Mark Alan Levy

Proteolytic Activity of Human Osteoclast Cathepsin K EXPRESSION, PURIFICATION, ACTIVATION, AND SUBSTRATE IDENTIFICATION

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F. H., Dodds, R., James, I., Connor, J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M.(1996) J. Biol. Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (k/[I] = 273,000 M•s). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type‐selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast‐mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast‐mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration‐dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast‐mediated assay in vitro. Cbz‐Leu‐Leu‐Leu‐H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)‐stimulated resorption in the FRLB assay with an IC‐50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC‐50 of 100 nM. In the adjuvant‐arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz‐Leu‐Leu‐Leu‐H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz‐Leu‐Leu‐Leu‐H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast‐mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Structure-activity studies of synthetic FKBP ligands as peptidyl-prolyl isomerase inhibitors

Abstract A series of non-macrocyclic pipecolyl α-ketoamides were prepared and evaluated as FKBP cis-trans peptidyl-prolyl isomerase inhibitors. These compounds exhibited inhibition constants as low as 2 nM. Their design was based on a consideration of the common FKBP-binding elements of FK506 and rapamycin. Structure-activity relationships are discussed.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface

BACKGROUND Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Cloning, expression and functional characterization of type 1 and type 2 steroid 5α-reductases from Cynomolgus monkey: Comparisons with human and rat isoenzymes

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.

Synthesis and structure-activity relationships of macrocyclic FKBP ligands

Abstract A number of pipecolinate dilactones have been synthesized as simplified macrocyclic mimics of the binding domains in rapamycin (1) and FK506 (2). Crystallographic studies of these compounds indicate that the conformation of the pipecolinyl α-ketoamide region is preorganized for binding to FKBP. This is confirmed by the ability of these analogs to inhibit the FKBP cis-trans peptidyl-prolyl isomerase activity.

Epristeride is a selective and specific uncompetitive inhibitor of human steroid 5α-reductase isoform 2

Specificity of an enzyme inhibitor can have profound implications upon the compounds therapeutic potential, utility and safety profile. As potent inhibitors of human steroid 5 alpha-reductase (SR) the 3-androstene-3-carboxylic acids, or steroidal acrylates, may be useful in treatment of diseases such as benign prostatic hyperplasia for which 5 alpha-dihydrotestosterone (DHT) appears to be a causative agent. To determine its specificity profile, the interactions of a representative compound from this class, N-(t-butyl)androst-3,5-diene-17 beta-carboxamide-3-carboxylic acid (epristeride, SK&F 105657), have been studied with 7 other steroid processing enzymes and 5 steroid hormone receptors. The affinity of epristeride for each of these 12 potential targets was found to be at least 1000-fold weaker than that for SR, the intended target. In addition, using samples of the individually expressed two known forms of human SRs, epristeride has been shown to be a selective inhibitor of the recombinant human SR type 2, the predominant activity found in the prostate of man. Nonetheless, the mechanisms of SR inhibition for both isoenzymes involve formation of a ternary complex with epristeride, NADP+, and enzyme. Epristeride, consequently, has been shown to be an uncompetitive inhibitor versus steroid substrate of both human SR isoenzymes. These results suggest that this 3-androstene-3-carboxylic acid is a specific and selective inhibitor of the human type 2 SR, and that epristeride is an attractive compound for further investigation as a safe and effective therapeutic agent in the potential treatment of disease states associated with DHT-induced tissue growth.

Structure-activity studies of nonmacrocyclic rapamycin derivatives

Abstract X-ray crystallography suggests the C23–C28 segment for rapamycin may act more as an element of the FKBP binding domain than as part of the immunosuppressant effector domain. Selective excision of this region from the natural product followed by minor reconstruction of the binding domain resulted in compounds with high affinity for FKBP but no immunosuppressive activity. This, along with data from other secorapamycin analogs, suggest the importance of C23–C28 in the orientation of the effector domain.

Design, synthesis and evaluation of dual domain FKBP ligands

Abstract A number of dual domain acyclic and macrocyclic pipecolyl α-ketoamide derivatives were prepared which possess the elements of previously described high-affinity FKBP binding domains as well as simplified mimics of the FK506 effector domain, a critical feature for immunosuppressive activity of the FKBP12-FK506 complex. Compounds of this study exhibited a range of FKBP cis-trans peptidyl-prolyl isomerase inhibitory activities but no activity in a splenocyte mitogenesis assay for immunosuppression.

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.