Exploiting high-energy hydration sites for the discovery of potent peptide aldehyde inhibitors of the SARS-CoV-2 main protease with cellular antiviral activity.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

Tryptanthrin Analogs Substoichiometrically Inhibit Seeded and Unseeded Tau4RD Aggregation.

Microtubule-associated protein tau is an intrinsically disordered protein (IDP) that forms characteristic fibrillar aggregates in several diseases, the most well-known of which is Alzheimer's disease (AD). Despite keen interest in disrupting or inhibiting tau aggregation to treat AD and related dementias, there are currently no FDA-approved tau-targeting drugs. This is due, in part, to the fact that tau and other IDPs do not exhibit a single well-defined conformation but instead populate a fluctuating conformational ensemble that precludes finding a stable "druggable" pocket. Despite this challenge, we previously reported the discovery of two novel families of tau ligands, including a class of aggregation inhibitors, identified through a protocol that combines molecular dynamics, structural analysis, and machine learning. Here we extend our exploration of tau druggability with the identification of tryptanthrin and its analogs as potent, substoichiometric aggregation inhibitors, with the best compounds showing potencies in the low nanomolar range even at a ~100-fold molar excess of tau4RD. Moreover, conservative changes in small molecule structure can have large impacts on inhibitory potency, demonstrating that similar structure-activity relationship (SAR) principles as used for traditional drug development also apply to tau and potentially to other IDPs.

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Optimization of 3-Cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines toward the Development of an In Vivo Chemical Probe for CSNK2A.

3-Cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines, including the chemical probe SGC-CK2-1, are potent and selective inhibitors of CSNK2A in cells but have limited utility in animal models due to their poor pharmacokinetic properties. While developing analogues with reduced intrinsic clearance and the potential for sustained exposure in mice, we discovered that phase II conjugation by GST enzymes was a major metabolic transformation in hepatocytes. A protocol for codosing with ethacrynic acid, a covalent reversible GST inhibitor, was developed to improve the exposure of analogue 2h in mice. A double codosing protocol, using a combination of ethacrynic acid and irreversible P450 inhibitor 1-aminobenzotriazole, increased the blood level of 2h by 40-fold at a 5 h time point.

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

Optimization of 3-Cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines Toward the Development of an In Vivo Chemical Probe for CSNK2A.

3-cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines, including the chemical probe SGC-CK2-1, are potent and selective inhibitors of CSNK2A in cells but have limited utility in animal models due to their poor pharmacokinetic properties. While developing analogs with reduced intrinsic clearance and the potential for sustained exposure in mice, we discovered that Phase II conjugation by GST enzymes was a major metabolic transformation in hepatocytes. A protocol for co-dosing with ethacrynic acid, a covalent reversible GST inhibitor, was developed to improve the exposure of analog 2h in mice. A double co-dosing protocol, using a combination of ethacrynic acid and irreversible P450 inhibitor 1-aminobenzotriazole increased the blood level of 2h by 40-fold at a 5 h time point.

Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like ß-Coronaviruses.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like ß-coronavirus drugs.

Host kinase CSNK2 is a target for inhibition of pathogenic ß-coronaviruses including SARS-CoV-2.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-ß-coronavirus drugs.

Sphingolipid biosynthesis in pathogenic fungi: identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus.

An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p (TSC10 (YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.

Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox.

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.

NMR-based structural characterization of large protein-ligand interactions.

Genomic research on target identification and validation has created a great need for methods that rapidly provide detailed structural information on protein-ligand interactions. We developed a suite of NMR experiments as rapid and efficient tools to provide descriptive structural information on protein-ligand complexes. The methods work with large proteins and in particular cases also without the need for a complete three-dimensional structure. We will show applications with two tetrameric enzymes of 120 and 170 kDa.