M. David Percival

The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K.

Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.

A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyclooxygenase-1 inactivation by aspirin

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697 and NS-398 block the inhibition of Cox-1 by aspirin in vitro. However, clinical studies have shown that the Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at therapeutic doses, do not interfere with the antiplatelet effect of aspirin, in contrast to ibuprofen. Here, we have evaluated the relative potential of ibuprofen and various coxibs to interfere with the inactivation of Cox-1 by aspirin by using purified enzyme and calcium ionophore-activated human platelets. The irreversible inactivation of Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit with widely different potencies. The rank order of potencies for this process (ibuprofen > celecoxib > valdecoxib > rofecoxib > etoricoxib) parallels that obtained for the inhibition of Cox-1-mediated thromboxane B2 production by calcium ionophore-stimulated platelets. The antagonism of aspirin therefore likely involves a competition at the enzyme active site. The EC50 value for the antagonism against 10 μM aspirin for each drug is ≈10- to 40-fold lower than the corresponding IC50 value for inhibition of platelet Cox-1 activity, consistent with the much weaker initial binding of aspirin to Cox-1 as compared with arachidonic acid. These results show that a low affinity for Cox-1 and a high degree of Cox-2 selectivity confers a low potential to block aspirin inhibition of platelet Cox-1, consistent with the results of clinical studies.

Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K

The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds are 10- to 20-fold more potent than the corresponding amide derivatives. Compound 8 is a 5 pM inhibitor of human cathepsin K with >10,000-fold selectivity over other cathepsins.

Cleavage of type II collagen by cathepsin K in human osteoarthritic cartilage.

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

Structure-based design of COX-2 selectivity into flurbiprofen.

Comparative computer modeling of the X-ray crystal structures of cyclooxygenase isoforms COX-1 and COX-2 has led to the design of COX-2 selectivity into the nonselective inhibitor flurbiprofen. The COX-2 modeling was based on a postulated binding mode for flurbiprofen and took advantage of a small alcove in the COX-2 active site created by different positions of the Leu384 sidechain between COX-1 and COX-2. The design hypothesis was tested by synthesis and biological assay of a series of flurbiprofen analogs, culminating in the discovery of several inhibitors having up to 78-fold selectivity for COX-2 over COX-1.

The Consequences of Lysosomotropism on the Design of Selective Cathepsin K Inhibitors

Many drug candidates contain a basic functional group that results in lysosomotropism—the accumulation of drug in the acidic lysosomes of a cell. When evaluating inhibitors of lysosomal enzymes, such as the cathepsins, this physical property can have a dramatic impact on the functional selectivity of the test compounds. A basic P3 substituent in cathepsin K inhibitors provides a means of achieving potent and selective enzyme inhibition. To evaluate the whole‐cell selectivity of the basic cathepsin K inhibitor L‐006235, we identified the irreversible pan‐selective cathepsin probe BIL‐DMK and used it to design whole‐cell enzyme‐occupancy assays. These cell‐based assays showed a dramatic reduction in selectivity against cathepsins B, L, and S relative to the selectivities observed in enzyme assays. Two‐photon confocal fluorescence microscopy showed punctated subcellular localization of L‐006235, which colocalized with BODIPY‐labelled Lysotracker, consistent with compound lysosomotropism. To address this potential problem, a series of potent cathepsin K inhibitors was developed by replacing the P2P3 amide bond with a metabolically stable trifluoroethylamine moiety. X‐ray crystallography has identified the binding of this functional group to active‐site residues in cathepsin K. This modification resulted in increased potency and selectivity that allowed the removal of the basic P3 substituent. The resulting nonbasic inhibitor L‐873724 is a 0.2 nM inhibitor of cathepsin K with cathepsin B, L, and S potencies that were not shifted between purified enzyme and whole‐cell assays; thus indicating that this compound is not lysosomotropic. L‐873724 exhibits excellent pharmacokinetics and is orally active in a monkey model of osteoporosis at 3 mg kg−1 q.d.

Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.

