Metalloprotease inhibitors are cellular inhibitors of the matrix metalloproteinases (MMPs). MMPs belong to a family of zinc-dependent neutral endopeptidases. These enzymes have the ability to break down connective tissue. The expression of MMPs is increased in various pathological conditions like inflammatory conditions, metabolic bone disease, to cancer invasion, metastasis and angiogenesis.
Examples of diseases are periodontitis, hepatitis, glomerulonephritis, atherosclerosis, emphysema, asthma, autoimmune disorders of skin and dermal photoaging, rheumatoid arthritis, osteoarthritis, multiple sclerosis, Alzheimer's disease, chronic ulcerations, uterine involution, corneal epithelial defects, bone resorption and tumor progression and metastasis. Due to the role of MMPs in pathological conditions, inhibitors of MMPs may have therapeutic potential. Several other proteins have similar inhibitory effects, however none as effective (netrins, procollagen C-terminal proteinase enhancer (PCPE), reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and tissue factor pathway inhibitor (TFPI-2)). They might have other biological activities which have yet been fully characterised.
MMP inhibitors can broadly be subdivided into non-synthetic (e.g. endogenous) or synthetic. Several potent MMP inhibitors have been identified, including hydroxymates, thiols, carbamoylphosphonates, hydroxyureas, hydrazines, β-lactams, squaric acids and nitrogenous ligands.
There are three classes of commonly used inhibitors for metalloproteinases.
In vitro, EDTA, 1,10-phenanthroline and other chelating compounds lower the concentration of metal to the point where the metal is removed from the enzyme active site. Classical lock and key inhibitors such as phosphoramidon and bestatin bind tightly by approximating the transition state of the hydrolysis of the peptide, preventing it from acting on other substrates. Protein inhibitors such as α2-macroglobulin are known to work with metalloproteinases.
History The first generation of MMP inhibitors were based on the structure of the collagen molecule. This group of inhibitors contain a hydroxamate (-CONHOH) group that binds the zinc atom in the active site of the MMP enzyme. The first MMP inhibitors that were tested in patients were Ilomastat and Batimastat, hydroxamate-based MMP inhibitors. However, neither compound showed good oral bioavailability.
Thus far, Periostat (active ingredient is doxycycline hyclate) is the only MMP inhibitor that has been approved by the U.S. Food and Drug Administration (FDA). It is used for the treatment of periodontitis. Other MMP inhibitors have exhibited serious side effects during preclinical trials. These side effects are caused by insufficient selectivity. Most MMP inhibitors are unable to target specific MMPs connected to specific pathological conditions. Instead, they inhibit multiple MMPs, some of which have protective functions or are not related to pathology.
MMPs have been regarded as promising targets for cancer therapy. Preclinical studies investigating the efficacy of MMP suppression in tumor models were encouraging. Following these results, clinical studies were conducted but turned out to be disappointing. Recent studies have shown that MMPs may even have paradoxical roles in tumor progression. MMPs seem to have tumor-promoting effects as well as tumor suppressive effects dependent on different contexts.
Mechanism of action
Most MMP inhibitors are chelating agents. The inhibitor binds to the zinc at the active center of the enzyme, thereby blocking its activity. Other inhibitor mechanisms are possible.
α2-Macroglobulin (α2M) is a protease inhibitor which inhibits activated MMPs. α2M and MMP form a complex which is able to inactivate the MMP.
MMPs are associated with the cell surface or bound to the extracellular matrix which prevents them from diffusing away and keeps the MMP under control of the cell. One mechanism to inhibit MMP activity is by dislodging the enzymes from their receptors. Gold salts bind to a heavy metal site distinct form the zinc-containing active center, which inhibits their activity. MMP activity can be decreased by binding to the cleavage site on the substrate e.g. catechin.
Two molecular features of most MMP inhibitors are responsible for the affinity. One is a chelating moiety that interacts with the zinc ion and the other is a hydrophobic extension from the catalytic site that project into S1’ pocket (P1’ group) of the metalloproteinase. The structural difference MMPs’ is mainly in the S1’ side and by modifying the P1’ group, inhibitor selectivity can be developed.
Drug development Various potential MMP inhibitors will be explained in the following sections, including information on their development, structure-activity relationship and pharmacokinetics.
Pioneering hydroxamate structures The first generation of MMP inhibitors were based on the structure of the collagen molecule. In the design of these inhibitors, the basic protein backbone of collagen is maintained but the amide bond is replaced with a zinc-binding group. This group of inhibitors contain a hydroxamate (-CONHOH) group that binds the zinc atom in the active site of the MMP enzyme, therefore this group is called "hydroxamate-based MMP inhibitors“. An example can be seen in Marimastat, a first generation inhibitor, which has a similar backbone and sidechain format to collagen.
Ilomastat and batimastat were the first two MMP inhibitors to be tested in patients. These are both hydroxamate-based MMP inhibitors and have similar overall structures.
The hydroxamate-based MMP inhibitors display an excellent anticancer activity in tumor cells but the clinical performances of these compounds were disappointing. A factor contributing to this disappointment was that they are broad-spectrum inhibitors of many MMP sub-types that can in many cases also inhibit members of the ADAMs protease family. When they were tested in patients they induced dose-limiting muscular and skeletal pain in a number of the patients. Only when the structures of the MMP inhibitors could be adjusted to impart selectivity and abolish toxicity, would they achieve clinical impact in cancer chemotherapy.
