Current Pharmaceutical Design - Volume 13, Issue 3, 2007

Proteases have been known for many years as protein-degrading enzymes. However, this view has dramatically changed and proteases are now considered as extremely important signalling molecules, involved in numerous vital processes. Their activity requires strict regulation and regulation defects can lead to pathologies, often associated with excessive proteolysis. The number of diseases, where proteases were identified to be important, if not essential, is increasing almost exponentially and proteases are therefore seriously considered as important drug targets in the areas of cardiovascular diseases, cancer, neurodegenerative diseases (Alzheimer, ...), osteoporosis, diabetes type II, pancreatitis, inflammation, arthritis and rheumatoid arthritis, to list just some of them. In addition, there has been a boom also in the area of infectious diseases, which were less seriously considered with few exceptions such as AIDS and hepatitis C. The recent determination of human, mouse, monkey and rat genomes, as well as the development of new technologies set new standards for more rapid target identification and validation, which is reflected also in the protease field. The major progress in the field resulted in successful development of drugs targeting new targets, such as dipeptidyl peptidase IV inhibitors (Novartis, Merck) for diabetes type II and a renin inhibitor for hypertension (Novartis), which are expected to be launched in 2007. Moreover, the number of new compounds being currently tested in advanced clinical trials suggests a major increase of new therapies based on protease inhibition in the coming years, which is also expected to largely increase the current ∼$11 billion market for protease-targeted drugs [1]. The goal of this issue is to review some of the current advances in the field. In the first paper, Fonovi and Bogyo describe development of activity based probes for proteases, and their applications to biomarker discovery, molecular imaging and drug screening [2]. In the next paper, Butler and Overall describe the power of proteomics in validation of protease drug targets, illustrated on an example from metalloprotease inhibition [3]. This is followed by a paper from Eder et al. [4], who describe the development of inhibitors against different aspartic proteases, including the development of renin inhibitors, where an NDA has been filed in this year. Pre elj et al. discuss the recent advances in anticoagulant therapy based on the inhibition of serine protease inhibition [5]. In another paper based on serine proteases, Sommerhoff and Schaschke describe current advances in the development of tryptase inhibitors for treatment of allergic diseases, including bronchial asthma [6]. After the failure of broad spectrum MMP inhibitors in cancer and rheumatoid arthritis, the focus of MMP inhibition has shifted as described by Fingleton [7]. In addition to MMPs, also metalloprotease exopeptidases can be attractive targets, as described by Arolas et al. [8]. There are also important targets among cysteine proteases, as shown by the papers on caspase [9] and cysteine cathepsin [10] inhibition, where the compounds have also progressed into the clinical studies. Finally, the issue is concluded by a paper from Kido et al. on development of drugs against human influenza virus based on protease inhibition [11]. At the end, I would like to thank all the authors for their contributions, as well as Mr. Mirza Kazim Ali Baig from Bentham Science Publishers for all his help and support. References [1] Turk B. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Disc 2006; 5: 785-99. [2] Fonovi M, Bogyo M. Activity based probes for proteases: applications to biomarker discovery, molecular imaging and drug screening. Curr Pharm Des 2007; 13(3): 253-261. [3] Butler GS, Overall CM. Proteomic validation of protease drug targets. Curr Pharm Des 2007; 13(3): 263-270. [4] Eder J, Hommel U, Cumin F, Martoglio B, Gerhartz B. Aspartic proteases in drug discovery. Curr Pharm Des 2007; 13(3): 271-285. [5] Pre elj A, tefani Anderluh P, Peternel L, Urleb U. Recent advances in serine protease inhibitors as anticoagulant agents. Curr Pharm Des 2007; 13(3): 287-312. [6] Sommerhoff CP, Schaschke N. Mast cell tryptase as a target in allergic inflammation: an evolving story. Curr Pharm Des 2007; 13(3): 313-332. [7] Fingleton B. Matrix metalloproteinases as valid clinical targets. Curr Pharm Des 2007; 13(3): 333-346.....

Recent advances in global genomic and proteomic methods have lead to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.

We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and γ-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.

The drawbacks and limitations of existing anticoagulant therapy which may result in serious adverse effects and a high mortality rate, have given rise to many anticoagulant development programmes in the last decade, focusing mainly at development of thrombin and FXa low-molecular weight inhibitors. A detailed understanding of blood coagulation pathways, functioning of the serine proteases thrombin, FXa, FVIIa and FIXa and elucidation of their crystal structures resulted in many potent compounds, among which some have entered the clinical phase or have been approved for use in clinical practice. Recently, the focus of anticoagulant research turned to inhibition of the TF:FVIIa complex, with some promising clinical candidates on the horizon. This article provides an overview of the current development status of serine protease inhibitors as anticoagulants, including new trends such as dual coagulation factor inhibitors.

Tryptases comprise a group of trypsin-like serine proteases that are highly and selectively expressed in mast cells and to a lesser extent in basophils. Among them interest has been focused on tryptase β, primarily because it was the first tryptase identified and because it is the predominant protease and protein component of mast cells. Subsequent studies have provided convincing evidence that tryptase β is not only a clinically useful marker of mast cells and their activation but that it contributes to the pathogenesis of allergic inflammatory disorders, most notably asthma. The pathogenetic relevance together with the apparent lack of overt physiological functions has caused considerable interest in β-tryptase as a potential therapeutic target. Meanwhile diverse tryptase inhibitors have been synthesized whose design in part was fostered by the structural analysis of the enzymatically active β tryptase tetramer. Various compounds have been studied both in animal models and in man, providing proof of principle that tryptase inhibitors have therapeutic potential in asthma. Here we review the rationale to develop tryptase inhibitors and the approaches pursued, and also try to pinpoint some of the problems that hamper the development of clinically applicable drugs.

