Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

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.

Identification of Potential Modulators of the RGS7/Gß5/R7BP Complex.

Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein ß subunit (Gß5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gß5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gß5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gß5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity.

Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropin-releasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC50 ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility.

Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 µM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

Effects of high-dose modified-release nicotinic acid on atherosclerosis and vascular function: a randomized, placebo-controlled, magnetic resonance imaging study.

OBJECTIVES: Our aim was to determine the effects of high-dose (2 g) nicotinic acid (NA) on progression of atherosclerosis and measures of vascular function. BACKGROUND: NA raises high-density lipoprotein cholesterol (HDL-C) and reduces low-density lipoprotein cholesterol and is widely used as an adjunct to statin therapy in patients with coronary artery disease. Although changes in plasma lipoproteins suggest potential benefit, there is limited evidence of the effects of NA on disease progression when added to contemporary statin treatment. METHODS: We performed a double-blind, randomized, placebo-controlled study of 2 g daily modified-release NA added to statin therapy in 71 patients with low HDL-C (<40 mg/dl) and either: 1) type 2 diabetes with coronary heart disease; or 2) carotid/peripheral atherosclerosis. The primary end point was the change in carotid artery wall area, quantified by magnetic resonance imaging, after 1 year. RESULTS: NA increased HDL-C by 23% and decreased low-density lipoprotein cholesterol by 19%. At 12 months, NA significantly reduced carotid wall area compared with placebo (adjusted treatment difference: -1.64 mm(2) [95% confidence interval: -3.12 to -0.16]; p = 0.03). Mean change in carotid wall area was -1.1 +/- 2.6 mm(2) for NA versus +1.2 +/- 3.0 mm(2) for placebo. In both the treatment and placebo groups, larger plaques were more prone to changes in size (r = 0.4, p = 0.04 for placebo, and r = -0.5, p = 0.02 for NA). CONCLUSIONS: In statin-treated patients with low HDL-C, high-dose modified-release NA, compared with placebo, significantly reduces carotid atherosclerosis within 12 months. (Oxford Niaspan Study: Effects of Niaspan on Atherosclerosis and Endothelial Function; NCT00232531).