Current Pharmaceutical Design - Volume 19, Issue 6, 2013

Human neutrophil proteinase 3 (PR3) and elastase (HNE) are homologous serine proteinases involved in the proteolytic events associated with inflammation and infection. Their close structural and functional resemblance makes it difficult to understand their respective biological functions. Thus, all natural inhibitors of PR3 identified to date preferentially target HNE, and only recently have inhibitors that target PR3 selectively been described. This review describes how differences in the structures of the extended active sites of PR3 and HNE can be exploited to produce selective inhibitors of PR3.

The HtrA proteases degrade damaged proteins and thus control the quality of proteins and protect cells against the consequences of various stresses; they also recognize specific protein substrates and in this way participate in regulation of many pathways. In many pathogenic bacteria strains lacking the HtrA function lose virulence or their virulence is decreased. This is due to an increased vulnerability of bacteria to stresses or to a decrease in secretion of virulence factors. In some cases HtrA is secreted outside the cell, where it promotes the pathogen's invasiveness. Thus, the HtrA proteases of bacterial pathogens are attractive targets for new therapeutic approaches aimed at inhibiting their proteolytic activity. The exported HtrAs are considered as especially promising targets for chemical inhibitors. In this review, we characterize the model prokaryotic HtrAs and HtrAs of pathogenic bacteria, focusing on their role in virulence. In humans HtrA1, HtrA2(Omi) and HtrA3 are best characterized. We describe their role in promoting cell death in stress conditions and present evidence indicating that HtrA1 and HtrA2 function as tumor suppressors, while HtrA2 stimulates cancer cell death induced by chemotherapeutic agents. We characterize the HtrA2 involvement in pathogenesis of Parkinson's and Alzheimer's diseases, and briefly describe the involvement of human HtrAs in other diseases. We hypothesize that stimulation of the HtrA's proteolytic activity might be beneficial in therapies of cancer and neurodegenerative disorders, and discuss the possibilities of modulating HtrA proteolytic activity considering the present knowledge about their structure and regulation.

The giant proteolytic factory called the proteasome came a long way from a biochemical curio to a major regulator of cellular physiology and a renowned drug target within the ubiquitin proteasome pathway (UPP). Thanks to availability of highly specific inhibitors of the proteasome, in less than twenty years it was possible to identify major transcription factors, cyclins, and products of oncogenes as crucial substrates for the UPP. Nine years passed since the FDA speedily approved bortezomib, the inhibitor of proteasome, for treatment of multiple myeloma. One year after its approval, the field was honored by awarding the Nobel Prize to Hershko, Ciechanover and Rose for introducing the concept of controlled proteolysis of ubiquitin-tagged substrates, with proteasome as the intracellular recycling facility. Taking into consideration the universal involvement of the proteasome in the life of all cells in human body, it comes to no surprise that the enzyme is deeply implicated in etiology, progression, diagnosis or cure of multiple diseases. Below we discuss some aspects of the involvement: from direct causative links to changes in proteasome properties that correlate with pathological conditions. We start with diseases collectively known as cancer, and with immune system-related pathologies. Here, the proteasome inhibitors are either already used in clinics, or undergo advanced preclinical screening. Then, we will continue with cardiovascular disorders, followed by aging. Changes of the proteasome make-up during aging may be a priming factor for neurodegenerative diseases, described last. We discuss the potential for proteasome regulation: inhibition, activation or specificity modulation, to successfully enter the clinical setting.

Processing of antigens within antigen presenting cells (APCs) is necessary for an immune response. Two pathways exist to present antigens to T cells: the major histocompatibility complex class I (MHC I) pathway to activate cytotoxic T cells (CTLs) and the MHC II route to stimulate T helper cells (Ths). Prior to efficient antigen presentation to MHC II, antigens have to be proteolytically degraded by proteases, the cathepsins, inside the endocytic compartment of APCs. Cathepsins process both antigens and self-antigens to antigenic peptides; the latter are critical for autoimmunity. Remarkably, distribution, substrate specificity, and function of cathepsins located in the antigen processing machinery depend on the cell type, primary or cultured cells, or species analyzed. However, a precise understanding of the antigen processing and presentation machinery is needed to generate specific immune modulators since the MHC antigen- processing pathway is subsequently regulated during tumorigenesis, infection, or autoimmunity. In this review, the latest finding regarding function and regulation of the MHC II proteolytic machinery and its possible target for immunomodulation will be discussed.

