Hydroxamate Inhibitors for Blocking Tumors

While active site inhibitors of collagenase would be of interest in treating any of the diseases in which the enzyme is over-active, one focus of clinical drug development and trials has been in oncology. The elaboration of various MMPs by malignant cells and their role in breaching basement membranes and degrading connective tissue is one mechanism by which tumor invasion, local spread and metastasis are thought to occur.99 In this context, there is little evidence to support the notion that these inhibitors alone could serve as monotherapy against malignancy since they have not been shown to be cytotoxic to a variety of malignant cells grown in cell culture. Rather, they may have a role in adjuvant therapy, delaying or preventing tumor growth, invasion and metastasis. Of the inhibitors which have been developed so far, the hydroxamates have been the most extensively studied in vitro and in vivo, and several are currently in clinical trials. Batimastat (BB-94, 4-(N-hydroxyamino)-2(R)-isobutyl-3(S)-[(2-thienylthomethyl)succinyl]-L-phenylalanine-N-methylamide) is the prototype of the majority of hydroxamate inhibitors. It possesses a broad spectrum of inhibition with IC50 values for collagenase (MMP-1), both 72 and 92 kilodalton gelatinases (MMP-2 and MMP-9), and matrilysin (MMP-7) in the 2-6 nM range, and for stromelysin (MMP-3) at 20 nM.6 Although it is poorly soluble in aqueous solution (< 3 |ig/ml), depot and intraperitoneal injections have nevertheless been demonstrated to result in systemic levels of the drug well within the range of the major MMPs.6 A second generation of hydroxamate inhibitors such as Marimastat (BB-2516) has been developed and possesses improved solubility and are orally bioavailable. Marimastat is as effective as Batimastat in inhibiting MMP-1, MMP-2, MMP-7 and MMP-9 in vitro, and an order of magnitude worse at inhibiting stromelysin.96

In animal models, batimastat has been reported to inhibit tumor growth and spread (modest effect in the latter) of B16-BL6 melanoma cells injected in mice,6,101 inhibit angio-genesis and reduce tumor doubling time in hemangiomas in mice by inhibiting recruitment and organization of vascular cells and structures,102 human breast cancer regrowth and metastasis in a nude mouse xenograft model,103 reduce human colon cancer growth and local, regional and distant spread as well as reduce ascites volume and improve survival in a mouse model,104-106 and in human ovarian cancer.107 However, the drug was ineffective in preventing colonization in multiple organs following intraperitoneal injection of a Burkitt lymphoma cell line into SCID mouse model, using PCR to detect the presence of the malignant clone.6 Recently, AG3340, a nonpolypeptide MMP inhibitor whose design was based on the crystallographic data accumulated so far, has shown picomolar K;s against MMP-2, MMP-3, and MMP-9. In a Lewis lung carcinoma murine model, this inhibitor was associated with modest reductions in the sizes of primary tumors and a more impressive reduction in metastases following intraperitoneal administration.108 Interestingly, other similarly designed inhibitors with in vitro inhibition of MMPs had a wide range of effects in the animal model, including enhanced tumor growth. AG3340 is orally bioavailable and a phase I study in healthy volunteers has been announced in abstract form.109

Some hydroxamate-based inhibitors have also been developed for other clinical applications. Galardin or GM6001, another hydroxamate inhibitor, is one of the most potent peptide inhibitors of human skin fibroblast collagenase with a K of 0.4 nM.110 While it is effective in inhibiting MMP-1, it is not specific since similar Ki values have been measured for neutrophil collagenase, and gelatinases A and B.111 GM6001 has been shown to inhibit angiogenesis in rats and chick chorioallantoic membranes,112,113 corneal ulceration due to chemical and infectious insults in rabbits113 and phorbol ester-induced skin inflammation and thickening when applied topically to the ears of hairless mice.111 Interestingly, GM1489, in which a carboxylic acid group replaces the hydroxamate and a phenylmethyl group replaces methyl at the P3' position, was as effective as GM6001 in reducing skin thickening, although GM1489 is specific for MMP-1 over MMP-2, MMP-3, MMP-8 and MMP-9 by at least several orders of magnitude.111 Another inhibitor, Ro-31-7467, inhibits collagenase with an IC50 of 16.8 nM, compare to 208.8 nM for gelatinase A, and 238.8 nM for stromelysin.114 Ro-31-7467 was able to inhibit bone resorption in a cultured neonatal mouse calvarial system at 10-8 M assayed in a bone resorption model, although not completely at concentrations at which its specificity for collagenase was retained.114 Ro 32-3555 inhibits collagenases fairly specifically with Kj values of 3-4 nM, one to two orders of magnitude better than its inhibition of stromelysin or either of the gelatinases.115 In a rat monoarthritis model in which Propionibacterium acnes was injected intra-articularly, Ro 32-3555 administered orally was capable of inhibiting cartilage degradation.

