Current Topics in Medicinal Chemistry - Volume 4, Issue 12, 2004

Proteases play crucial roles for the normal functioning of biological systems. Proteins that are generated in ribosome must be degraded in order for them to carry out their functions in the biological systems and these protein degradations are catalyzed by a variety of proteolytic enzymes. A myriad of metabolic reactions involved in diverse physiological processes such as protein turnover, digestion, blood coagulation and wound healing, fertilization, cell differentiation and growth, immune response, and apoptosis are effected and controlled by proteases. Uncontrolled, unregulated or undesired proteolyses cause aberrant physiological processes, leading to many disease states such as emphysema, viral infection, stroke, cancer, Alzheimer's disease, inflammation, and arthritis. Inhibitors of proteases thus have potential utility for therapeutic intervention in a variety of disease states, and in fact numerous protease inhibitors are currently being used as clinical agents for treatment of a variety of states. The proteolytic enzymes can be divided into four major classes on the basis of the mode of their catalytic actions: serine-, cysteine-, aspartic- and metallo-proteases. In this special issue, recent developments in design protocols of small molecule inhibitors that are effective against these enzymes have been reviewed by leading scientists in their respective field. The review by Zhang and Groutas focuses the design of mechanism-based and alternate substrate inhibitors of serine proteases, and Kim reviewes recent progresses on the design of irreversible inhibitors that covalently modify carboxypeptidase A, a protopytical zinc proteases. Mobsahery and associates derscribe the design strategy for a novel type of irreversible inhibitors for matrix metalloproteases (MMPs), which show an excellent selectivity toward specific MMPs. In light of the fact that there is mounting evidences that broad-spectrum inhibition of MMPs is highly undesirable due to diverse physiological activities these enzymes manifest, selective inhibition of MMPs is currently of prime concern with regard to obtaining therapeutically viable inhibitors of MMPs. Vederas and collegues report strategies used for designing inhibitors effective against 3C cysteine proteases from picornaviridae, viruses comprising one of the most important families of human pathogens. Since excellent review articles related to aspartic proteases have appeared recently, no inclusion for inhibitors of the enzymes is made in this issue. It is hoped that the present reviews would serve to provide new impetus for efficient design and discovery of new medicinal agents. Finally, I would like to express my sincere thanks to all the contributors, whose hard work and dedication made this special issue possible.

The matrix metalloproteinases (MMPs) are a family of more than 20 zinc-dependent mammalian enzymes, both membrane bound and secreted, most of which are involved in the cleavage of some component of the extracellular matrix (ECM), including collagen and gelatin. Related enzyme families include the adamalysins (ADAMs) and the ADAM-TS enzymes. The discovery of these important enzymes and their physiological functions began in the 1960s and still continues 40 years later. Furthermore, the apparent importance of the MMPs and ADAMs in the etiology of several debilitating diseases for which only palliative treatments had been available has made them exceedingly attractive targets for drug discovery programs at virtually every major pharmaceutical company and many academic institutions. From the 1990s to the present an explosion of patent applications and publications has delivered potent inhibitors of MMPs, TACE, and aggrecanases evolving from peptidic scaffolds bearing a variety of zinc chelators to non-peptides, some even devoid of any zinc ligating moiety. Unfortunately, clinical trials of MMP and TACE inhibitors have so far failed to deliver on the great promise envisioned for this class of molecules. Drug candidates in oncology, osteoarthritis and rheumatoid arthritis have almost uniformly failed to provide efficacy, or an acceptable risk-benefit profile, for advancing past Phase III clinical studies. In this context research into inhibitors of MMPs and ADAMs awaits advances to determine the best enzyme targets for particular therapeutic areas, and the degree of subtype specificity required of these inhibitors to allow their safe use as chronically administered drugs. In this issue are presented several different approaches to the design and synthesis of MMP and TACE inhibitors. In the first review, by Wada, the progression from peptidic hydroxamate, broad spectrum MMP inhibitors to more selective non-peptide, reverse hydroxamate inhibitors of gelatinases is described. This program resulted in a clinical candidate in oncology. In the second review, Hanessian and Moitessier present their elegant combination of structure-based design and organic synthesis in exploring potent and selective sulfonamide hydroxamate inhibitors. The third review, by Levin, describes the MMP / TACE activity of aryl hydroxamic acids. These scaffolds run the gamut from broad spectrum inhibitors, to more selective inhibitors of MMP-9 and MMP-13 with no TACE activity, to potent MMP / TACE inhibitors. In the fourth review, Rush and Powers report on the application and importance of structural information accumulated from X-ray, NMR and molecular modeling to guide the design of potent and diverse inhibitors. Finally, I would like to offer my thanks to all the authors who took the time and made the effort to contribute to this issue.

A wide range of human diseases are associated with the aberrant activity of mammalian, viral, bacterial or parasitic proteases. These include members of all four classes of proteases, namely, serine, cysteine, aspartic and metalloproteases. The involvement of proteases in disease states has provided the impetus behind studies related to the design of potent and selective inhibitors and their use as either therapeutic agents and / or pharmacological probes to gain a better understanding of the pathophysiology of a particular disease. This review focuses on recent developments related to the design of mechanism-based and alternate substrate inhibitors of serine proteases of mammalian and non-mammalian origin. Numerous examples are cited that illustrate the fundamental principles and subtleties associated with the design of covalent and non-covalent inhibitors of these enzymes. This is an exciting and promising area of investigation that will undoubtedly continue unabated in the future.

