The design and synthesis of high-affinity FKBP 12 ligands is described. These compounds potently inhibit the m-rrans-peptidylprolyl isomerase (rotamase) activity catalyzed by FKBP 12 with inhibition constants (Ki,app) as low as 1 nM, yet they possess remarkable structural simplicity relative to FK506 and rapamycin, from which they are conceptually derived. The atomic structures of three FKBP12-ligand complexes and of one unbound ligand were determined by X-ray crystallography and are compared to the FKBP12-FK506 and FKBP12-rapamycin complexes.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-Å resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S 2 subsite, and do not bind in the caspase primary aspartic acid binding pocket (S 1 ). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

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Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the response to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, -13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBPI (for rapamycin-binding protein). The RBPI protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.Agents that inhibit T-cell activation include cyclosporin A (CsA) (19) and the recently discovered macrolide FK506 (31, 36). CsA was originally discovered as an antifungal agent, and FK506 was identified as an inhibitor of interleukin-2 (IL-2) production (20). Despite the structural dissimilarity between these two immunosuppressive drugs, recent reports suggest that the targets for both agents, cyclophilin and FK506-binding protein (FKBP), respectively, are peptidylprolyl cis-trans isomerases (PPlases), enzymes that promote protein folding in vitro (12,15,34,35,37,40,41). Although the endogenous function of PPlases is not known, the fact that the immunosuppressive action of CsA and FK506 is linked to inhibition of PPIase activity suggests that they may be required in the regulation of intracellular signaling events leading to T-cell activation (8, 12, 37).Rapamycin, a macrolide antifungal agent with structural similarity to FK506 (32, 42), also exhibits immunosuppressive (3, 26, 38) as well as antineoplastic (9, 18) properties. Rapamycin and FK506 act as reciprocal antagonists in vivo (murine T cell activation [6]) and compete for binding to FKBP (15). Given these similarities, the mechanism of action of rapamycin remains enigmatic because, whereas FK506 (like CsA) acts to inhibit IL-2 transcription, rapamycin has no effe...

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 K i,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 osteoclastmediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or

surements will be reported in detail elsewhere.Finally, one could question the physiological relevance of these experiments and observations. However, Pr3+ is an often-used Ca2+ substitute.43 Also, and although free-acid acids are minor components of biological membranes, exogeneous additions alter several membrane-mediated cellular functions such as cell permeability44 or activity of membrane-bound enzymes.45-47

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups f lanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

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