Current Topics in Medicinal Chemistry - Volume 10, Issue 3, 2010

Members of the protease families discussed in this issue are of great interest for different therapeutic areas tackling contemporary afflictions of mankind such as cardiovascular, infective, or respiratory diseases as well as cancer. The proteases dealt with not only act as protein-degrading enzymes, their important function within signaling pathways makes them also highly attractive as valid drug targets. The search for as well as the identification of potent and selective inhibitors for this protein family has been very popular for decades and still is a highly competitive field in industrial and academic drug research development. This special issue of Current Topics in Medicinal Chemistry “The Medicinal Chemistry of Protease Inhibitors” focuses on inhibitors of different protease classes and is divided into three major parts: the first part discusses inhibitors for particular members of the threonine, serine, and cysteine protease family, the second gives an overview of proteases and their inhibitors involved in infectious diseases, and the third part reviews hit validation, conducting as well as pitfalls frequently occurring in protease assays. The issue starts with a review of Genin, Reboud-Ravaux, and Vidal on recent advances and new perspectives of proteasome inhibitors. The well-known covalent inhibitors for this member of the threonine protease family as well as recent developments towards first non-covalent derivatives are discussed. In the following, Straub, Rohrig, and Hillisch highlight the field of factor Xa, representing one example of the serine protease family, and its corresponding inhibitors. Their review concentrates on different non-basic P1-site inhibitors and describes in particular the development of Rivaroxaban (Xarelto™). The family of cysteine proteases and their inhibitors are presented within two contributions. Pietsch, Chua, and Abell introduce into the field of calpains as attractive drug targets and inhibitors thereof, followed by an article of Frizler, Stirnberg, Sisay, and Gütschow discussing nitrile-based peptidic inhibitors for the subfamily of cathepsins. The subsequent part of this issue focuses on proteases that are suitable drug targets to combat infectious diseases. The aim of this part is to give a survey of active compounds inhibiting proteases of different classes but share their quality of being active in only one therapeutic category. Steuber and Hilgenfeld describe the class of viral proteases which are investigated within the antiviral drug discovery. A second topic, summarized in a review of Wegscheid-Gerlach, Gerber and Diederich, deals with different malarial proteases of plasmodium falciparum and inhibitors thereof. In the final part of this issue, Ludewig, Kossner, Schiller, Baumann, and Schirmeister discuss in their contribution entitled “Enzyme kinetics and hit validation in fluorimetric protease assays” common problems frequently occurring in fluorimetric assays. They furthermore provide methods and strategies concerning problem solving as well as circumventive measures prior to assay determination. We very much appreciated the valuable contributions of all authors to this special issue of Current Topics in Medicinal Chemistry entitled “The Medicinal Chemistry of Protease Inhibitors”. We would also like to thank all reviewers for their very constructive comments. We are also grateful to the editor-in-chief, Dr. Allen Reitz, for the invitation and the opportunity to compile this special issue as guest editors and for all his support concerning the preparation of this special issue.

The search for proteasome inhibitors began fifteen years ago. These inhibitors proved to be powerful tools for investigating many important cellular processes regulated by the ubiquitin-proteasome pathway. Targeting the proteasome pathway can also lead to new treatments for disorders like cancer, muscular dystrophies, inflammation and immune diseases. This is already true for cancer; the FDA approved bortezomib, a potent proteasome inhibitor, for treating multiple myeloma in 2003, and mantle cell lymphoma in 2006. The chemical structures identified in some of the early proteasome inhibitors have led to the development of new anti-cancer drugs (CEP-18770, Carfilzomib, NPI-0052). All these molecules are covalent bonding inhibitors that react with the catalytic Thr1-Oγ of the three types of active site. This review covers recent developments in medicinal chemistry of natural and synthetic proteasome inhibitors. Advances in non-covalent inhibitors that have no reactive group will be highlighted as they should minimize side-effects. New structures and new modes of action have been recently identified that open the door to new drug candidates for treating a range of diseases.

Several clinical candidates have now emerged as a result of an intense search for orally available, antithrombotic factor Xa inhibitors. This review highlights the discovery of Xarelto™ (Rivaroxaban) starting from an initial tetrahydrophthalimide screening hit. The major breakthrough was the finding that a chlorothiophene moiety can undergo an interaction in the S1 binding site thus leading to high potency combined with favorable oral bioavailability. The binding mode of this P1 moiety is discussed, and further non-basic S1 binders of this new type are reviewed.

