James R. Kiefer

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Cyclooxygenase enzymes: catalysis and inhibition

Scientists working in the field of cyclooxygenase enzymes have witnessed several major advances in the past two years. Crystal structures of fatty acid substrate and prostaglandin product complexes have been elucidated. Elegant site-directed mutagenesis studies have pinpointed the roles of key amino acids within the active site. Together, these results have provided key insights into the overall reaction mechanism. Detailed kinetics, spectroscopic and crystallographic studies have shed new light on the complex mechanism of inhibition of these fascinating enzymes. Finally, novel substrates of cyclooxygenase-2 have been identified.

Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6.

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The second clinical candidate having a shorter and favorable human half-life.

In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently substituted members of this class. The challenge of a undesirably long human half-life for the first clinical candidate 1t(1/2)=360 h was addressed by multiple strategies, leading to the discovery of 29b-(S) (SC-75416) with t(1/2)=34 h.

Molecular Basis for Cyclooxygenase Inhibition by the Non-steroidal Anti-inflammatory Drug Naproxen

Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH2 for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

High Resolution Crystal Structure of the Catalytic Domain of ADAMTS-5 (Aggrecanase-2)

Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4Å resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.

Orally Active MMP-1 Sparing α-Tetrahydropyranyl and α-Piperidinyl Sulfone Matrix Metalloproteinase (MMP) Inhibitors with Efficacy in Cancer, Arthritis, and Cardiovascular Disease

α-Sulfone-α-piperidine and α-tetrahydropyranyl hydroxamates were explored that are potent inhibitors of MMPs-2, -9, and -13 that spare MMP-1, with oral efficacy in inhibiting tumor growth in mice and left-ventricular hypertrophy in rats and in the bovine cartilage degradation ex vivo explant system. α-Piperidine 19v (SC-78080/SD-2590) was selected for development toward the initial indication of cancer, while α-piperidine and α-tetrahydropyranyl hydroxamates 19w (SC-77964) and 9i (SC-77774), respectively, were identified as backup compounds.

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).

Minor Structural Change to Tertiary Sulfonamide RORc Ligands Led to Opposite Mechanisms of Action.

A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity. Compounds behaving as agonists in our biochemical assay also gave rise to an increased production of IL-17 in human PBMCs whereas inverse agonists led to significant suppression of IL-17 under the same assay conditions. The most potent inverse agonist compound showed >180-fold selectivity over the ROR isoforms as well as all other nuclear receptors that were profiled.