Identification of a potent and selective non-basic cathepsin K inhibitor

Based on our previous study with trifluoroethylamine as a P2-P3 amide isostere of cathepsin K inhibitor, further optimization led to identification of compound 22 (L-873724) as a potent and selective non-basic cathepsin K inhibitor. This compound showed excellent pharmacokinetics and efficacy in an ovariectomized (OVX) rhesus monkey model. The volumes of distribution close to unity were consistent with this compound not being lysosomotropic, which is a characteristic of basic cathepsin K inhibitors.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

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.     

Structure-based design of non-peptide, carbohydrazide-based cathepsin K inhibitors.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

An enormously active and selective azapeptide inhibitor of cathepsin B.

The peptidomimetic Z-Arg-Leu-Arg-Agly-Ile-Val-OMe (where Agly means alpha-aza-glycyl, -NHNHCO-) is the strongest (K(i) = 480 pM) and the most selective inhibitor of cathepsin B to date, being approximately 2310 times as active to cathepsin B as to cathepsin K. In this paper we introduce the peptide and seek to rationalize its structure-activity relationships using molecular dynamics (MD) and NMR. It is shown that the -Agly-moiety restrains the peptide backbone to a bent shape, contrary to its parent peptide (with Gly in position 4), having its backbone extended and flexible. This fold is maintained in the plug covalently bound to the cathepsin B Cys29, in compliance with similar bends already observed in two other azapeptides attached to the active sites of cathepsin B. The MD simulation of the Z-Arg-Leu-Arg-Agly approximately cathepsin B complex suggests that, contrary to other potent inhibitors of cathepsin B, the current double Arg(1)/Arg(3) inhibitor, while maintaining the fold is able to form a unique ion cluster involving both Arg residues on the inhibitor part and two acidic Glu171 and Glu245 on the cathepsin B part, thus enhancing the affinity and subsequently the inhibiting power and selectivity of Z-Arg-Leu-Arg-Agly-Ile-Val-OMe to the observed extreme extent.     

Discovery of selective and nonpeptidic cathepsin S inhibitors

Nonpeptidic, selective, and potent cathepsin S inhibitors were derived from an in-house pyrrolopyrimidine cathepsin K inhibitor by modification of the P2 and P3 moieties. The pyrrolopyrimidine-based inhibitors show nanomolar inhibition of cathepsin S with over 100-fold selectivity against other cysteine proteases, including cathepsin K and L. Some of the inhibitors showed cellular activities in mouse splenocytes as well as oral bioavailabilities in rats.     

A cytochemical assay for osteoclast cathepsin K activity

Cathepsin K is a member of the papain superfamily of cysteine proteases and plays a pivotal role in osteoclast-mediated bone resorption. This enzyme is an excellent target for antiresorptive therapies for osteopenic disorders such as osteoporosis.(1) Although isolated inhibitor studies on purified enzymes is required to discover potent and selective inhibitors of cathepsin K, a quantitative cytochemical assay(2) for cathepsin K would allow inhibitors to be tested on actual osteoclasts within sections of bone. Furthermore cathepsin K activity could be used to identify and analyse osteoclasts at definitive stages of their lifespan. A cytochemical assay is described that localizes osteoclast cathepsin K activity in unfixed, undecalcified cryostat sections of animal and human bone.     

Investigation of ketone warheads as alternatives to the nitrile for preparation of potent and selective cathepsin K inhibitors

Amino ketone warheads were explored as alternatives to the nitrile group of a potent and selective cathepsin K inhibitor. The resulting compounds were potent and selective inhibitors of cathepsin K and these nitrile replacements had a significant effect on metabolism and pharmacokinetics.     

Orally bioavailable small molecule ketoamide-based inhibitors of cathepsin K

An orally available series of ketoamide-based inhibitors of cathepsin K has been identified. Starting from a potent inhibitor with poor oral bioavailability, modifications to P1 and P1' elements led to enhancements in solubility and permeability. These improvements resulted in orally available cathepsin K inhibitors.     

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Novel and potent cyclic cyanamide-based cathepsin K inhibitors

Starting from a PDE IV inhibitor hit derived from high throughput screening of the compound collection, a key pyrrolidine cyanamide pharmacophore was identified. Modifications of the pyrrolidine ring produced enhancements in cathepsin K inhibition. An X-ray co-crystal structure of a cyanamide with cathepsin K confirmed the mode of inhibition.     

