Discovery of DS44470011: An oral hypoxia-inducible factor prolyl hydroxylase inhibitor for the treatment of renal anemia.

Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We identified a pyrimidine core with HIF-PHD inhibitory activity based on scaffold hopping of FG-2216 using crystal structures of HIF-PHD2 in complex with compound. By optimizing the substituents at the 2- and 6- positions of the pyrimidine core, we discovered DS44470011, which improves the effectiveness of erythropoietin (EPO) release in cells. Oral administration of DS44470011 to cynomolgus monkeys increased plasma EPO levels.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production.

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4(+) T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.

Discovery of a Novel Class of ERRα Agonists.

A novel class of estrogen-related receptor α (ERRα) agonists has been discovered. A structure-activity relationship study of high-throughput screening hits 1 and 2 led to the discovery of benzimidazole 3d (DS20362725) and acetophenone analogue 5c (DS45500853). The X-ray crystal structure of the ERRα ligand-binding domain in complex with 5c and PGC-1α coactivator peptide revealed conformational changes in the ligand-binding pocket to accommodate 5c and the key interaction between the protein and ligand. Since both analogues avoided PPARγ transcriptional activity, they can be useful tool compounds for investigating biological ERRα functions.

Discovery of DS-1558: A Potent and Orally Bioavailable GPR40 Agonist.

GPR40 is a G protein-coupled receptor that is predominantly expressed in pancreatic ß-cells. GPR40 agonists stimulate insulin secretion in the presence of high glucose concentration. On the basis of this mechanism, GPR40 agonists are possible novel insulin secretagogues with reduced or no risk of hypoglycemia. The improvement of in vitro activity and metabolic stability of compound 1 led to the discovery of 13, (3S)-3-ethoxy-3-(4-{[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]oxy}phenyl)propanoic acid, as a potent and orally available GPR40 agonist. Compound 13 (DS-1558) was found to have potent glucose lowering effects during an oral glucose tolerance test in ZDF rats.

Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.

Crystallization and preliminary x-ray studies of the Fab fragment from a humanized version of the mouse anti-human fas antibody HFE7a.

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.

Inhibiting RORγt/Th17 axis for autoimmune disorders.

The recent success reported in late-stage clinical trials for the treatment of psoriasis by antibodies directed against interleukin (IL)-17 or its receptor has validated and strongly supports the development of inhibitors of the IL-17 pathway as a new therapeutic modality in chronic inflammation and autoimmunity. These results also encourage the drug discovery of orally available small molecules that can modulate down the production of IL-17 by Th17 cells (the major IL-17 producers) or the downstream signaling of the IL-17 receptor. Here, we review these strategies with an emphasis on inhibiting the retinoic-acid-related orphan nuclear receptor RORγt, which is the master regulator of Th17 cells and a promising therapeutic target for the treatment of multiple autoimmune disorders.

Terpenoids Produced by Actinomycetes: Napyradiomycins from Streptomyces antimycoticus NT17.

Napyradiomycin SR ( 1), 16-dechloro-16-hydroxynapyradiomycin C2 ( 2), 18-hydroxynapyradiomycin A1 ( 3), 18-oxonapyradiomycin A1 ( 4), 16-oxonapyradiomycin A2 ( 5), 7-demethyl SF2415A3 ( 6), 7-demethyl A80915B ( 7), and ( R)-3-chloro-6-hydroxy-8-methoxy-alpha-lapachone ( 8) were isolated from the culture broth of Streptomyces antimycoticus NT17. These compounds are derivatives of the napyradiomycins isolated previously from Chainia rubra or Streptomyces aculeolatus. The structures of the new compounds, some of which exhibit antibacterial activities, were established by comparing their NMR data with data of related known compounds. The unique structure of 1, containing a highly strained ring, was established by NMR and was confirmed by X-ray analysis. Two of the compounds are C-16 stereoisomers of napyradiomycin A2 and are named napyradiomycins A2a ( 9a) and A2b ( 9b).

Structural origins of efficient proton abstraction from carbon by a catalytic antibody.

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Crystal structure of RANK ligand involved in bone metabolism.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Recently, an essential cytokine system for osteoclast biology has been identified and extensively characterized. This system consists of a ligand, receptor activator of NF-kappaB ligand (RANKL), a receptor, RANK, and its soluble decoy receptor, osteoprotegerin (OPG). RANKL, a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with RANK, a member of the TNF receptor family. Because members of the TNF family have the same topology and the extracellular domains of the TNF receptor family members also adopt the same structural scaffold, in addition to their rapid increase in the number, this poses an intriguing question of how recognition between cognate ligands and receptors is achieved in a highly specific manner. Structural studies on the mouse RANKL extracellular domain showed that the RANKL is trimeric, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF-beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, revealed conserved and specific features of RANKL in the TNF superfamily. Residues important for receptor binding and activation have also been confirmed by mutagenesis experiments. Further structural and mutational studies on the RANKL/RANK/OPG system will provide useful information for developing drug candidates that inhibit osteoclastogenesis and mediate problems of bone metabolism.

Crystal structure of the extracellular domain of mouse RANK ligand at 2.2-A resolution.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.

Humanization of the mouse anti-Fas antibody HFE7A and crystal structure of the humanized HFE7A Fab fragment.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. We have previously obtained the mouse anti-Fas antibody HFE7A (m-HFE7A), which specifically induces apoptosis in inflammatory cells. In order to apply m-HFE7A for human therapy, we performed antibody humanization of m-HFE7A by grafting the mouse complementarity-determining regions (CDRs) to a human antibody. Five versions of humanized HFE7A (h-HFE7A) demonstrated the same antigen-binding affinity and same competition-binding activity against Fas as the chimeric HFE7A. Furthermore, these h-HFE7As induced the same degree of apoptosis in WR19L12a cells that express human Fas on their surface as chimeric HFE7A does. To further probe the structural basis for antibody humanization, we determined the three-dimensional structure of the h-HFE7A antigen-binding fragment (Fab) by X-ray crystallography and compared it with the crystal structure of the parent m-HFE7A Fab previously determined. The main-chain conformation in each h-HFE7A CDR is almost identical to that in m-HFE7A with root mean square (rms) deviations of 0.14-0.77 A. However, a significant segmental shift was observed in the CDR-L1 loop. Together with the high temperature factors of the CDR-L1 residues, both the loops are flexible, suggesting that the CDR-L1 loop would undergo conformational change upon binding to the antigen. Our results indicate that the humanization of m-HFE7A succeeded in maintaining the main-chain conformation as well as the flexibility of the CDR loop.