The matrix metalloproteinase family of enzymes has been a pharmaceutical target for over 20 years. In that time, many drugs have been developed but none have successfully passed clinical trials. A significant problem has been development of dose-limiting side-effects that were revealed during long-term clinical trials in diseases such as arthritis and various cancers. There are, however, other clinical settings where evidence for MMP function contributing to the pathophysiology of disease is strong. A number of these settings will be discussed here together with evidence from animal models that MMP inhibition is a valid strategy to be considered. A major advantage with many of these settings is that drug exposure may not have to be long-term and/or systemic thus reducing the possibility that side-effects will stymie MMPI-based therapy.

Metallocarboxypeptidases (MCPs) are commonly regarded as exopeptidases that actively participate in the digestion of proteins and peptides. In the recent years, however, novel MCPs comprising a wide range of physiological roles have been found in different mammalian extra-pancreatic tissues and fluids. Among them, CPU, also known as thrombinactivatable fibrinolysis inhibitor (TAFI), has been shown to cleave C-terminal Lys residues from partially degraded fibrin, acting as inhibitor of clot fibrinolysis and therefore constituting an important drug target for thrombolytic therapies. Other MCPs such as CPE, CPN, CPM, and CPD function as pro-hormone and neuropeptide processors and display several structural differences with the pancreatic-like enzymes. In addition, important advances have been made in the discovery and characterization of new endogenous and exogenous proteinaceous inhibitors; the structural determination of their complexes with several MCPs has revealed novel binding modes. Finally, the use of MCPs in antibody-directed enzyme pro-drug therapy (ADEPT) has proved to be an efficient approach for the delivery of lethal levels of chemotherapeutic drugs specifically at tumor tissues. Taken together, these recent developments may help to understand potential biomedical implications of MCPs. Future perspectives for the regulation of these enzymes through the use of more selective and potent inhibitors are also discussed in this review and combined with earlier observations in the field.

This review provides an overview of the biochemistry and activation of inflammatory caspases, and focuses on their therapeutic potential as disease targets in pathologies such as sepsis, Crohn´s disease, rheumatoid arthritis, traumatic brain injury and amyotrophic lateral sclerosis (ALS). We summarize the proof-of-principal evidence obtained by studies in several corresponding experimental disease models confirming the validity of strategies targeting inflammatory caspases. We discuss the use of inflammatory caspase inhibitors, such as VX-740 (Pralnacasan) and VX-765, in clinical studies for rheumatoid arthritis and osteoarthritis. Finally, we point out recent approaches identifying novel peptidomimetic or non-peptide caspase inhibitors with suitable clinical profiles.

The general view on cysteine cathepsins, which were long believed to be primarily involved in intracellular protein turnover, has dramatically changed in last 10 to 15 years. The discovery of new cathepsins, such as cathepsins K, V, X, F and O, and their tissue distribution suggested that at least some of them are involved in very specific cellular processes. Moreover, gene ablation experiments revealed that cathepsins play a vital role in numerous physiological processes, such as antigen processing and presentation, bone remodelling, prohormone processing and wound healing. Their involvement in several pathologies, including osteoporosis, rheumatoid arthritis, osteoarthritis, bronchial asthma and cancer have also been confirmed and today several of them have been validated as relevant targets for therapies. Compounds targeting cathepsins S and K are already in clinical evaluation, whereas others are in experimental phases. The cathepsin K inhibitor AAE-581 (balicatib) as the most advanced of them passed Phase II clinical trials in 2005. In this review, we discuss the current view on cathepsins as an emerging group of targets for several diseases and the development of cathepsin K and S inhibitors for treatment of osteoporosis and various immune disorders.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since IVA genome does not have the processing protease for the viral membrane fusion glycoprotein precursors, entry of this virus into cells is determined primarily by host cellular, trypsin-type, processing proteases that proteolytically activate the fusion glycoprotein precursors of IAV. At least five different processing proteases have been identified in the airways of animals and humans. These proteases determine the infectious organ tropism of IAV infection as well as the efficiency of viral multiplication in the airway, and sometimes in the brain. Proteases in the upper respiratory tract are suppressed by secretory leukoprotease inhibitor, and those in the lower respiratory tract are suppressed by pulmonary surfactant which, by adsorption, inhibits the interaction between the proteases and viral membrane proteins. Since protease activities predominate over those of endogenous inhibitory compounds under normal airway conditions, administration of protease inhibitors in the early-stage of infection significantly suppresses viral entry and viral multiplication. Several viral neuraminidase inhibitors are used clinically as anti-influenza virus agents, based on their inhibitory action on viral release from infected cells. Furthermore, protease inhibitors of viral entry could be potentially useful against influenza virus as well as neuraminidase inhibitor- resistant viruses. We also found that ambroxol, a mucolytic and anti-oxidant agent, up-regulates the levels of endogenous protease inhibitory compounds in the airway fluids in early-phase infection, and that clarithromycin, a macrolide antibiotic, increases IgA levels and mucosal immunity through augmentation of interleukin-12 levels in the airway. The combination of neuraminidase inhibitors and protease inhibitors, clarithromycin or ambroxol, could be potentially used as a potent anti-influenza therapy to minimize the emergence of drug-resistant mutant viruses.