Carboxypeptidase A (CPA) is one of the most extensively studied zinc proteases and serves as a prototypical enzyme for a large family of metalloproteases that play important roles in biological systems. CPA has been used as a model enzyme for developing design strategies of inhibitors that restrain the catalytic activity of zinc proteases. Recently, there has been made a remarkable progress in designing small molecule inactivators that inhibit the enzymic activity of CPA irreversibly by chemically modifying a functional group at the active site of the enzyme. Of these irreversible inhibitors mechanism-based inactivators are of special interest due to their high selectivity for target enzyme and long duration of enzyme inhibition. These inactivators have been designed rationally on the basis of established topology of the active site and catalytic mechanism of the enzyme. Chemistry inherent to the zinc ion at the active site of the enzyme has been exploited in the design. The present review covers the progress in the CPA inactivator design strategy. The design strategy developed with CPA may be transferred to other zinc proteases of medicinal interest, leading to discovery of a novel type of therapeutically useful enzyme inhibitors.

Matrix metalloproteinases (MMPs), of which at least 26 are known in humans, have been linked to a number of pathological conditions including tumor metastasis, inflammation, neurological and cardiovascular diseases. Inhibition of MMPs has been widely sought as a strategy in intervention of these disease processes. Whereas a large number of broadspectrum MMP inhibitors have been developed over the past decade, these inhibitors have not met the promise and expectations in clinical trials. The broad-spectrum inhibition, which besides MMPs often targets other metalloproteinases, has been considered one of the potential problems that affects the therapeutic efficacy of MMPs inhibitors. Several MMP inhibitors that show selectivity for various MMPs have been reported in the past few years. This report describes the structural and inhibitory properties of these novel inhibitors, which hold considerable promise for effective targeting of these important enzymes.

The Picornaviridae are among the smallest icosahedral positive-sense single stranded RNA containing viruses known, and comprise one of the largest and most important families of human and animal pathogens. The hepatitis A virus (HAV) and human rhinovirus (HRV) are important pathogens that belong to the picornavirus family. All picornaviruses have a 3C proteinase that processes an initially biosynthesized precursor protein and is crucial for viral maturation and replication. Although it is a cysteine proteinase, this 3C enzyme has a topology similar to those of the chymotrypsin-like serine proteinases. A series of inhibitors of HAV and HRV 3C proteinases were synthesized and tested as potential lead compounds for the design of therapeutic agents for human picornaviral pathogens. This research shows that thiol-reactive groups or “warheads” such as iodoacetamides, β-lactones, Michael acceptors, ketones and pseudoxazolones can be used as effective tools to inhibit the HAV and HRV 3C proteinase enzymes. In addition, studies based on enzyme-inhibitor kinetics, mass spectrometry and NMR spectroscopy were effectively used to gain knowledge concerning enzyme-inhibitor mechanism of action and enzyme-inhibitor regiospecific reactivity.

Matrix metalloproteinases (MMPs) have been implicated in several pathologies. At Abbott Laboratories, the matrix metalloproteinases inhibitor drug discovery program has focused on the discovery of a potent, selective, orally bioavailable MMP inhibitor for the treatment of cancer. The program evolved from early succinate-based inhibitors to utilizing in-house technology such as SAR by NMR to develop a novel class of biaryl hydroxamate MMP inhibitors. The metabolic instability of the biaryl hydroxamates led to the discovery of a new class of N-formylhydroxylamine (retrohydroxamate) biaryl ethers, exemplified by ABT-770 ( 16). Toxicity issues with this pre-clinical candidate led to the discovery of another novel class of retrohydroxamate MMP inhibitors, the phenoxyphenyl sulfones such as ABT-518 ( 19j). ABT-518 is a potent, orally bioavailable, selective inhibitor of MMP-2 and 9 over MMP-1 that has been evaluated in Phase I clinical trials in cancer patients.

The present account relates to our studies in the computer assisted design and synthesis of acyclic and cyclic MMP inhibitors. Our early efforts focused on the preparation of cyclopropane and tetrahydrofuran-based mimics of batimastat which were not active. The discovery of subnanomolar sulfonamide-based acyclic inhibitors instigated the design of novel target compounds. Thus, with the help of a fully automated and reliable docking program, we embarked on the design and synthesis of enantiopure inhibitors incorporating cyclic scaffolds. This ultimately led to compounds exhibiting inhibitory activities in the nanomolar range. Interestingly, the qualitative ranking prediction was found to be in good agreement with the observed activities.

Three different classes of aryl hydroxamic acid scaffolds have been explored and provided potent inhibitors of MMP-1, -2, -9, -13 and TACE. Structure-based design has allowed the evolution of these inhibitors from broad spectrum inhibitors into compounds that are more selective for MMPs relevant to particular disease states. Aryl hydroxamates selective for MMP-9, MMP-13 and TACE have been disclosed that may aid in the study of the physiological role of these enzymes. Furthermore, the different selectivity profiles offered by these MMP / TACE inhibitors may allow the determination of which metalloprotease, or group of metalloproteases, must be inhibited for the safe, long-term treatment of osteoarthritis, rheumatoid arthritis and cancer. Some of these compounds have demonstrated useful biological activity in efficacy models relevant to osteoarthritis and rheumatoid arthritis and are therefore potential clinical candidates.

The following review discusses the successful application of X-ray, NMR, and molecular modeling in the design of potent and selective inhibitors of matrix metalloproteinases (MMPs) and TNFα-converting enzyme (TACE) from Wyeth. The importance of protein and ligand mobility as it impacts structure-based design is also discussed. The MMPs are an active target for a variety of diseases, including cancer and arthritis.