Novel, potent P2-P3 pyrrolidine derivatives of ketoamide-based cathepsin K inhibitors

Starting from a potent pantolactone ketoamide cathepsin K inhibitor discovered from structural screening, conversion of the lactone scaffold to a pyrrolidine scaffold allowed exploration of the S(3) subsite of cathepsin K. Manipulation of P3 and P1' groups afforded potent inhibitors with drug-like properties.     

Acyclic cyanamide-based inhibitors of cathepsin K

Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate bone resorption in the rat calvarial model.     

Benzodioxocin-3-ones and N-acyl-3-amino-3-buten-2-ones: novel classes of cathepsin K cysteine protease inhibitors.

The design, synthesis, enzymologic, and protein mass spectrometric characterization of benzodioxocin-3-one and N-acyl-3-amino-3-buten-2-one inhibitors of the cysteine protease cathepsin K are described. The benzodioxocin-3-one ring system is chemically unstable giving rise to a mixture of N-acyl-3-amino-3-buten-2-one and hemiketals. This mixture of N-acyl-3-amino-3-buten-2-one and hemiketals potently inhibits recombinant, human cathepsin K (IC50 = 36 nM) by a time-independent, irreversible mechanism. Formation of a covalent adduct between cathepsin K and inhibitor has been confirmed by mass spectrometry.     

The slow-binding inhibition of cathepsin K by its propeptide

A peptide corresponding to the full-length proregion (amino acids 16-114) of human cathepsin K was expressed and purified from Escherichia coli. This recombinant propeptide was investigated for its ability to inhibit the activity of three cysteine proteinases: cathepsins K, L, and B. Kinetic studies showed the propeptide to be a potent slow-binding inhibitor of its parent enzyme with a K(i) = 2. 61 nM at pH 6. This inhibition was pH-dependent, with a decrease in pH from 6 to 4 leading to a concomitant increase in K(i) to 147 nM. The propeptide also inhibited cathepsin L with a K(i) = 26.1 nM at pH 6, but showed little inhibition of cathepsin B at concentrations up to 400 nM.     

Cathepsin K and matrix metalloprotease activities in bone tissue of the OXYS rats during the development of osteoporosis

The activity of osteoclast-specific cysteine protease, cathepsin K, and matrix metalloproteases (MMPs) has been investigated in bone tissue of senescence-accelerated OXYS rats and in Wistar rats. At the age of 3 month (the period preceding manifestation of osteoporosis in OXYS rats) cathepsin K activity was higher whereas MMP activity was lower in Wistar rats. At the age of 14 months Wistar rats cathepsin K activity increased and MMP activity decreased. The age-related changes in bone cathepsin K and MMP activity of OXYS rats had opposite direction. Thus, despite of marked manifestations of osteoporosis previously found by us in OXYS rats (the decrease in mineralization density of the bone tissue and its resorption) no interstrain differences in cathepsin K and MMPs were found between Wistar and OXYS rats. Activity of a universal protease inhibitor, α₂-macroglobulin, was higher in serum of 14-month old OXYS rats than in Wistar rats of the same age. The role of cathepsin K activation in resorption of bone tissue in the development of osteoporosis in senescence-accelerated OXYS rats is discussed.     

Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors

Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonylleucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohydrazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water-soluble polymers [alpha-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I]. The conjugation of inhibitor I to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K (k(obs)/[I] = 1.3 x 10(6) M(-1) s(-1)) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 x 10(5) M(-1) s(-1)). The k(obs)/[I] values for I attached to the side chains of HPMA copolymers (PHPMA-GG-I and PHPMA-I) were about 3 x 10(4) M(-1) s(-1). When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k(obs)/[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization.     

pH Dependence of inhibitors targeting the occluding loop of cathepsin B

Potent and selective cathepsin B inhibitors have previously been synthesized based upon the natural product cysteine protease inhibitor E-64. X-ray crystal data indicates that these compounds interact through their free carboxylate with the positively charged histidine residues located on the prime-side of the active site within the occluding loop of cathepsin B. Herein, we examine the pH dependence of two prime-side-binding compounds. In each case there is a dramatic decrease in k(inact)/K(I) as the pH is raised from 4 to 7.8 corresponding to a single ionization of pK(a) 4.4. These results suggest that targeting of the occluding loop of cathepsin B may be a poor inhibitor design strategy if the enzyme environment has a pH greater than 5.5. However, this type of inhibitor may be a useful tool to help elucidate the role and the environment of cathepsin B in invading tumors.