log10 and approximately 1‐log10, as measured in both Caco‐2 cells and VeroE6‐eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS‐441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3‐log10 drop and >4‐log10 drop in Caco‐2 cells and VeroE6‐eGFP cells, respectively. Itraconazole and 17‐OH itraconazole exert in vitro low micromolar activity against SARS‐CoV‐2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID‐19 patients in a clinical study (EudraCT Number: 2020‐001243‐15).
Highlights
Itraconazole exerted in vitro low micromolar activity against SARS‐CoV‐2
Remdesivir demonstrated potent antiviral activity, confirming validity of the assay
Itraconazole has since shown no efficacy (study in hospitalized COVID‐19 patients)
•Darunavir showed no in vitro antiviral activity against SARS-CoV-2 (EC50 > 100 μM).•Remdesivir demonstrated potent antiviral activity, confirming validity of the assay.•Overall, the data do not ...support use of darunavir for treatment of COVID-19.
Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 SARS-CoV-2), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives.
Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed.
DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 μM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 μM).
Overall, the data do not support the use of DRV for the treatment of COVID-19.
Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on ...the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for ...SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest ...as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.
The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health ...emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.
•We established a high-throughput, high-content imaging platform to identify coronavirus antivirals.•This platform can simultaneously measure antiviral activity and other cellular features to identify compound liabilities.•The platform can be rapidly adapted to target other cell-based infection models.•We successfully used this method to screen a large library of compounds and to further prioritize relevant compounds.
Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and ...therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.
Multidrug resistance-associated protein (MRP; ABCC gene family) mediated efflux transport plays an important role in the systemic and tissue exposure profiles of many drugs and their metabolites, and ...also of endogenous compounds like bile acids and bilirubin conjugates. However, potent and isoform-selective inhibitors of the MRP subfamily are currently lacking. Therefore, the purpose of the present work was to identify novel rat Mrp3 inhibitors. Using 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) as a model-(pro)substrate for Mrp3 in an oil-spin assay with primary rat hepatocytes, the extent of inhibition of CDF efflux was determined for 1584 compounds, yielding 59 hits (excluding the reference inhibitor) that were identified as new Mrp3 inhibitors. A naive Bayesian prediction model was constructed in Pipeline Pilot to elucidate physicochemical and structural features of compounds causing Mrp3 inhibition. The final Bayesian model generated common physicochemical properties of Mrp3 inhibitors. For instance, more than half of the hits contain a phenolic structure. The identified compounds have an AlogP between 2 and 4.5, between 5 to 8 hydrogen bond acceptor atoms, a molecular weight between 260 and 400, and 2 or more aromatic rings. Compared to the depleted dataset (i.e. 90% remaining compounds), the Mrp3 hit rate in the enriched set was 7.5-fold higher (i.e. 17.2% versus 2.3%). Several hits from this first screening approach were confirmed in an additional study using Mrp3 transfected inside-out membrane vesicles. In conclusion, several new and potent inhibitors of Mrp3 mediated efflux were identified in an optimized in vitro rat hepatocyte assay and confirmed using Mrp3 transfected inside-out membrane vesicles. A final naive Bayesian model was developed in an iterative way to reveal common physicochemical and structural features for Mrp3 inhibitors. The final Bayesian model will enable in silico screening of larger libraries and in vitro identification of more potent Mrp3 inhibitors.
Display omitted
Analysis of the scientific literature based on the Eidogen-Sertanty Kinase Knowledgebase shows that there are 164 and 235 human kinases (from a total of 518) for which more than 10 ligands per kinase ...are known with potencies better than 10 or 100nm, respectively. This explored kinome was extended with kinase broad profiling data using the DiscoveRx and Millipore kinase assay panels. The detailed analysis of the profiling of 3368 selected kinase inhibitors on 456 kinases in the DiscoveRx KINOMEscan format delivers a number of insights. Selective compounds identified through broad kinase inhibitor profiling can be used in target validation, enable jumpstarting new kinase drug discovery projects, and chart new biological space via phenotypic screening.
•Kinome coverage augmented to 331 kinases.•Analysis of KINOMEscan data.•Insights into kinase library design.
The explored kinome was extended with broad profiling using the DiscoveRx and Millipore assay panels. The analysis of the profiling of 3368 selected inhibitors on 456 kinases in the DiscoveRx format delivered several insights. First, the coverage depended on the threshold of the selectivity parameter. Second, the relation between hit confirmation rates and inhibitor selectivity showed unexpectedly that higher selectivity can increase the likelihood of false positives. Third, comparing the coverage of a focused to that of a random library showed that the design based on a maximum number of scaffolds was superior to a limited number of scaffolds. Therefore, selective compounds can be used in target validation, enable the jumpstarting of new kinase drug discovery projects, and chart new biological space via phenotypic screening.
PDF