Few human trials of the synthetic peptide inhibitors have been reported so far in the peer-reviewed literature. Batimastat (BB-94) has been in Phase II clinical trials for malignant pleural fluid effusions and ascites6 and a phase I trial in patients with various advanced malignancies without ascites has recently been reported.116 This trial involved 9 patients stratified into three dose levels 600, 1200 and 1800 mg/m2; batimastat was administered every four weeks. The principal side effects were abdominal discomfort and cramping at 1200 and 1800 mg/m2, which were severe and prolonged at the highest dose requiring premedication with analgesics, including narcotics. Due to the side effect profile, the poor bioavailability necessitating administration as a suspension, and the development of marimastat, batimastat has not been pursued as systemic therapy and has reportedly been discontinued as a treatment for cancer.107,117 Marimastat, a second generation, orally bioavailable hydroxamate inhibitor which followed Batimastat, is now in clinical trials for lung, ovarian, colorectal, pancreatic, and small cell lung cancers and glioblastomas.9,116 Galardin or GM6001 has been in phase III trial for treatment of corneal ulceration.9 The complete and published results of the various clinical trials of these inhibitors are awaited with anticipation.

In contrast to inhibitors based on similarities to the collagen substrate of MMPs, an alternative approach has exploited the fact that the prodomain sequence of MMPs contains a highly conserved twelve amino acid sequence, MRKPRCGVPDVG, containing a cysteine residue whose free thiol moiety is thought to form a coordinating ligand with the catalytic zinc, thus preventing collagenolytic activity.114,115 The sequence interestingly is found to be oriented in the active site of the enzyme running in the opposite direction than the collagen substrate and the peptide analog inhibitors discussed above.86 A screen of various peptides based on this sequence resulted in the identification of the hexapeptide Ac-RCGVPD-NH2 and the pentapeptide Ac-RCGVP-NH2 with IC50 values of 4.5 and 10 |iM, respectively. These compounds were active against both stromelysin and collagenase. Subsequently, it was shown that only Cys-75, Gly-76 and Val-77 are essential for inhibition, and that replacement of Cys-75 with isocysteine improved potency by 5-fold. These complexes suffer a potential problem with stability due to autoxidation, and because the sequences are common to both stromelysin and collagenase, they are not completely specific for a single type of MMP. The details of interactions will have to await a high-resolution structure with the prodomain sequence, and it may be possible to substitute other residues or moieties which will both allow discrimination between the large S1' pocket of stromelysin and the more restrictive collagenase as well as enhance stability of the molecule.

In addition to active site inhibitors based on oligopeptide derivatives, nonpeptide compounds have also been developed which retain the hydroxamate moiety as a zinc-coordinating ligand and have been shown to inhibit various MMPs, including MMP-1. These include aryl sulfonamides, alkyl sulfonamides and futenones.9

In addition to crystallography and NMR, other methods are being developed which promise to aid in the design and optimization of active site inhibitors. Molecular dynamics simulations and other computational methods which search conformational space will be important as additional structural information is provided by more traditional methods.120 The contribution of molecular dynamics may be particularly relevant in understanding the time-dependent aspects of collagenase activity and pointing attention to residues or parts of the protein which may significantly determine key vibrational or phonon modes necessary for catalysis. It may also possibly explain how an entire collagen triple helix is cleaved by an active site which appears to be able to accommodate only a single polypeptide at a time. In a further acknowledgment that our present understanding of protein structure-function relationships is incomplete, it is worth noting that there is evidence from random mutagenesis studies that sites of enzymes and proteins which are not obviously related to their functional sites are indeed important for function.121 Because these may be in more exposed surfaces of the protein, they may constitute another, more accessible target for therapeutic peptides, antibodies and small molecules.

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