Cathepsin K Null Mice Show Reduced Adiposity during the Rapid Accumulation of Fat Stores

Growing evidences indicate that proteases are implicated in adipogenesis and in the onset of obesity. We previously reported that the cysteine protease cathepsin K (ctsk) is overexpressed in the white adipose tissue (WAT) of obese individuals. We herein characterized the WAT and the metabolic phenotype of ctsk deficient animals (ctsk−/−). When the growth rate of ctsk−/− was compared to that of the wild type animals (WT), we could establish a time window (5–8 weeks of age) within which ctsk−/−display significantly lower body weight and WAT size as compared to WT. Such a difference was not observable in older mice. Upon treatment with high fat diet (HFD) for 12 weeks ctsk−/− gained significantly less weight than WT and showed reduced brown adipose tissue, liver mass and a lower percentage of body fat. Plasma triglycerides, cholesterol and leptin were significantly lower in HFD-fed-ctsk−/− as compared to HFD-fed WT animals. Adipocyte lipolysis rates were increased in both young and HFD-fed-ctsk−/−, as compared to WT. Carnitine palmitoyl transferase-1 activity, was higher in mitochondria isolated from the WAT of HFD treated ctsk−/− as compared to WT. Together, these data indicate that ctsk ablation in mice results in reduced body fat content under conditions requiring a rapid accumulation of fat stores. This observation could be partly explained by an increased release and/or utilization of FFA and by an augmented ratio of lipolysis/lipogenesis. These results also demonstrate that under a HFD, ctsk deficiency confers a partial resistance to the development of dyslipidemia.

Inhibition of the Activation of Multiple Serine Proteases with a Cathepsin C Inhibitor Requires Sustained Exposure to Prevent Pro-enzyme Processing

Cathepsin C is a cysteine protease required for the activation of several pro-inflammatory serine proteases and, as such, is of interest as a therapeutic target. In cathepsin C-deficient mice and humans, the N-terminal processing and activation of neutrophil elastase, cathepsin G, and proteinase-3 is abolished and is accompanied by a reduction of protein levels. Pharmacologically, the consequence of cathepsin C inhibition on the activation of these serine proteases has not been described, due to the lack of stable and non-toxic inhibitors and the absence of appropriate experimental cell systems. Using novel reversible peptide nitrile inhibitors of cathepsin C, and cell-based assays with U937 and EcoM-G cells, we determined the effects of pharmacological inhibition of cathepsin C on serine protease activity. We show that indirect and complete inhibition of neutrophil elastase, cathepsin G, and proteinase-3 is achievable in intact cells with selective and non-cytotoxic cathepsin C inhibitors, at concentrations ∼10-fold higher than those required to inhibit purified cathepsin C. The concentration of inhibitor needed to block processing of these three serine proteases was similar, regardless of the cell system used. Importantly, cathepsin C inhibition must be sustained to maintain serine protease inhibition, because removal of the reversible inhibitors resulted in the activation of pro-enzymes in intact cells. These findings demonstrate that near complete inhibition of multiple serine proteases can be achieved with cathepsin C inhibitors and that cathepsin C inhibition represents a viable but challenging approach for the treatment of neutrophil-based inflammatory diseases.

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Inhibition of cathepsin C, a dipeptidyl peptidase that activates many serine proteases, represents an attractive therapeutic strategy for inflammatory diseases with a high neutrophil burden. We recently showed the feasibility of blocking the activation of neutrophil elastase, cathepsin G, and proteinase-3 with a single cathepsin C selective inhibitor in cultured cells. Here we measured the fractional inhibition of cathepsin C that is required for blockade of downstream serine protease processing, in cell-based assays and in vivo. Using a radiolabeled active site probe and U937 cells, a 50% reduction of cathepsin G processing required ∼50% of cathepsin C active sites to be occupied by an inhibitor. In EcoM-G cells, inhibition of 50% of neutrophil elastase activity required ∼80% occupancy. Both of these serine proteases were fully inhibited at full cathepsin C active site occupancy, whereas granzyme B processing in TALL-104 cells was partially inhibited, despite complete occupancy. In vivo, leukocytes from cathepsin C+/- mice exhibited comparable levels of neutrophil elastase activity to wild-type animals, even though their cathepsin C activity was reduced by half. The long-term administration of a cathepsin C inhibitor to rats, at doses that resulted in the nearly complete blockade of cathepsin C active sites in bone marrow, caused significant reductions of neutrophil elastase, cathepsin G and proteinase-3 activities. Our results demonstrate that the inhibition of cathepsin C leads to a decrease of activity of multiple serine proteases involved in inflammation but also suggest that high fractional inhibition is necessary to reach therapeutically significant effects.