Development and optimization of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: Evaluation of 5676 Phase 1 Passed Structures.

Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

In vitro activity of itraconazole against SARS-CoV-2.

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).

A new modeling approach for quantifying expert opinion in the drug discovery process.

Expert opinion plays an important role when choosing clusters of chemical compounds for further investigation. Often, the process by which the clusters are assigned to the experts for evaluation, the so-called selection process, and the qualitative ratings given by the experts to the clusters (chosen/not chosen) need to be jointly modeled to avoid bias. This approach is referred to as the joint modeling approach. However, misspecifying the selection model may impact the estimation and inferences on parameters in the rating model, which are of most scientific interest. We propose to incorporate the selection process into the analysis by adding a new set of random effects to the rating model and, in this way, avoid the need to model it parametrically. This approach is referred to as the combined model approach. Through simulations, the performance of the combined and joint models was compared in terms of bias and confidence interval coverage. The estimates from the combined model were nearly unbiased, and the derived confidence intervals had coverage probability around 95% in all scenarios considered. In contrast, the estimates from the joint model were severely biased under some form of misspecification of the selection model, and fitting the model was often numerically challenging. The results show that the combined model may offer a safer alternative on which to base inferences when there are doubts about the validity of the selection model. Importantly, thanks to its greater numerical stability, the combined model may outperform the joint model even when the latter is correctly specified.

Impact of selection bias on the evaluation of clusters of chemical compounds in the drug discovery process.

Expert opinion plays an important role when selecting promising clusters of chemical compounds in the drug discovery process. Indeed, experts can qualitatively assess the potential of each cluster, and with appropriate statistical methods, these qualitative assessments can be quantified into a success probability for each of them. However, one crucial element often overlooked is the procedure by which the clusters are assigned to/selected by the experts for evaluation. In the present work, the impact such a procedure may have on the statistical analysis and the entire evaluation process is studied. It has been shown that some implementations of the selection procedure may seriously compromise the validity of the evaluation even when the rating and selection processes are independent. Consequently, the fully random allocation of the clusters to the experts is strongly advocated.

A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

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.

Identification of Z-Tyr-Ala-CHN2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

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.

Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor.

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.

Library enhancement through the wisdom of crowds.

We present a novel approach for enhancing the diversity of a chemical library rooted on the theory of the wisdom of crowds. Our approach was motivated by a desire to tap into the collective experience of our global medicinal chemistry community and involved four basic steps: (1) Candidate compounds for acquisition were screened using various structural and property filters in order to eliminate clearly nondrug-like matter. (2) The remaining compounds were clustered together with our in-house collection using a novel fingerprint-based clustering algorithm that emphasizes common substructures and works with millions of molecules. (3) Clusters populated exclusively by external compounds were identified as "diversity holes," and representative members of these clusters were presented to our global medicinal chemistry community, who were asked to specify which ones they liked, disliked, or were indifferent to using a simple point-and-click interface. (4) The resulting votes were used to rank the clusters from most to least desirable, and to prioritize which ones should be targeted for acquisition. Analysis of the voting results reveals interesting voter behaviors and distinct preferences for certain molecular property ranges that are fully consistent with lead-like profiles established through systematic analysis of large historical databases.

Identification of novel inhibitors of rat Mrp3.

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.

Lack of antiviral activity of darunavir against SARS-CoV-2.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: Overall, the data do not support the use of DRV for the treatment of COVID-19.

Monte Carlo simulations using PELE to identify a protein-protein inhibitor binding site and pose.

In silico binding site location and pose prediction for a molecule targeted at a large protein surface is a challenging task. We report a blind test with two peptidomimetic molecules that bind the flu virus hemagglutinin (HA) surface antigen, JNJ7918 and JNJ4796 (recently disclosed in van Dongen et al., Science, 2019, 363). Tests with a series of conventional approaches such as rigid (receptor) docking against available X-ray crystal structures or against an ensemble of structures generated by quick methodologies (NMA, homology modeling) gave mixed results, due to the shallowness and flexibility of the binding site and the sheer size of the target. However, tests with our Monte Carlo platform PELE in two protocols involving either exploration of the whole protein surface (global exploration), or the latter followed by refinement of best solutions (local exploration) yielded remarkably good results by locating the actual binding site and generating binding modes that recovered all native contacts found in the X-ray structures. Thus, the Monte Carlo scheme of PELE seems promising as a quick methodology to overcome the challenge of identifying entirely unknown binding sites and modes for protein-protein disruptors.

Design, Synthesis, and Biological Evaluation of Novel Indoles Targeting the Influenza PB2 Cap Binding Region.

In the search for novel influenza inhibitors we evaluated 7-fluoro-substituted indoles as bioisosteric replacements for the 7-azaindole scaffold of Pimodivir, a PB2 (polymerase basic protein 2) inhibitor currently in clinical development. Specifically, a 5,7-difluoroindole derivative 11a was identified as a potent and metabolically stable influenza inhibitor. 11a demonstrated a favorable oral pharmacokinetic profile and in vivo efficacy in mice. In addition, it was found that 11a was not at risk of metabolism via aldehyde oxidase, an advantage over previously described inhibitors of this class. The crystal structure of 11a bound to influenza A PB2 cap region is disclosed here and deposited to the PDB.

A small-molecule fusion inhibitor of influenza virus is orally active in mice.

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.

Electrophile-induced bromocyclization of gamma,delta-unsaturated ketimines to intermediate 1-pyrrolinium salts and their selective conversion into novel 5-alkoxymethyl-2-aryl-3-chloropyrroles and 2-aroylpyrroles.

N-(1-Aryl-2,2-dichloropent-4-enylidene)amines were efficiently transformed into 5-bromomethyl-1-pyrrolinium bromides via electrophile-induced bromocyclization. The latter pyrrolinium salts were converted into novel 5-alkoxymethyl-2-aryl-3-chloropyrroles by reaction with alkoxides in the corresponding alcohol or in THF. This chemistry clearly deviates from the corresponding gamma,delta-unsaturated alpha,alpha-dialkylaldimines under similar conditions. Furthermore, treatment of 5-bromomethyl-1-pyrrolinium bromides with sodium hydroxide in water afforded a new entry into 2-aroylpyrroles by an unexpected ring transformation of intermediate aziridine derivatives, which could be isolated as well.

Protocols for the Design of Kinase-focused Compound Libraries.

Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.

Potent peptidic fusion inhibitors of influenza virus.

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.

Extending kinome coverage by analysis of kinase inhibitor broad profiling data.

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.

2'-Deoxy-2'-spirocyclopropylcytidine revisited: a new and selective inhibitor of the hepatitis C virus NS5B polymerase.

The current therapy for hepatitis C virus (HCV) infection has limited efficacy, in particular against the genotype 1 virus, and a range of side effects. In this context of high unmet medical need, more efficacious drugs targeting HCV nonstructural proteins are of interest. Here we describe 2'-deoxy-2'-spirocyclopropylcytidine (5) as a new inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC(50) of 7.3 µM measured in the Huh7-Rep cell line and no associated cytotoxicity (CC(50) > 98.4 µM). Computational results indicated high similarity between 5 and related HCV inhibiting nucleosides. A convenient synthesis was devised, facilitating synthesis of multigram quantities of 5. As the exposure measured after oral administration of 5 was found to be limited, the 3'-mono- and 3',5'-diisobutyryl ester prodrugs 20 and 23, respectively, were evaluated. The oral dosing of 23 led to substantially increased exposure to 5 in both rats and dogs.