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.

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.

Discovery of Novel and Highly Selective Cyclopropane ALK Inhibitors through a Fragment-Assisted, Structure-Based Drug Design.

Fragment screening is frequently used for hit identification. However, there was no report starting from a small fragment for the development of an anaplastic lymphoma kinase (ALK) inhibitor, despite the number of ALK inhibitors reported. We began our research with the fragment hit F-1 and our subsequent linker design, and its docking analysis yielded novel cis-1,2,2-trisubstituted cyclopropane 1. The fragment information was integrated with a structure-based approach to improve upon the selectivity over tropomyosin receptor kinase A, leading to the potent and highly selective ALK inhibitor, 4-trifluoromethylphenoxy-cis-1,2,2-trisubstituted cyclopropane 12. This work shows that fragments become a powerful tool for both lead generation and optimization, such as the improvement of selectivity, by combining them with a structure-based drug design approach, resulting in the fast and efficient development of a novel, potent, and highly selective compound.

Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]Pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part†2.

We identified novel potent inhibitors of p38 mitogen-activated protein (MAP) kinase using a structure-based design strategy, beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroanilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To enhance the inhibitory activity of 1 against production of tumor necrosis factor-α (TNF-α) in human whole blood (hWB) cell assays, we designed and synthesized hybrid compounds in which the imidazo[4,5-b]pyridin-2-one core was successfully linked with the p-methylbenzamide fragment. Among the compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)benzamide (25) exhibited potent p38 inhibition, superior suppression of TNF-α production in hWB cells, and also significant in vivo efficacy in a rat model of collagen-induced arthritis (CIA). In this paper, we report the discovery of potent, selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.

Discovery of Potent, Selective, and Brain-Penetrant 1 H-Pyrazol-5-yl-1 H-pyrrolo[2,3- b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors.

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is predominantly expressed in the brain and implicated in neuronal development and cognition. However, the detailed function of ALK in the central nervous system (CNS) is still unclear. To elucidate the role of ALK in the CNS, it was necessary to discover a potent, selective, and brain-penetrant ALK inhibitor. Scaffold hopping and lead optimization of N-(2,4-difluorobenzyl)-3-(1 H-pyrazol-5-yl)imidazo[1,2- b]pyridazin-6-amine 1 guided by a cocrystal structure of compound 1 bound to ALK resulted in the identification of (6-(1-(5-fluoropyridin-2-yl)ethoxy)-1-(5-methyl-1 H-pyrazol-3-yl)-1 H-pyrrolo[2,3- b]pyridin-3-yl)((2 S)-2-methylmorpholin-4-yl)methanone 13 as a highly potent, selective, and brain-penetrable compound. Intraperitoneal administration of compound 13 significantly decreased the phosphorylated-ALK (p-ALK) levels in the hippocampus and prefrontal cortex in the mouse brain. These results suggest that compound 13 could serve as a useful chemical probe to elucidate the mechanism of ALK-mediated brain functions and the therapeutic potential of ALK inhibition.

TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

Identification of PARP14 inhibitors using novel methods for detecting auto-ribosylation.

Poly(ADP-ribose) polymerases (PARPs) use nicotinamide adenine dinucleotide (NAD+) as a co-substrate to transfer ADP-ribose when it releases nicotinamide as the metabolized product. Enzymes of the PARP family play key roles in detecting and repairing DNA, modifying chromatin, regulating transcription, controlling energy metabolism, and inducing cell death. PARP14, the original member of the PARP family, has been reported to be associated with the development of inflammatory diseases and various cancer types, making it a potential therapeutic target. In this study, we purified the macrodomain-containing PARP14 enzyme and established an assay for detecting the auto-ribosylation activity of PARP14 using RapidFire high-throughput mass spectrometry and immunoradiometric assay using [3H]NAD+. Subsequently, we performed high-throughput screening using the assays and identified small-molecule hit compounds, which showed NAD+-competitive and PARP14-selective inhibitory activities. Co-crystal structures of PARP14 with certain hit compounds revealed that the inhibitors bind to the NAD+-binding site. Finally, we confirmed that the hit compounds interacted with intracellular PARP14 by a cell-based protein stabilization assay. Thus, we successfully identified primary candidate compounds for further investigation.

A fragment-based approach to identifying S-adenosyl-l-methionine -competitive inhibitors of catechol O-methyl transferase (COMT).

Catechol O-methyl transferase belongs to the diverse family of S-adenosyl-l-methionine transferases. It is a target involved in the treatment of Parkinson's disease. Here we present a fragment-based screening approach to discover noncatechol derived COMT inhibitors which bind at the SAM binding pocket. We describe the identification and characterization of a series of highly ligand efficient SAM competitive bisaryl fragments (LE = 0.33-0.58). We also present the first SAM-competitive small-molecule COMT co-complex crystal structure.

Functional proteomic and structural insights into molecular recognition in the nitrilase family enzymes.

Nitrilases are a large and diverse family of nonpeptidic C-N hydrolases. The mammalian genome encodes eight nitrilase enzymes, several of which remain poorly characterized. Prominent among these are nitrilase-1 (Nit1) and nitrilase-2 (Nit2), which, despite having been shown to exert effects on cell growth and possibly serving as tumor suppressor genes, are without known substrates or selective inhibitors. In previous studies, we identified several nitrilases, including Nit1 and Nit2, as targets for dipeptide-chloroacetamide activity-based proteomics probes. Here, we have used these probes, in combination with high-resolution crystallography and molecular modeling, to systematically map the active site of Nit2 and identify residues involved in molecular recognition. We report the 1.4 A crystal structure of mouse Nit2 and use this structure to identify residues that discriminate probe labeling between the Nit1 and Nit2 enzymes. Interestingly, some of these residues are conserved across all vertebrate Nit2 enzymes and, conversely, not found in any vertebrate Nit1 enzymes, suggesting that they are key discriminators of molecular recognition between these otherwise highly homologous enzymes. Our findings thus point to a limited set of active site residues that establish distinct patterns of molecular recognition among nitrilases and provide chemical probes to selectively perturb the function of these enzymes in biological systems.

Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3.

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center.

BACKGROUND: The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. RESULTS: The structure of SA1388 has been solved to 2.0A resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. CONCLUSION: SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric "lids" that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins.

Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs.

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 A as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular beta-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme.

Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-A crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitin-like N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin-aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.

Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3.

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1''-phosphate (Appr-1''-p). The SARS nsP3 domain readily removes the 1'' phosphate group from Appr-1''-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1''-p.