This chapter examines the activity, specificity and structural chemistry of clostridium collagenases. The clostridial collagenases are distinguished by their ability to digest native, triple-helical types I, II and III collagens into a mixture of small peptides under physiological conditions. A variety of collagen-based assays is available to quantify these activities. Clostridial collagenases also digest other types of collagen, but these reactions have not been characterized in detail. The initial proteolytic events in the hydrolysis of type I, II, and III collagens by the class I and II collagenases have been delineated and the kinetic parameters for these reactions measured. The class I and II enzymes initially attack all three collagens at distinct hyper-reactive sites whose sequences have been identified. Clostridium histolyticum is a pathogenic anaerobe that causes gas gangrene. All strains of the bacterium elaborate a collagenase, but the amount depends on the strain and the culture medium. The bacterium presumably uses the collagenases as a means to invade the host and possibly to degrade host protein for nutritional purposes. The clostridial collagenases also appear able to digest most if not all of the other collagen types, while the vertebrate collagenases cannot.

A novel series of TNF inhibitors was identified based on the screening of existing MMP inhibitor libraries. Further SAR optimization led to the discovery of a novel lead compound. Its synthesis, efficacy in experimental animal models, and pharmacokinetic data are discussed.

The parallel synthesis of novel inhibitors of procollagen C-terminal proteinase is described. The synthetic strategy allowed for the facile synthesis of a large number of side-chain diversified diamino acid hydroxamates, of which the D-diaminopropionic acid derivatives were shown to be single digit nanomolar PCP inhibitors.

Glutamic acid derived hydroxamates were identified as potent and selective inhibitors of procollagen C-proteinase, an essential enzyme for the processing of procollagens to fibrillar collagens. Such compounds have potential therapeutic application in the treatment of fibrosis.

Optimization of the amino acid side chain and the N-alkyl group of the sulfonamide of amino acid derived sulfonamide hydroxamates is discussed. The solid-phase synthesis of these potent inhibitors of procollagen C-proteinase (PCP) is presented. In addition, novel carboxylic acid sulfonamides were discovered to be PCP inhibitors.

A discussion of the solid-phase synthesis of ornithine derived sulfonamide hydroxamic acids is illustrated. These analogues are shown to be potent, non-peptide inhibitors of procollagen C-proteinase (PCP).

Each of the three known human collagenases has been implicated in the degradation of the type II collagen matrix of cartilage in arthritis. RS-113,456 is a synthetic MMP inhibitor that is selective for collagenase-3 (K-I = 0.17 nM) and collagenase-2 (0.13 nM) over collagenase-1 (70 nM). RS-113,456 inhibits the IL-1 alpha-induced degradation of live bovine nasal cartilage explants in vitro, as measured by hydroxyproline loss, with an IC50 of 50 nM. To ascertain the effect of RS-113,456 on cartilage degradation in vivo, bovine nasal cartilage explants were placed in the center of a cellulose sponge, treated with a suspension of killed M. tuberculosis and implanted subcutaneously into rats. The implants were removed after 16 days, at which time the extent of collagen degradation in vehicle-treated animals was >40%. Given orally from days 1 to 15 at 5 to 30 mg/kg/day BID, RS-113,456 prevented cartilage degradation by 24 to 103%. To assess efficacy in a model of arthritis, the effect of RS-113,456 on type II collagen-induced arthritis was tested in DBA/1 mice. The maximal arthritic score was reduced in all treated groups between 23 and 70%, with p<0.05 at 20 and 40 mg/kg/day. These observations suggest that synthetic inhibitors of human collagenases may prevent cartilage degradation in human arthritis.

Objective To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). Methods Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. Results As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. Conclusion These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

A solid-phase approach to the rapid synthesis of di- and tripeptide-like hydroxamic acids is presented. These compounds are shown to be potent inhibitors of procollagen C-proteinase (PCP).

The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether. In contrast, the collagenase-1 S1' pocket must undergo a conformational change to accommodate comparable P1' groups. The selectivity of the diphenylether series of inhibitors for collagenase-3 is largely determined by their affinity for the preformed S1' pocket of collagenase-3, as compared to the induced fit in collagenase-1.

