Discovery and SAR of JTE-151: A Novel RORγ Inhibitor for Clinical Development.

A number of RORγ inhibitors have been reported over the past decade. There were also several examples advancing to human clinical trials, however, none of them has reached the market yet, suggesting that there could be common obstacles for their future development. As was expected from the general homology of nuclear receptor ligands, insufficient selectivity as well as poor physicochemical properties were identified as potential risks for a RORγ program. Based on such considerations, we conducted a SAR investigation by prioritizing drug-like properties to mitigate such potential drawbacks. After an intensive SAR exploration with strong emphasis on "drug-likeness" indices, an orally available RORγ inhibitor, JTE-151, was finally generated and was advanced to a human clinical trial. The compound was confirmed to possess highly selective profiles along with good metabolic stability, and most beneficially, no serious adverse events (SAE) and good PK profiles were observed in the human clinical trial.

Ternary complex of human RORγ ligand-binding domain, inverse agonist and SMRT peptide shows a unique mechanism of corepressor recruitment.

Retinoid-related orphan receptor gamma (RORγ) directly controls the differentiation of Th17 cell and the production of interleukin-17, which plays an integral role in autoimmune diseases. To obtain insight into RORγ, we have determined the first crystal structure of a ternary complex containing RORγ ligand-binding domain (LBD) bound with a novel synthetic inhibitor and a repressor peptide, 22-mer peptide from silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Comparison of a binary complex of nonliganded (apo) RORγ-LBD with a nuclear receptor co-activator (NCoA-1) peptide has shown that our inhibitor displays a unique mechanism different from those caused by natural inhibitor, ursolic acid (UA). The compound unprecedentedly induces indirect disruption of a hydrogen bond between His479 on helix 11 (H11) and Tyr502 on H12, which is crucial for active conformation. This crystallographic study will allow us to develop novel synthetic compounds for autoimmune disease therapy.

SAR Exploration Guided by LE and Fsp(3): Discovery of a Selective and Orally Efficacious RORγ Inhibitor.

A novel series of RORγ inhibitors was identified starting with the HTS hit 1. After SAR investigation based on a prospective consideration of two drug-likeness metrics, ligand efficiency (LE) and fraction of sp(3) carbon atoms (Fsp(3)), significant improvement of metabolic stability as well as reduction of CYP inhibition was observed, which finally led to discovery of a selective and orally efficacious RORγ inhibitor 3z.

Structurally similar estradiol analogs uniquely alter the regulation of intracellular signaling pathways.

Ligand structure can affect the activation of nuclear receptors, such as estrogen receptors (ERs), and their control of signaling pathways for cellular responses including death and differentiation. We hypothesized that distinct biological functions of similar estradiol (E(2)) analogs could be identified by integrating gene expression patterns obtained from human tumor cell lines with receptor binding and functional data for the purpose of developing compounds for treatment of a variety of diseases. We compared the estrogen receptor subtype selectivity and impact on signaling pathways for three distinct, but structurally similar, analogs of E(2). Modifications in the core structure of E(2) led to pronounced changes in subtype selectivity for estrogen receptors, ER-α or ER-ß, along with varying degrees of ER dimerization and activation. While all three E(2) analogs are predominantly ER-ß agonists, the cell growth inhibitory activity commonly associated with this class of compounds was detected for only two of the analogs and might be explained by a ligand-specific pattern of gene transcription. Microarray studies using three different human tumor cell lines demonstrated that the analogs distinctly affect the transcription of genes in signaling pathways for chromosome replication, cell death, and oligodendrocyte progenitor cell differentiation. That the E(2) analogs could lower tumor cell viability and stimulate neuronal differentiation confirmed that gene expression data could accurately distinguish biological activity of the E(2) analogs. The findings reported here confirm that cellular responses can be regulated by making key structural alterations to the core structure of endogenous ER ligands.

Design, synthesis, and biological evaluation of peptidomimetic inhibitors of factor XIa as novel anticoagulants.

Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with >1000-fold FXa selectivity and >100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 microM and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.

Synthesis and in vitro biological evaluation of aryl boronic acids as potential inhibitors of factor XIa.

A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa.

Synthesis, SAR exploration, and X-ray crystal structures of factor XIa inhibitors containing an alpha-ketothiazole arginine.

Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents.

Mutation of surface residues to promote crystallization of activated factor XI as a complex with benzamidine: an essential step for the iterative structure-based design of factor XI inhibitors.

Activated factor XI (FXIa) is a key enzyme in the amplification phase of the blood-coagulation cascade. Thus, a selective FXIa inhibitor may have lesser bleeding liabilities and provide a safe alternative for antithrombosis therapy to available drugs on the market. In a previous report, the crystal structures of the catalytic domain of FXIa (rhFXI(370-607)) in complex with various ecotin mutants have been described. However, ecotin forms a matrix-like interaction with rhFXI(370-607) and is impossible to displace with small-molecule inhibitors; ecotin crystals are therefore not suitable for iterative structure-based ligand design. In addition, rhFXI(370-607) did not crystallize in the presence of small-molecule ligands. In order to obtain the crystal structure of rhFXI(370-607) with a weak small-molecule ligand, namely benzamidine, several rounds of surface-residue mutation were implemented to promote crystal formation of rhFXI(370-607). A quadruple mutant of rhFXI(370-607) (rhFXI(370-607)-S434A,T475A,C482S,K437A) readily crystallized in the presence of benzamidine. The benzamidine in the preformed crystals was easily exchanged with other FXIa small-molecule inhibitors. These crystals have facilitated the structure-based design of small-molecule FXIa inhibitors.

Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2.

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.

Crystal structures of the FXIa catalytic domain in complex with ecotin mutants reveal substrate-like interactions.

Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370-607 (rhFXI370-607). Here we report the first crystal structure of rhFXI370-607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 A resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2' in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI370-607-ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.

Conversion of human-selective PPARalpha agonists to human/mouse dual agonists: a molecular modeling analysis.

To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform.

The three-dimensional structure of the ZAP-70 kinase domain in complex with staurosporine: implications for the design of selective inhibitors.

The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.