Neuropeptides play crucial, mediatory roles in many processes that occur in both CNS and PNS. The commonly accepted dogma for the release of bioactive peptides involves cleavage of inactive precursor by sequential action of proteinases recognizing dibasic stretches, followed by truncation of C-terminal Arg/Lys by the carboxypeptidase-like enzyme(s). Dynorphin convertases play an important role in CNS by regulating dynorphins level and also releasing enkephalins, thus maintaining a balance between these neuropeptides and their distinct functions (dynorphins are preferentially bound to kappa receptors and enkephalins are directed toward delta receptors). Knowledge on the cleavage fragments of dynorphins is important for understanding the pharmacological activity of these peptides. As some new data emerged in the literature, we would like to update recent achievements and progress concerning functions of such convertases, inhibitors, and their potential role in future pharmacotherapy.

Matriptase-2 is a cell surface serine protease with a modular structure. The exploration of its function in iron homeostasis was of significance for the understanding of the regulation of hepcidin expression, the master protein in iron control. Mutations in matriptase- 2 cause iron-refractory iron deficiency anemia (IRIDA), an iron deficiency disorder where the level of hepcidin is inappropriately high. Matriptase-2 controls hepcidin expression through the suppression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic homologue protein (SMAD) signaling, probably by cleaving the BMP co-receptor hemojuvelin. Since prospective studies revealed that genetic inactivation of matriptase-2 reduces iron loading in different mouse models, matriptase-2 becomes highly attractive as a novel target for the design of low-molecular weight inhibitors. The first synthetic peptidomimetic matriptase-2 inhibitors have been reported. A computational model of the active site of matriptase-2 based on the X-ray crystal structure of the close homologue matriptase was generated and mutational studies were performed in order to identify critical amino acids specifying the preferred recognition site of matriptase-2. So far, the only known putative natural substrates of matriptase-2 are hemojuvelin and matriptase-2 itself, as this protease undergoes complex auto-processing during zymogen activation. Cleavage sites within both natural substrates were identified.

Proteolysis is doubtlessly the most widespread mechanism of biological regulation. By controlling protein synthesis, turnover and activity, it is involved in fundamental physiological processes including apoptosis, cell differentiation, growth and signaling, fertilization, immune response, blood coagulation and digestion. Yet, uncontrolled proteolysis can be harmful for organisms, causing - amongst others - such diseases as cancer, emphysema, inflammation, and neurodegenerative, immunological, and cardiovascular disorders. This paper briefly describes recent advances in the development of methodological design to follow up protease activity. Novel methods of protease sensing are described and evaluated. A variety of fluorescent reporter molecules including nanoparticles, and rare metal chelates are also characterized.

The deregulated proteolysis is associated with various diseases in humans. Proteases are commonly regarded as the therapeutic targets. Almost one-third of all proteolytic enzymes in humans are serine proteases. This work provides a brief characteristic of the proteinaceous natural inhibitors, mostly of serine proteases. The examples of some classical and recently identified canonical and noncanonical inhibitors as well as serpins are described. Their actual and potential therapeutic applications are discussed.

Matrix metalloproteinases (MMPs) are either secreted or membrane-bound proteases, capable to degrade extracellular matrix (ECM) proteins as well as a large number of non-ECM proteins, such as growth factors, cytokines, chemokines, cell surface receptors, serine proteinase inhibitors and other MMPs. MMPs play major physiological roles in reproduction, growth, development, angiogenesis, immune response, wound healing and brain physiology. MMPs, and especially MMP-2 and -9 were considered to be targets for drug development (especially in oncology) and over fifty MMP inhibitors have been pursued in clinical trials that, however, failed mainly for the reason of insufficient knowledge about complexity of the biology. Recent studies implicating MMP-9 in aberrant synaptic plasticity underlying neuropsychiatric disorders, as well as in aggravating detrimental effects of the brain stroke, appear to offer a new hope for application of MMP inhibitors in treatment of those conditions.

