Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.

A novel fluorogenic substrate for the measurement of endothelial lipase activity.

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

Enhancing recombinant protein quality and yield by protein stability profiling.

The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

Potency variation of small-molecule chymase inhibitors across species.

Chymases (EC 3.4.21.39) are mast cell serine proteinases that are variably expressed in different species and, in most cases, display either chymotryptic or elastolytic substrate specificity. Given that chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, and cardiovascular disorders, it is important to understand interspecies differences of the enzymes as well as the behavior of inhibitors with them. We have expressed chymases from humans, macaques, dogs, sheep (MCP2 and MCP3), guinea pigs, and hamsters (HAM1 and HAM2) in baculovirus-infected insect cells. The enzymes were purified and characterized with kinetic constants by using chromogenic substrates. We evaluated in vitro the potency of five nonpeptide inhibitors, originally targeted against human chymase. The inhibitors exhibited remarkable cross-species variation of sensitivity, with the greatest potency observed against human and macaque chymases, with K(i) values ranging from approximately 0.4 to 72nM. Compounds were 10-300-fold less potent, and in some instances ineffective, against chymases from the other species. The X-ray structure of one of the potent phosphinate inhibitors, JNJ-18054478, complexed with human chymase was solved at 1.8A resolution to further understand the binding mode. Subtle variations in the residues in the active site that are already known to influence chymase substrate specificity can also strongly affect the compound potency. The results are discussed in the context of selecting a suitable animal model to study compounds ultimately targeted for human chymase.

Structural basis for elastolytic substrate specificity in rodent alpha-chymases.

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors.

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies.

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity.

MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4-10mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1-370, removal of the C-terminal autoinhibitory peptide in 1-338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1-338 by p38alpha further increased activity 12-fold. A putative constitutively active mutant, 1-370/T222E/T334E, was 6.3-fold less active than phosphorylated 1-370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg(2+)-dependent. Residues 1-40 were required for high-affinity binding of ADP, ATPgammaS, staurosporine, and K252a. A mutation M138A rendered 1-370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 microM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.

Dihydroquinone ansamycins: toward resolving the conflict between low in vitro affinity and high cellular potency of geldanamycin derivatives.

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.

Discovery, SAR, and X-ray structure of novel biaryl-based dipeptidyl peptidase IV inhibitors.

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors.

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.