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.     

Design and synthesis of 6-substituted amino-4-oxa-1-azabicyclo[3,2,0]heptan-7-one derivatives as cysteine proteases inhibitors

A series of 6-substituted amino-4-oxa-1-azabicyclo[3,2,0]heptan-7-one compounds was designed and synthesized as a new class of inhibitors for cysteine proteases cathepsins B, L, K, and S. One compound (5S,6S)-6-(N-benzyloxycarbonyl-L-phenylalanyl) amino-4-oxa-1-azabicyclo[3,2,0]heptan-7-one showed excellent cathepsin L and K inhibition activity with IC(50) at a low nanomolar range.     

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.     

Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases.     

Identification of new peptide amides as selective cathepsin L inhibitors: The first step towards selective irreversible inhibitors?

A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.     

Peptide ketobenzoxazole inhibitors bound to cathepsin K

Potent inhibitors of human cysteine proteases of the papain family have been made and assayed versus a number of relevant family members. We describe the synthesis of peptide alpha-ketoheterocyclic inhibitors that occupy binding subsites S1'-S3 of the cysteine protease substrate recognition cleft and that form a reversible covalent bond with the Cys 25 nucleophile. X-ray crystal structures of cathepsin K both unbound and complexed with inhibitors provide detailed information on protease/inhibitor interactions and suggestions for the design of tight-binding, selective molecules.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

6-Acylamino-penam derivatives: synthesis and inhibition of cathepsins B,L, K,and S

The synthesis of a new series of 6-acylamino penam derivatives and their inhibition of cysteine proteases cathepsins B, L, K, and S is described. The 6-acylamino-penam sulfone compounds showed excellent cathepsin L, K, and S inhibition activity with IC(50) values in the nanomolar and subnanomolar range.     

The alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.     

Development of calpain-specific inactivators by screening of positional scanning epoxide libraries

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.     

Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.     

Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.     

Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?

Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.     

Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.     

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design,synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (K_i = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (K_i = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.     

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.     

Crystal structure of human cathepsin V

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.