Targeting SARS-CoV-2 M3CLpro by HCV NS3/4a Inhibitors: In Silico Modeling and In Vitro Screening.

Currently the entire human population is in the midst of a global pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2). This highly pathogenic virus has to date caused >71 million infections and >1.6 million deaths in >180 countries. Several vaccines and drugs are being studied as possible treatments or prophylactics of this viral infection. M3CLpro (coronavirus main cysteine protease) is a promising drug target as it has a significant role in viral replication. Here we use the X-ray crystal structure of M3CLpro in complex with boceprevir to study the dynamic changes of the protease upon ligand binding. The binding free energy was calculated for water molecules at different locations of the binding site, and molecular dynamics (MD) simulations were carried out for the M3CLpro/boceprevir complex, to thoroughly understand the chemical environment of the binding site. Several HCV NS3/4a protease inhibitors were tested in vitro against M3CLpro. Specifically, asunaprevir, narlaprevir, paritaprevir, simeprevir, and telaprevir all showed inhibitory effects on M3CLpro. Molecular docking and MD simulations were then performed to investigate the effects of these ligands on M3CLpro and to provide insights into the chemical environment of the ligand binding site. Our findings and observations are offered to help guide the design of possible potent protease inhibitors and aid in coping with the COVID-19 pandemic.

Discovery of Novel Small-Molecule Inhibitors of SARS-CoV-2 Main Protease as Potential Leads for COVID-19 Treatment.

The main protease of SARS-CoV-2 virus, Mpro, is an essential element for viral replication, and inhibitors targeting Mpro are currently being investigated in many drug development programs as a possible treatment for COVID-19. An in vitro pilot screen of a highly focused collection of compounds was initiated to identify new lead scaffolds for Mpro. These efforts identified a number of hits. The most effective of these was compound SIMR-2418 having an inhibitory IC50 value of 20.7 µM. Molecular modeling studies were performed to understand the binding characteristics of the identified compounds. The presence of a cyclohexenone warhead group facilitated covalent binding with the Cys145 residue of Mpro. Our results highlight the challenges of targeting Mpro protease and pave the way toward the discovery of potent lead molecules.

Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease patient-derived cells.

Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme ß-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.

Ribosome-Templated Azide-Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry.

Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

Extracellular Acetylated Histone 3.3 Induces Inflammation and Lung Tissue Damage.

Extracellular histones, part of the protein group known as damage-associated molecular patterns (DAMPs), are released from damaged or dying cells and can instigate cellular toxicity. Within the context of chronic obstructive pulmonary disease (COPD), there is an observed abundance of extracellular histone H3.3, indicating potential pathogenic implications. Notably, histone H3.3 is often found hyperacetylated (AcH3.3) in the lungs of COPD patients. Despite these observations, the specific role of these acetylated histones in inducing pulmonary tissue damage in COPD remains unclear. To investigate AcH3.3's impact on lung tissue, we administered recombinant histones (rH2A, rH3.3, and rAcH3.3) or vehicle solution to mice via intratracheal instillation. After 48 h, we evaluated the lung toxicity damage and found that the rAcH3.3 treated animals exhibited more severe lung tissue damage compared to those treated with non-acetylated H3.3 and controls. The rAcH3.3 instillation resulted in significant histological changes, including alveolar wall rupture, epithelial cell damage, and immune cell infiltration. Micro-CT analysis confirmed macroscopic structural changes. The rAcH3.3 instillation also increased apoptotic activity (cleavage of caspase 3 and 9) and triggered acute systemic inflammatory marker activation (TNF-α, IL-6, MCP-3, or CXCL-1) in plasma, accompanied by leukocytosis and lymphocytosis. Confocal imaging analysis confirmed lymphocytic and monocytic/macrophage lung infiltration in response to H3.3 and AcH3.3 administration. Taken together, our findings implicate extracellular AcH3.3 in inducing cytotoxicity and acute inflammatory responses, suggesting its potential role in promoting COPD-related lung damage progression.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

Patient-Derived Phenotypic High-Throughput Assay to Identify Small Molecules Restoring Lysosomal Function in Tay-Sachs Disease.

Tay-Sachs disease is an inherited lysosomal storage disease resulting from mutations in the lysosomal enzyme, ß-hexosaminidase A, and leads to excessive accumulation of GM2 ganglioside. Tay-Sachs patients with the infantile form do not live beyond 2-4 years of age due to rapid, progressive neurodegeneration. Enzyme replacement therapy is not a therapeutic option due to its inability to cross the blood-brain barrier. As an alternative, small molecules identified from high-throughput screening could provide leads suitable for chemical optimization to target the central nervous system. We developed a new high-throughput phenotypic assay utilizing infantile Tay-Sachs patient cells based on disrupted lysosomal calcium signaling as a monitor of diseased phenotype. The assay was validated in a pilot screen on a collection of Food and Drug Administration-approved drugs to identify compounds that could reverse or attenuate the disease. Pyrimethamine, a known pharmacological chaperone of ß-hexosaminidase A, was identified from the primary screen. The mechanism of action of pyrimethamine in reversing the defective lysosomal phenotype was by improving autophagy. This new high-throughput screening assay in patient cells will enable the screening of larger chemical compound collections. Importantly, this approach could lead to identification of new molecular targets previously unknown to impact the disease and accelerate the discovery of new treatments for Tay-Sachs disease.

High-Throughput Ca2+ Flux Assay To Monitor Cyclic Nucleotide-Gated Channel Activity and Characterize Achromatopsia Mutant Channel Function.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.

Optimization of the beta-aminoester class of factor Xa inhibitors. Part 1: P(4) and side-chain modifications for improved in vitro potency.

A systematic modification of the C(3) side-chain of the beta-aminoester class of factor Xa inhibitors and a survey of P(4) variations is described. These changes have resulted in the identification of sub-nanomolar inhibitors with improved selectivity versus related proteases. Coagulation parameters (i.e., APTT doubling concentrations) are also improved.

Optimization of the beta-aminoester class of factor Xa inhibitors. Part 2: Identification of FXV673 as a potent and selective inhibitor with excellent In vivo anticoagulant activity.

Further optimization of the beta-aminoester class of factor Xa (fXa) inhibitors is described culminating in the identification of 9c (FXV673), a potent and selective factor Xa inhibitor with excellent in vivo anticoagulant activity. An X-ray structure of FXV673 bound to human fXa is also presented. Based on its selectivity, potent in vivo activity and favorable pre-clinical safety profile, FXV673 was selected for further development and is currently undergoing clinical trials.