Pathogenic bacteria have evolved multiple mechanisms aimed to evade host defenses. This review summarizes selected examples of how bacteria utilize proteolytic enzymes to efficiently establish and spread infection systemically. First, the role of proteases in intracellular survival and persistence – the primary means used by bacteria to endure phagocytosis and/or avoid the vigilance of the immune system – is discussed. Second, it is demonstrated how some bacteria escape entanglement in fibrin(ogen) meshes, by inducing their proteolytic dissolution while other species modify the proteolytic cascade of mesh formation to divert this important innate immune defense for their own benefit. Third, bacterial proteolytic toxins are introduced, which allow microorganisms to exert and take advantage of systemic effects already during primary, localized infection. Finally, it is discussed how viruses utilize bacterial proteases by taking advantage of concurrent infection, and how pathogens may even mutually benefit from the joint presence of other pathogens. The reviewed adaptations are often essential for pathogen survival in the hostile environment of a host organism. As such, the potential benefits of pharmacological interference in relevant pathways for the struggle against bacterial pathogens are also discussed.

Mast cells are critical effectors in inflammatory diseases, including cardiovascular and metabolic diseases and their associated complications. These cells exert their physiological and pathological activities by releasing granules containing histamine, cytokines, chemokines, and proteases, including mast cell-specific chymases and tryptases. Several recent human and animal studies have shown direct or indirect participation of mast cell-specific proteases in atherosclerosis, abdominal aortic aneurysms, obesity, diabetes, and their complications. Animal studies have demonstrated the beneficial effects of highly selective and potent chymase and tryptase inhibitors in several experimental cardiovascular and metabolic diseases. In this review, we summarize recent discoveries from in vitro cell-based studies to experimental animal disease models, from protease knockout mice to treatments with recently developed selective and potent protease inhibitors, and from patients with preclinical disorders to those affected by complications. We hypothesize that inhibition of chymases and tryptases would benefit patients suffering from cardiovascular and metabolic diseases.

In order to productively infect a host, viruses must enter the cell and force host cell replication mechanisms to produce new infectious virus particles. The success of this process unfortunately results in disease progression and, in the case of infection with many viral species, may cause mortality. The discoveries of Louis Pasteur and Edward Jenner led to one of the greatest advances in modern medicine - the development of vaccines that generate long-lasting memory immune responses to combat viral infection. Widespread use of vaccines has reduced mortality and morbidity associated with viral infection and, in some cases, has completely eradicated virus from the human population. Unfortunately, several viral species maintain a significant ability to mutate and “escape” vaccine-induced immune responses. Thus, novel anti-viral agents are required for treatment and prevention of viral disease. Targeting proteases that are crucial in the viral life cycle has proven to be an effective method to control viral infection, and this avenue of investigation continues to generate anti-viral treatments. Herein, we provide the reader with a brief history as well as a comprehensive review of the most recent advances in the design and synthesis of viral protease inhibitors.

α-Aminoalkylphosphonate diaryl esters are potent, irreversible, and highly selective site-directed inhibitors of serine proteases. The structure of the phosphonate group resembles the transition state observed during a peptide bond hydrolysis and therefore phosphonates are referred as transition state analogues. They react with the hydroxyl group of the active site serine residue leading to formation of a stable enzyme-inhibitor complex. Moreover, incorporation of a peptidyl chain at the N-terminus as well as an introduction of electron withdrawing or electron donating substituents within the ester ring structure allows for a generation of specific inhibitors that react only with target serine protease. The great advantage of the aminophosphonate diaryl esters over other classes of inhibitors is their stability in aqueous solutions, no toxicity and lack of reactivity with cysteine, threonine, aspartyl and metalloproteinases. The above resulted in their application as convenient tools to study proteases function and activity using in vivo and in vitro assays of different pathological disorders (diabetes, cancer metastasis, pulmonary diseases or hypertension); to determine the cellular localization of the proteinases (activity based probes), to be used in proteomic approach or as the reactive antigens to develop a catalytic function within the antibodies binding site. Herein we present the development of α-aminoalkylphosphonate diaryl esters as inhibitors of several serine proteases including dipeptidyl peptidase IV, cathepsin G, human neutrophil elastase, mast cell chymase and urokinase-type plasminogen activator. We have provided a historical perspective as well as a comprehensive report of the most recent studies in this field.