Drug repurposing screens identify chemical entities for the development of COVID-19 interventions.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis.

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.

Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH).

The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC.

Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant.

A variable region fusion strategy was used to generate an immunosuppressive antibody based on a novel "stalk-knob" structural motif in the ultralong complementary-determining region (CDR) of a bovine antibody. The potent Kv1.3 channel inhibitory peptides Moka1-toxin and Vm24-toxin were grafted into different CDRs of the humanized antibodies BVK and Synagis (Syn) using both ß-sheet and coiled-coil linkers. Structure-activity relationship efforts led to generation of the fusion protein Syn-Vm24-CDR3L, which demonstrated excellent selectivity and potency against effector human memory T cells (subnanomolar to picomolar EC50 values). This fusion antibody also had significantly improved plasma half-life and serum stability in rodents compared with the parent Vm24 peptide. Finally, this fusion protein showed potent in vivo efficacy in the delayed type hypersensitivity in rats. These results illustrate the utility of antibody CDR fusions as a general and effective strategy to generate long-acting functional antibodies, and may lead to a selective immunosuppressive antibody for the treatment of autoimmune diseases.

Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of lipogenesis and cholesterol homeostasis. Ligand activation of these receptors in vivo induces expression of the LXR target gene SREBP-1c and increases plasma triglyceride levels. Expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis and an established target of the SREBP-1 pathway, is also induced by LXR ligands. The effects of LXR ligands on FAS expression have been proposed to be entirely secondary to the induction of SREBP-1c. We demonstrate here that LXRs regulate FAS expression through direct interaction with the FAS promoter as well as through activation of SREBP-1c expression. Induction of FAS expression in HepG2 cells by LXR ligands is reduced, but not abolished, under conditions where SREBP processing is suppressed. Moreover, LXR ligands induce FAS expression in CHO-7 cells without altering expression of SREBP-1. We demonstrate that in addition to tandem SREBP sites, the FAS promoter contains a high affinity binding site for the LXR/RXR heterodimer that is conserved in diverse animal species including birds, rodents, and humans. The LXR and SREBP binding sites independently confer LXR responsiveness on the FAS promoter, and maximal induction requires both transcription factors. Transient elevation of plasma triglyceride levels in mice treated with a synthetic LXR agonist correlates with transient induction of hepatic FAS expression. These results indicate that the LXR signaling pathway modulates FAS expression through distinct but complementary mechanisms and suggest that the FAS gene may be a critical target in the control of lipogenesis by LXRs.