New generation hydroxamate-based inhibitors The pioneering hydroxamate-based inhibitors were followed by a set of 'new generation' molecules with features including a substituted aryl, a sulfonamide and a hydroxamate zinc-binding group.
In MMI-270 there is also an amino acid sidechain-type substituent on the carbon that is α to the hydroxamate, along with a sidechain on the sulfonamide (which was later shown to be unnecessary). The N-arylsulfonyl-α-aminoacid hydroxamate of MMI-270 mimics the marimastat succinate motif. Cipemastat, which was developed as an MMP-1, -3 and -9 collagenase inhibitor for the treatment of rheumatoid- and osteo-arthritis, also has the marimastat succinate motif. Its clinical trial was terminated prematurely.
MMI-166 has an N-arylsulfonyl-α-aminocarboxylate zinc-binding group, different from the hydroxamate-zinc binding group seen in MMI-270 and Cipemastat. It also has a triaryl substitution that the other structures didn't have. ABT-770 and Prinomastat also have an aryl substitution. In ABT-770 the two phenyl rings are directly connected but in Prinomastat the two phenyl rings are connected by an oxygen atom, forming a diphenylether. These three permutations direct the SAR away from MMP-1 and toward the „deep pocket“ MMPs such as the gelatinases. ABT-770 shows anticancer activity in animal models, but it is easily metabolised to an amine metabolite that causes phospholipidosis. MMI-166 has shown anticancer activity in numerous animal models, but there is no data available of its clinical performance. Prinomastat on the other hand, is one of the best studied MMP inhibitors. It showed excellent preclinical animal anticancer efficacy, but a recurring limitation to these hydroxamates (Prinomastat in particular) is drug metabolism including loss of the hydroxamate zinc-binding group.
These inhibitors were followed by the next group of hydroxamate-based inhibitors, which focus on the suppression of metabolism, minimization of MMP-1 inhibitory activity and the control of subtype selectivity, by structure-based design. The tetrahydropyran in RS-130830 introduces a steric block that suppresses metabolism, which would fix the problem that the previous generation of inhibitors showed. The outcome of its clinical evaluation has not yet been disclosed. 239796-97-5 has improved ADME and MMP-1 selectivity properties and has shown excellent oral efficacy in an animal model of osteoarthritis. Although, the therapeutic objectives for these inhibitors is not cancer, like it has been for most of the MMP inhibitors.
New generation thiol-based inhibitors
Rebimastat is a broad spectrum MMP inhibitor with a thiol zinc-binding group. It has oral bioavailability and is a collagen non-peptide mimetic. Rebimastat has some selectivity as it doesn't inhibit all the MMPs operations. The metalloproteinases that release TNF-alpha, TNF-II, L-selectin, IL-1-RII and IL-6 are for example not inhibited by Rebimastat.
In phase I of clinical trials, there was no sign of dose-dependent joint toxicity and a disease stabilization. Arthralgia was noted in phase II early breast cancer trials, which was connected to MMP inhibitor toxicity. Rebimastat was used in a Paclitaxel/Carboplatin treatment in phase III. The results of the trial was a higher incidence of adverse reactions, without survival benefit.
Clinical trials for Tanomastat, an alfa-((phenylthio)methyl)carboxylate, showed similar results. It showed good disease stability and tolerance in Phase I solid tumor trials and good tolerance in advanced cancer in combination with Etoposide. However, its efficacy was not proven to be adequate. Tanomastat showed significant hepatotoxicity in a cancer therapy combined with Cisplatin and Etoposide, although in a treatment with Doxorubicin it showed good tolerance and lowered toxicity with 5-fluorouracil and Leucovorin.
Many compounds in the thiol zinc-binding groups have good water solubility and are air stable in plasma and these groups will be continued in MMP inhibitor designing.
Pyrimidine-based inhibitors
Ro 28-2653 is highly selective for MMP-2, MMP-9 and membrane type 1 (MT-1)-MMP. It is an antitumor and antiangiogenic agent with oral bioavailability. Inhibition of TACE and MMP-1 are linked to the musculoskeletal side effects seen in hydroxamate metalloproteinase inhibitors, but this compound spares the enzymes. It has been shown to diminish tumor growth in nasal cancer in rats as well as prostate cancer cell cultures. The compound only has moderate effect on mice's adipose tissue and no alteration on joints. Based on this, it is concluded that class of inhibitors is less likely to trigger neuromuscular adverse effects. On the active site of the structure is a pyrimidinetrione chelation and the phenyl and piperidynil section occupy the S1’ and S2’ binding pockets of MMP-8.
Compound 556052-30-3 is similar to Ro 28-2653 but incorporates a 4-((2-methylquinolin-4-yl)methoxy)phenyl sidechain that is TACE selective.
5-(spiropyrrolidin-5-yl)pyrimidinetrione is a compound named 848773-43-3 that is a potent MMP-2, MMP-9 and MMP-13 inhibitor that spares MMP-1 and TACE.
By substituting 1,3,4-oxadiazol-2-yl heteroaryl at C-4’ of the diphenylether segment to accomplish MMP-13 selectivity over MT-1 MMP, made the compound 420121-84-2. The compound has IC50 (half maximal inhibitory concentration) of 1 nM for MMP-13.
I125-radioable pyrimidinetriones that have similar structure have been made to be used in MMP-9 elevated atherosclerosis and elevated MMP-2 and MMP-9 cancers.
This class of MMP inhibitors is easy to synthesize and are potent enough for clinical valuation.