Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

A gain-of-function mutation in adenylate cyclase 3 protects mice from diet-induced obesity.

In a screen for genes that affect the metabolic response to high-fat diet (HFD), we selected one line of N-ethyl-N-nitrosourea (ENU)-mutagenized mice, Jll, with dominantly inherited resistance to diet-induced obesity (DIO). Mutant animals had dramatically reduced body weight and fat mass, and low basal insulin and glucose levels relative to unaffected controls. Both white adipose tissue (WAT) and brown adipose tissue (BAT) depots were smaller in mutant animals. Mutant animals fed a HFD gained only slightly more weight than animals fed regular chow, and were protected from hepatic lipid accumulation. The phenotype was genetically linked to a 5.7-Mb interval on chromosome 12, and sequencing of the entire interval identified a single coding mutation, predicted to cause a methionine-to-isoleucine substitution at position 279 of the Adcy3 protein (Adcy3M279I, henceforth referred to as Adcy3Jll). The mutant protein is hyperactive, possibly constitutively so, producing elevated levels of cyclic AMP in a cell-based assay. These mice demonstrate that increased Adcy3 activity robustly protect animals from diet-induced metabolic derangements.

Inhibition of c-Kit is not required for reversal of hyperglycemia by imatinib in NOD mice.

AIM/HYPOTHESIS: Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice. METHODS: The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit(T670I) mice and NOD.c-Kit(wt) littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit(T670I) allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored. RESULTS: In vitro expansion of HSCs from NOD.c-Kit(wt) mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit(T670I) mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice. CONCLUSIONS/INTERPRETATION: The HSC experiment confirmed that, in NOD.c-Kit(T670I) mice, c-Kit is resistant to imatinib. As both NOD.c-Kit(T670I) and NOD.c-Kit(wt) mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.

Substituted 2-phenylimidazopyridines: a new class of drug leads for human African trypanosomiasis.

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogues were prepared, which led to the identification of 64, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity in vitro with an EC50 of 2 nM and displayed reasonable druglike properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent antiparasitic properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African trypanosomiasis.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis.

A search to identify new mechanisms of isoniazid resistance in Mycobacterium bovis led to the isolation of mutants defective in mycothiol biosynthesis due to mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants showed low-level resistance to isoniazid but were highly resistant to ethionamide. This study further illustrates that mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species.

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Polymorphisms of the scavenger receptor class B member 1 are associated with insulin resistance with evidence of gene by sex interaction.

BACKGROUND: Genetic variation in diabetes-associated genes cumulatively explain little of the overall heritability of this trait. We sought to determine whether polymorphisms of the scavenger receptor class B, member I (SCARB1), an estrogen-regulated chromosome 12q24 positional candidate diabetes gene, were associated with type 2 diabetes or insulin resistance in a sex-specific fashion. METHODS: We evaluated 34 haplotype-tagged single-nucleotide polymorphisms (SNPs) of SCARB1 for their association with type 2 diabetes and measures of insulin resistance in two populations: a clinic-based sample of 444 Mexican-American women from Proyecto SALSA and a community-based sample of 830 white women from the Rancho Bernardo Study. RESULTS: We identified significant associations between a tagged SNP in intron 9, rs9919713, and fasting glucose in the SALSA population (P = 2.3 x 10(-4)). In the Rancho Bernardo Study, the same SNP also showed significant association with the related traits homeostasis model assessment for insulin resistance (P = 3.0 x 10(-4)), fasting glucose (P = 1.1 x 10(-3)), and type 2 diabetes (P = 9.0 x 10(-3)). In men from the Rancho Bernardo population, the opposite effect was found (genotype by sex interaction in the Rancho Bernardo population P < 10(-3) for insulin resistance). CONCLUSIONS: Our data support an association between SCARB1 variants and insulin resistance, especially in women, with evidence of significant gene by sex interaction. These findings warrant further investigation in additional populations and prompt exploration of a role for SR-BI in the development of insulin resistance.

Stanniocalcin 2 is an estrogen-responsive gene coexpressed with the estrogen receptor in human breast cancer.

Differences in gene expression are likely to explain the phenotypic variation between hormone-responsive and hormone-unresponsive breast cancers. In this study, DNA microarray analysis of approximately 10,000 known genes and 25,000 expressed sequence tag clusters was performed to identify genes induced by estrogen and repressed by the pure antiestrogen ICI 182 780 in vitro that correlated with estrogen receptor (ER) expression in primary breast carcinomas in vivo. Stanniocalcin (STC) 2 was identified as one of the genes that fulfilled these criteria. DNA microarray hybridization showed a 3-fold induction of STC2 mRNA expression in MCF-7 cells in < or = 3 h of estrogen exposure and a 3-fold repression in the presence of antiestrogen (one-way ANOVA, P < 0.0005). In 13 ER-positive and 12 ER-negative breast carcinomas, the microarray-derived mRNA levels observed for STC2 correlated with tumor ER mRNA (Pearson's correlation, r = 0.85; P < 0.0001) and ER protein status (Spearman's rank correlation, r = 0.73; P < 0.0001). The expression profile of STC2 was further confirmed by in situ hybridization and immunohistochemistry on a larger cohort of 236 unselected breast carcinomas using tissue microarrays. STC2 mRNA and protein expression were found to be associated with tumor ER status (Fisher's exact test, P < 0.005). The related gene, STC1, was also examined and shown to be associated with ER status in breast carcinomas (Fisher's exact test, P < 0.05). This study demonstrates the feasibility of using global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance.