Structural basis for murine norovirus engagement of bile acids and the CD300lf receptor.

Murine norovirus (MNoV) is closely related to human norovirus (HNoV), an infectious agent responsible for acute gastroenteritis worldwide. Here we report the X-ray crystal structure of the dimeric MNoV VP1 protruding (P) domain in complex with its cellular receptor CD300lf. CD300lf binds the P domain with a 2:2 stoichiometry, engaging a cleft between the AB and DE loops of the P2 subdomain at a site that overlaps the epitopes of neutralizing antibodies. We also identify that bile acids are cofactors enhancing MNoV cell-binding and infectivity. Structures of CD300lf-P domain in complex with glycochenodeoxycholic acid (GCDCA) and lithocholic acid (LCA) reveal two bile acid binding sites at the P domain dimer interface distant from receptor binding sites. The structural determinants for receptor and bile acid binding are supported by numerous biophysical assays utilizing interface residue mutations. We find that the monomeric affinity of CD300lf for the P domain is low and is divalent cation dependent. We have also determined the crystal structure of CD300lf in complex with phosphocholine, revealing that MNoV engages its receptor in a manner mimicking host ligands including similar metal coordination. Docking of the cocomplex structures onto a cryo-EM-derived model of MNoV suggests that each virion can make multiple CD300lf engagements, and thus, infection may be driven by the avidity of cell surface clustered CD300lf. These studies identify multiple potential modulators of norovirus infection that may act to regulate the interaction between the viral capsid P domain and its cognate cellular receptor.

A quantitative assay reveals ligand specificity of the DNA scaffold repair protein XRCC1 and efficient disassembly of complexes of XRCC1 and the poly(ADP-ribose) polymerase 1 by poly(ADP-ribose) glycohydrolase.

The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.

Structural bioinformatics-based prediction of exceptional selectivity of p38 MAP kinase inhibitor PH-797804.

PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.

2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles: design and synthesis of a potent and isoform selective PKC-zeta inhibitor.

The inhibition of PKC-zeta has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-zeta). The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and analysis of the series. The most selective and potent analog was identified by variation of the aryl substituent at the 6-position of the indazole template to give a 4-NH(2) derivative. The analog displays good selectivity over other PKC isoforms (alpha, betaII, gamma, delta, epsilon, mu, theta, eta and iota/lambda) and CDK-2, however it displays marginal selectivity against a panel of other kinases (37 profiled).

Selective small molecule PARG inhibitor causes replication fork stalling and cancer cell death.

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.

Design, synthesis, and crystal structure of selective 2-pyridone tissue factor VIIa inhibitors.

Targeted 2-pyridones were selected as tissue Factor VIIa inhibitors and prepared from 2,6-dibromopyridine via a multistep synthesis. A variety of chemical transformations, including regioselective nucleophilic addition, selective nitrogen alkylation, and a Suzuki coupling, afforded the targeted tissue Factor VIIa inhibitors. The pyridone core was selected as a replacement for the pyrazinone core of noncovalent tissue Factor VIIa inhibitors and designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. These compounds were tested in several serine protease enzyme assays involved in the coagulation cascade exhibiting modest activity on tissue Factor VIIa with excellent selectivity over thrombin and Factor Xa. Finally, an X-ray crystal structure of inhibitor 14a bound to tissue Factor VIIa was obtained and will be described.

Synthesis and crystal structures of substituted benzenes and benzoquinones as tissue factor VIIa inhibitors.

Several multistep syntheses of substituted benzenes are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. A variety of chemical transformations including nucleophilic additions, reductive aminations, Stille couplings, and polymer-assisted solution-phase (PASP) techniques were used to prepare key intermediates and final products. The initial analogues identified some weakly active compounds which ultimately led to a 340 nM (IC(50)) tissue Factor VIIa inhibitor with selectivity over other related enzymes. The structure-activity relationship of these inhibitors and the synthetic progression from the discovery of the lead compound to the development of potent analogues will be discussed. The X-ray crystal structures of fluorobenzene 50c and benzoquinone 54 inhibitors complexed with the TF/VIIa enzyme will also be described.

Polymer-assisted solution-phase library synthesis and crystal structure of alpha-ketothiazoles as tissue factor VIIa inhibitors.

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification concepts, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep synthesis affords desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of tissue Factor VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10k bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue Factor VIIa, with some analogues demonstrating selectivity over thrombin.

Design, parallel synthesis, and crystal structures of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex.

Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-alpha-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with >6250 x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.

Structure of mammalian poly(ADP-ribose) glycohydrolase reveals a flexible tyrosine clasp as a substrate-binding element.

Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique 'tyrosine clasp' enabling endoglycosidic cleavage of branched PAR chains.

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa.

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.

Synthesis and X-ray crystal structures of substituted fluorobenzene and benzoquinone inhibitors of the tissue factor VIIa complex.

Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa (TF/VIIa) enzyme. The compounds exhibited modest potency on TF/VIIa with selectivity over Factor Xa and thrombin. The X-ray crystal structures of the targeted fluorobenzene 12a and benzoquinone 14 inhibitors bound to TF/VIIa were obtained and will be described.

Polymer-assisted solution-phase (PASP) parallel synthesis of an alpha-ketothiazole library as tissue factor VIIa inhibitors.

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex.

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.