The physiological roles of calpains are discussed, as are the associated pathological disorders that result from their over-activation. We also present practical information for establishing functional inhibition assays and an overview of X-ray crystal structures of calpain-inhibitor complexes to aid inhibitor design. These structures reveal the expected extended β-strand conformation for the inhibitor backbone, a geometry that has been engineered into inhibitors with the introduction of either an N-terminal heterocycle or a macrocycle that links the P1 and P3 residues. The structure and function of all the main classes of inhibitors are reviewed, with most examples being classified according to the nature of the C-terminal reactive warhead group that reacts with the active site cysteine of calpains. These inhibitor classes include epoxysuccinate derivatives, aldehydes, aldehyde prodrugs (hemiacetals) and α-keto carbonyl compounds. Inhibitors derived from the endogenous inhibitor calpastatin and examples lacking a warhead, are now known and these are also discussed.

It is now becoming clear that several papain-like cysteine cathepsins are involved in the pathophysiology of diseases such as osteoporosis, autoimmune disorders, and cancer. Therefore, the development of potent and selective cathepsin inhibitors is an attractive subject for medicinal chemists. New advances have been made for nitrile-based inhibitors, leading to the identification of the cathepsin K inhibitor odanacatib and other candidates with potential for therapeutic use. This review summarizes the development of peptidic and peptidomimetic compounds with an electrophilic nitrile ‘warhead’ as inhibitors of the cysteine cathepsins B, S, L, C, and K. Peptide nitriles have been shown to reversibly react with the active site cysteine under formation of a covalent thioimidate adduct. The structural optimization with respect to the positions P3, P2, P1, P1', and P2' resulted in the identification of potent and selective inhibitors of the corresponding cathepsins. The underlying structure-activity relationships are discussed herein.

The occurrence of life-threatening viral infections and the establishment of appropriate defense strategies exhibit major challenges to the disease management in our society. The unpredictable character of viral outbreaks will even be enhanced in the future due to human activities such as increasing international travel, deforestation, changes in social conditions, or influences induced by the climate change. The defense against these pathogenic agents requires preparedness, including successful drug design strategies. Viral proteases represent attractive targets for antiviral therapy because of their essential role in (+)-stranded RNA viruses and retroviruses. In addition, viral proteases can be involved in further processes relevant for viral replication. Numerous efforts have been made to develop potent small-molecule inhibitors of viral proteases; however, until now only a limited number reached the market. In the present contribution, functional aspects of the target proteases, their structural properties, drug design strategies, resulting inhibitors, and resistance management are reviewed and discussed by means of the representative cases of HIV, HCV, SARS coronavirus, and the flaviviruses Dengue and West Nile virus.

Malaria, caused by protozoa of the genus Plasmodium, remains one of the most dreadful infectious diseases worldwide killing more than 1 million people per year. The emergence of multidrug-resistant parasites highly demands a steadfast and continuous search not only for new targets but also for new anti-infectives addressing the known ones. As proteases in general have been proven to be excellent drug targets and the development of inhibitors has frequently resulted in approved drugs, this review will only focus on the proteases of Plasmodium falciparum as drug targets. The completion of the sequencing of the Plasmodium falciparum genome in 2002 lead to the discovery of nearly 100 putative proteases encoded therein. Within this review, only those proteases and inhibitors thereof will be discussed in more detail, in which their biological function has been determined undoubtedly or in those cases, in which the development of specific inhibitors has significantly contributed to the understanding of the underlying biological role of the respective protease thus validating the role as promising drug target.

Fluorimetric assays are convenient and efficient to determine the inhibitory potency of enzyme inhibitors. Since enzyme activity can be blocked in a number of ways, it is important to determine the exact mode of inhibition. The first part of the review deals with kinetic methods to distinguish among the different modes of inhibition. In addition to that, pitfalls are discussed that can be encountered if the mode of inhibition was not thoroughly investigated. The second part of the review deals with some basic techniques of hit validation. Specifically, three error sources that may result in misleadingly strong inhibitors are scrutinized and exemplified for two different typical protease assays (cathepsin B, chymotrypsin). The studied error sources are attenuation of the fluorescence signal, aggregation of the analysed molecules, and irreversible binding of the inhibitor to the enzyme. A simple experimental protocol to detect the aforementioned problems is proposed.