MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density ...modification, manual and semi‐automated or automated model building and rebuilding, real‐ and reciprocal‐space structure optimization and refinement, map calculations and various types of molecular structure validation. The prompt availability of various analytical tools and the immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed refined structure. The extraordinary depth perception of molecular objects in three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
The Future of Cysteine Cathepsins in Disease Management Kramer, Lovro; Turk, Dušan; Turk, Boris
Trends in pharmacological sciences (Regular ed.),
October 2017, 2017-10-00, 20171001, Letnik:
38, Številka:
10
Journal Article
Recenzirano
Since the discovery of the key role of cathepsin K in bone resorption, cysteine cathepsins have been investigated by pharmaceutical companies as drug targets. The first clinical results from ...targeting cathepsins by activity-based probes and substrates are paving the way for the next generation of molecular diagnostic imaging, whereas the majority of antibody–drug conjugates currently in clinical trials depend on activation by cathepsins. Finally, cathepsins have emerged as suitable vehicles for targeted drug delivery. It is therefore timely to review the future of cathepsins in drug discovery. We focus here on inflammation-associated diseases because dysregulation of the immune system accompanied by elevated cathepsin activity is a common feature of these conditions.
The newly discovered roles of secreted cysteine cathepsins are dominating preclinical research, with major developments in pain regulation and cancer chemotherapy resistance.
Cathepsin K inhibitors, including odanacatib, have all failed in osteoporosis clinical trials, but novel strategies to target cathepsin K are emerging.
The antibody–drug conjugate field is dominated by cathepsin-cleavable linkers.
First cathepsin-targeting imaging probes have entered clinical trials with favorable initial results.
A new concept for targeted drug delivery based on cathepsin-targeting is emerging.
Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in‐vivo imaging, as well ...as an extensive use of in‐vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein‐degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.
The identification of physiological protease substrates has resulted in a shift of paradigm from proteases as protein‐degrading enzymes to establishing their role as key signalling molecules.
It is more than 50years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among ...these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate “warhead”. The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50years after the discovery of lysosome.
► Current advances in the field of cysteine cathepsins and their regulation. ► Cysteine cathepsin activity as a delicate balance of various factors. ► Structure of cysteine cathepsins and their mechanism of interaction with inhibitors. ► Inhibition of cysteine cathepsins by protein and small-molecule inhibitors. ► The increased expression of cysteine cathepsins implicated in various diseases.
Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural ...product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-4,5'-bithiazole-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.
Macromolecular crystallography and electron microscopy (single-particle and in situ tomography) are merging into a single approach used by the two coalescing scientific communities. The merger is a ...consequence of technical developments that enabled determination of atomic structures of macromolecules by electron microscopy. Technological progress in experimental methods of macromolecular structure determination, computer hardware, and software changed and continues to change the nature of model building and visualization of molecular structures. However, the increase in automation and availability of structure validation are reducing interactive manual model building to fiddling with details. On the other hand, interactive modeling tools increasingly rely on search and complex energy calculation procedures, which make manually driven changes in geometry increasingly powerful and at the same time less demanding. Thus, the need for accurate manual positioning of a model is decreasing. The user's push only needs to be sufficient to bring the model within the increasing convergence radius of the computing tools. It seems that we can now better than ever determine an average single structure. The tools work better, requirements for engagement of human brain are lowered, and the frontier of intellectual and scientific challenges has moved on. The quest for resolution of new challenges requires out-of-the-box thinking. A few issues such as model bias and correctness of structure, ongoing developments in parameters defining geometric restraints, limitations of the ideal average single structure, and limitations of Bragg spot data are discussed here, together with the challenges that lie ahead.
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.
Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel‐based label‐free proteomic approach (DIPPS—direct in‐gel profiling ...of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in‐gel digestion of the gel‐separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length‐limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase‐7, and legumain) to broadly specific (matrix‐metalloproteinase‐3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH‐dependent change in the specificity of this protease, further supporting its broad applicability.
Synopsis
Direct In‐gel Profiling of Protease Specificity (DIPPS) enables quick and reliable determination of protease cleavage specificities under a large variety of experimental conditions, offering an excellent starting point for protease characterization and substrate design.
Gel‐based, label‐free whole‐proteome degradomics provides an easy‐to‐use approach applicable under a broad range of assay conditions.
DIPPS validation on 10 distinct proteases from three major mechanistic classes shows an excellent correlation with published protease specificities, without observable experimental biases.
DIPPS profiling of legumain reveals a pH‐dependent specificity switch between Asn and Asn/Asp cleavage specificity.
Direct In‐gel Profiling of Protease Specificity (DIPPS) under a large variety of experimental conditions offers a quick and reliable starting point for protease functional characterization and substrate design.
The cystatin superfamily comprises a large group of the cystatin domain containing proteins, present in a wide variety of organisms, including humans. Cystatin inhibitory activity is vital for the ...delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases such as the papain (C1) family, including cysteine cathepsins. Some of the cystatins also inhibit the legumain (C13) family of enzymes. Failures in biological mechanisms controlling protease activities result in many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis, arthritis, and cancer. Cystatins have been classified into three types: the stefins, the cystatins and the kininogens, although other cystatin-related proteins, such as CRES proteins, are emerging. The stefins are mainly intracellular proteins, whereas the cystatins and the kininogens are extracellular. The cystatins are tight binding and reversible inhibitors. The basic mechanism of interaction between cystatins and their target proteases has been established, based mainly on the crystal structures of various cathepsins, stefins and cystatins and their enzyme-inhibitor complexes. Cystatins, as rather non-selective inhibitors, discriminate only slightly between endo- and exopeptidases. They are also prone to form amyloids. The levels of some stefins and cystatins in tissue and body fluids can serve as relatively reliable markers for a variety of diseases. In this review we summarize present knowledge about cystatins and their role in some diseases.
Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed ...the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.
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