Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and In Vitro Metabolic Stability.

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.

Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Patterns of liver gene expression governed by TRbeta.

Several metabolic processes in the liver are regulated by thyroid hormone (T3). Gene expression profiles of livers from normal and TRbeta-deficient mouse strains should allow the classification of rapid and sustained effects of T3, as well as identification of target genes that are dependent on TRbeta. The immediate and long-term T3 regulation of about 4000 genes in livers from hypo- and hyperthyroid wild-type and TRbeta-deficient mice was analyzed using cDNA microarrays. T3 was found to regulate more than 200 genes, and among these, more than 100 were previously not described. Sixty percent of all these genes show dependence on the TRbeta gene for T3 regulation, indicating that TRalpha1 may have previously unknown functions in the liver. Analysis of the gene expression patterns showed a clear functional distinction between rapid (2 h) actions of T3 and late effects, seen after 5 d of sustained T3 treatment. Many metabolic actions were rapidly executed, whereas effects on mitochondrial function, for example, were seen after the sustained T3 treatment. As compared with wild-type controls, TRbeta-/-mice exhibited elevated expression of some target genes and reduced levels of others, indicating that both direct and indirect gene regulation by TRs in liver is complex and involves both ligand-dependent and -independent actions by the major TR isoforms.

Requirement for thyroid hormone receptor beta in T3 regulation of cholesterol metabolism in mice.

T3 potently influences cholesterol metabolism through the nuclear thyroid hormone receptor beta (TRbeta), the most abundant TR isoform in rodent liver. Here, we have tested if TRalpha1, when expressed at increased levels from its normal locus, can replace TRbeta in regulation of cholesterol metabolism. By the use of TRalpha2-/-beta-/- animals that overexpress hepatic TRalpha1 6-fold, a near normalization of the total amount of T3 binding receptors was achieved. These mice are similar to TRbeta-/- and TRalpha1-/-beta-/- mice in that they fail to regulate cholesterol 7alpha-hydroxylase expression properly, and that their serum cholesterol levels are unaffected by T3. Thus, hepatic overexpression of TRalpha1 cannot substitute for absence of TRbeta, suggesting that the TRbeta gene has a unique role in T3 regulation of cholesterol metabolism in mice. However, examination of T3 regulation of hepatic target genes revealed that dependence on TRbeta is not general: T3 regulation of type I iodothyronine deiodinase and the low density lipoprotein receptor were partially rescued by TRalpha1 overexpression. These in vivo data show that TRbeta is necessary for the effects of T3 on cholesterol metabolism. That TRalpha1 only in some instances can substitute for TRbeta indicates that T3 regulation of physiological and molecular processes in the liver occurs in an isoform-specific fashion.

Depressed thermogenesis but competent brown adipose tissue recruitment in mice devoid of all hormone-binding thyroid hormone receptors.

We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression.

A novel assay of cellular stearoyl-CoA desaturase activity of primary rat hepatocytes by HPLC.

The stearoyl-CoA desaturase (SCD) activity is involved in regulation of metabolism, energy storage, and membrane fluidity. However, only few cellular assays have been developed. We describe a simple and robust method to quantitate SCD activity and its inhibition in primary rat hepatocytes. Hepatocytes assimilate stearic acid, with or without modification by SCD, into its lipid pool. To measure the extent of this conversion primary rat hepatocytes were cultivated 4h or overnight with [1-(14)C]18:0 and extracellular fatty acids were washed out. Total cell lipids were then hydrolyzed and extracted. Recoveries of 18:0 were secured with a modified Folch method by addition of 0.1% Triton X-114 to the samples. The extracted fatty acids were dissolved in 85% ethanol and separated by reverse phase HPLC, which took 10 min including column recovery time. [1-(14)C]18:0 and [1-(14)C]18:1(n9) were detected and quantified by on-line flow scintillation analysis. Incubation of the cells with SCD inhibitors resulted in decreased ratios of 18:1/18:0 in dose-dependent manners. The improvements enabled us to establish a novel robust assay based solely on HPLC analysis of cellular SCD activity, which was developed in 12-well format.