Highly potent inhibitors of methionine aminopeptidase-2 based on a 1,2,4-triazole pharmacophore.

High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.

Structure activity relationships of 5-, 6-, and 7-methyl-substituted azepan-3-one cathepsin K inhibitors.

The syntheses, in vitro characterizations, and rat and monkey in vivo pharmacokinetic profiles of a series of 5-, 6-, and 7-methyl-substituted azepanone-based cathepsin K inhibitors are described. Depending on the particular regiochemical substitution and stereochemical configuration, methyl-substituted azepanones were identified that had widely varied cathepsin K inhibitory potency as well as pharmacokinetic properties compared to the 4S-parent azepanone analogue, 1 (human cathepsin K, K(i,app) = 0.16 nM, rat oral bioavailability = 42%, rat in vivo clearance = 49.2 mL/min/kg). Of particular note, the 4S-7-cis-methylazepanone analogue, 10, had a K(i,app) = 0.041 nM vs human cathepsin K and 89% oral bioavailability and an in vivo clearance rate of 19.5 mL/min/kg in the rat. Hypotheses that rationalize some of the observed characteristics of these closely related analogues have been made using X-ray crystallography and conformational analysis. These examples demonstrate the potential for modulation of pharmacological properties of cathepsin inhibitors by substituting the azepanone core. The high potency for inhibition of cathepsin K coupled with the favorable rat and monkey pharmacokinetic characteristics of compound 10, also known as SB-462795 or relacatib, has made it the subject of considerable in vivo evaluation for safety and efficacy as an inhibitor of excessive bone resorption in rat, monkey, and human studies, which will be reported elsewhere.

4-Aryl-1,2,3-triazole: a novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo.

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.

In situ data collection and structure refinement from microcapillary protein crystallization.

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.

Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinase.

The X-ray crystal structure of the proform of human matrix metalloproteinase MMP9 has been solved to 2.5 A resolution. The construct includes the prodomain, the catalytic domain and three FnII (fibronectin type II) domains. The prodomain is inserted into the active-site cleft, blocking access to the catalytic zinc. Comparison with the crystal structure of the most closely related MMP, MMP2, indicates that the conformations of residues in the active-site cleft and in the cysteine-switch peptide of the prodomain are highly conserved and that design of MMP9-specific inhibitors will be challenging. In common with MMP2, the MMP9 S1' inhibitor-binding pocket is large compared with that of other MMPs. One small point of difference in the S1' binding pockets of MMP9 and MMP2 may provide an opportunity to explore the design of specific inhibitors. The side chain of Arg424 in MMP9 is angled slightly away from the S1' pocket when compared with the corresponding residue in MMP2, Thr424. The secondary structure of the FnII domains is conserved between the two closely related MMPs, although the second FnII domain makes no contact with the catalytic domain in MMP9, while the same domain in MMP2 has a substantial area of interaction with the catalytic domain.