Choosing an appropriate performance measure is important for fund investors, nevertheless, many researchers find empirically that the choice of measures does not matter because those measures generate identical rank ordering, even though the distribution of fund returns is non-normal. In this paper we certify their findings by proving the monotonicity of several widely used performance measures when the distribution is a location-scale family. The mutual fund monthly return data from 1997 to 2015, together with simulation results, collaborate with our proof.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4°c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k 1) was 2.3 × 107 m −1 s−1 for PGHS-1 and 2.5 × 107 m −1s−1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k 2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 102–103 s−1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that thek 2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.

A plot of the pH dependence of k(cat)/K-M for human stromelysin-1 (HS) exhibits a narrow range of maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.5-8.5. In contrast, the pH profiles that have been reported for other members of the matrix metalloproteinase (MMP) family are bell-shaped and exhibit neutral pH optima. We hypothesized that the anomalous pH dependence of HS reflects the ionization of His-224, a residue located in a flexible loop that contributes to the S-1' binding pocket of the enzyme. HS is the only known MMP that has a histidine in this position. To test this hypothesis, the H224Q mutant of the short form (lacking the C-terminal hemopexin-like domain) of HS (sHS) has been prepared and studied. The pH profile of H224Q sHS is bell-shaped and similar to those reported for other MMPs. Although H224Q and wild-type sHS possess similar activities at pH <6, the k(cat)/K-M of H224Q sHS is more than 5-fold greater than that of the wild-type enzyme at pH >7. These data strongly suggest that the deprotonation of His-224 attenuates the activity of HS, thereby accounting for its low pH optimum and the characteristic shoulder in its pH profile. This attenuation of activity appears to be predominantly a K-M effect, reflecting a decrease in the affinity of the enzyme for the peptide substrate.

The aminopeptidases (APs) constitute a diverse group of proteinases that share the property that they catalyze the hydrolysis of amino acid residues from the N-terminus of peptide and protein substrates.1,2 It has become clear that the great majority of APs are metalloenzymes. Although our understanding of the metallobiochemistry of the APs has generally lagged behind that of the metallo-endopeptidases and -carboxypeptidases, recent advances in our knowledge of the sequence, structure and function of a number of prototypic APs have greatly clarified the picture. In this chapter, the APs will be discussed from the perspective of their metal dependencies with an emphasis on the role(s) of the active site metal atom(s) in catalysis. In particular, these roles will be related to the structures of the metal centers for prototypic APs.

The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgC12 is a moderately effective activator, while p-chloromercuribenzoate and NaOCI are poor activators. It is also shown that human matrilysin and human fibroblast-type collagenase can activate pro-HNG by a mechanism that is very similar to that of stromelysin. Initially, these proteinases hydrolyze the Glu40-Met41 bond in the propeptide domain to generate an 88 kDa inactive HNG species. Collagenase also generates a 68 kDa HNG species through hydrolysis of the Ala74-Met75 bond. Ultimately, treatment with either matrilysin, collagenase or trypsin results in the production of a 65 kDa active form of HNG that arises from hydrolysis of the Arg87-Phe88 bond. This is the same active species produced on activation by stromelysin. This cleavage site is downstream of the ‘cysteine-switch’ residue located at position 80 and releases it, accounting for the permanent activation of the enzyme. These results suggest that matrilysin and collagenase may be physiologically relevant activators of pro-HNG and/or other progelatinase B species. Activation by HgCl2 produces an active 68 kDa enzyme due to autolytic hydrolysis of the Ala14-Met75 bond. This species retains the cysteine switch residue; however, it is shown that it is only active in the continued presence of HgCl2. Removal of the HgCl2 restores latency, indicating that this species is reversibly activated by HgCl2, which functions by complexing the sulfhydryl group of the cysteine switch residue and keeping it dissociated from the active site zinc atom. Thus, in spite of reports to the contrary, the cysteine switch mechanism can account for the latency and activation of pro-HNG.