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

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.

IntroductionInhibitors of bone resorption can preserve bone mass and maintain normal bone architecture, thereby preventing fractures in later life (1).The bone-resorption process requires secretion of protons by the osteoclasts (2). This allows the dissolution of the bone mineral and the maintenance of the acidic environment required by proteolytic enzymes to degrade the bone matrix (3). Protons are extruded by a proton pump belonging to the class of vacuolar-type H + -ATPases (V-H + -ATPases (4) that is present in high concentration on the ruffled border (apical surface) of the resorbing osteoclast.The osteoclast V-H + -ATPase therefore represents a previously unexploited molecular target for the design of novel agents for reducing osteoclast activity. However, since V-H + -ATPase is an ubiquitous component of eukaryotic organisms and is the major electrogenic pump of endomembranes (5), the selective inhibition of the osteoclast V-H + -ATPase must be achieved in order to afford novel drugs that may constitute useful and safe agents for the treatment of osteoporosis and related osteopenic diseases.Bafilomycin A 1 , a potent and specific inhibitor of V-H + -ATPases (6), inhibits bone resorption both in vitro (7) and in vivo (8). However, since bafilomycin is not selective for any particular type of V-H + -ATPases, when administered to animals it inhibits all the essential V-H + -ATPases leading to systemic alteration of cellular physiology and to unacceptable toxicity (9). Recently, a novel class of small molecules endowed with potent osteoclast V-H + -ATPase inhibitory activity has been discovered (10), whose optimization resulted in (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide (SB 242784; Figure 1) (11). The in vitro V-H + -ATPase inhibitory profile of SB 242784 has been reported previously (11). SB 242784 was a low-nanomolar inhibitor (IC 50 = 26.3 nM) of the bafilomycin-sensitive (vacuolar) Mg 2+ -ATPase in membrane preparations of osteoclasts obtained from egg-laying hens. In addition, SB 242784 was a very potent inhibitor of bone resorption (IC 50 = 3.4 nM) in an assay measuring the collagen fragments released from bone slices after a 48-hour incubation with human osteoclasts.In this paper we characterize further the biochemical and pharmacological actions of SB 242784 and demonstrate that it is highly selective for the V-H + -ATPase of human osteoclasts when compared with V-H + -ATPases from a number of other human tissues. Most importantly, we show that oral administration of SB 242784 reduces the retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats and prevents the A potent and selective inhibitor of the osteoclastic V-H + -ATPase, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl) -2,4-pentadienamide (SB 242784), was evaluated in two animal models of bone resorption. SB 242784 completely prevented retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats when administered orally ...

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Twelve patients with vertebral fracture osteoporosis were recruited into a trial of treatment with hPTH 1-34 by daily injection for 1 year combined (from the 5th month) with an anti-resorptive agent (oestrogen, n = 9; nandrolone, n = 3). Treatment outcomes were monitored by biochemical and radiotracer measurements together with histomorphometry of transiliac biopsies before and at the end of treatment following double in vivo pre-labelling with demethylchlortetracycline. Indices of whole body bone formation, obtained from the analysis of 85Sr data, showed substantial increases (P less than 0.005) for all three indices measured) while biochemical (hydroxyproline) and kinetic measurements of bone resorption showed modest and equivocal changes only. As a result calcium balance improved. Gastrointestinal calcium absorption showed a tendency to improve, while urine calcium decreased; but these changes were statistically not significant except for radiocalcium absorption in the oestrogen treated subgroup. Histomorphometry revealed substantial increases in cancellous bone volume as reported previously with hPTH 1-34 given alone. However, iliac (as distinct from whole body) indices related to bone formation and resorption appeared to have returned towards pre-treatment values by the time of the second biopsy under the influence of the anti-resorptive agent given with the hPTH 1-34. It is confirmed that hPTH 1-34 therapy can increase iliac cancellous bone mass (as well as spinal cancellous bone mass as reported earlier) without a long-term increment in whole body bone resorption, providing the hPTH is combined with an anti-resorptive agent.