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5-Alkyl and aryl-pyrazole–tetrazoles have been identified as a new class of selective, small-molecule, agonists of the human G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.

A library of 48 di- and trisubstituted guanidines was synthesized on a soluble, tetravalent support. Support-bound intermediates and cleaved products were isolated in parallel by size exclusion chromatography using a semiautomated system. Products were generally obtained in good yield and purity.

A combinatorial library of N-carboxyalkyl tripeptides was prepared to generate new leads against metalloproteinases. Using the base labile TentaGel S HMB resin, an Fmoc strategy was employed to yield 100 mixtures of 200 compounds each of the general structure 5. A synthetic protocol combining both mix and split and indexed combinatorial strategies was used, and selected inhibition data against MMP-3 is reported.

A series of N-carboxyalkyl dipeptides were synthesized to evaluate their inhibitory activities against human stromelysin-1(MMP-3), collagenase(MMP-1), and gelatinase-A(MMP-2). Structures with a homophenylalanine residue at P1′ substituted at the para position with small alkyl groups are potent inhibitors of (MMP-3) and (MMP-2) (Ki′ s 2–40 nM), but weak inhibitors of (MMP-1).

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.

A series of N-carboxyalkyl peptides were prepared to test their inhibitory activity against human stromelysin (MMP-3), collagenase (MMP-1), and gelatinase A (MMP-2). Linear alkyl and ω-aminoalkyl residues were employed as replacements for a phenethyl group yielding inhibitors with in vitro activities comparable to their corresponding aromatic analogs.

An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.

Amino-anthranilic acid derivatives have been identified as a new class of low serum shifted, high affinity full agonists of the human orphan G-protein-coupled receptor GPR109a with improved ADME properties.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

The chirality of biological receptors often requires syntheses of therapeutic compounds in single enantiomer form. The field of asymmetric catalysis addresses enantioselective synthesis with chiral catalysts. Chemical differentiation of sites within molecules that are separated in space by long distances presents special challenges to chiral catalysts. As the distance between enantiotopic sites increases within a substrate, so too may the requirements for size and complexity for the catalyst. The extreme of catalyst complexity could be defined by macromolecular enzymes and their amazing capacity to effect stereospecific reactions over long distances between reactive sites and enzyme-substrate contacts. We report here a synthetic, miniaturized enzyme mimic that catalyzes a desymmetrization reaction over a very long distance.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

Stromelysin-1, a member of the matrix metalloendoprotease family, is a zinc protease involved in the degradation of connective tissue in the extracellular matrix. As a step toward determining the structure of this protein, multidimensional heteronuclear NMR experiments have been applied to an inhibited truncated form of human stromelysin-1. Extensive 1H, 13C, and 15N sequential assignments have been obtained with a combination of three- and four-dimensional experiments. On the basis of sequential and short-range NOEs and 13C alpha chemical shifts, two helices have been delineated, spanning residues Asp-111 to Val-127 and Leu-195 to Ser-206. A third helix spanning residues Asp-238 to Gly-247 is characterized by sequential NOEs and 13C alpha chemical shifts, but not short-range NOEs. The lack of the latter NOEs suggests that this helix is either distorted or mobile. Similarly, sequential and interstrand NOEs and 13C alpha chemical shifts characterize a four-stranded beta-sheet with three parallel strands (Arg-100 to Ile-101, Ile-142 to Ala-147, Asp-177 to Asp-181) and one antiparallel strand (Ala-165 to Tyr-168). Two zinc sites have been identified in stromelysin [Salowe et al. (1992) Biochemistry 31, 4535-4540]. The NMR spectral properties, including chemical shift, pH dependence, and proton coupling of the imidazole nitrogens of six histidine residues (151, 166, 179, 201, 205, and 211), invariant in the matrix metalloendoprotease family, suggest that these residues are zinc ligands. NOE data indicate that these histidines form two clusters: one ligates the catalytic zinc (His-201, -205, and -211), and the other ligates a structural zinc (His-151, -166, and -179). Heteronuclear multiple quantum correlated spectra and specific labeling experiments indicate His-151, -179, -201, -205, and -211 are in the N delta 1H tautomer and His-166 is in the N epsilon 2H tautomer.