István Hajdú

Matrix metalloproteinase inhibitors: A critical appraisal of design principles and proposed therapeutic utility

Matrix metalloproteinases (MMPs) play an important role in tissue remodelling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation and cancer metastasis. As a result, MMPs are considered to be viable drug targets in the therapy of these diseases. Despite the high therapeutic potential of MMP inhibitors (MMPIs), all clinical trials have failed to date, except for doxycycline for periodontal disease. This can be attributed to (i) poor selectivity of the MMPIs, (ii) poor target validation for the targeted therapy and (iii) poorly defined predictive preclinical animal models for safety and efficacy. Lessons from previous failures, such as recent discoveries of oxidative/nitrosative activation and phosphorylation of MMPs, as well as novel non-matrix related intra- and extracellular targets of MMP, give new hope for MMPI development for both chronic and acute diseases. In this article we critically review the major structural determinants of the selectivity and the milestones of past design efforts of MMPIs where 2-/3-dimensional structure-based methods were intensively applied. We also analyse the in vitro screening and preclinical/clinical pharmacology approaches, with particular emphasis on drawing conclusions on how to overcome efficacy and safety problems through better target validation and design of preclinical studies.

Structural Evidence for Non-Canonical Binding of Ca2+ to a Canonical EF-Hand of a Conventional Myosin.

We have previously identified a single inhibitory Ca2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399–27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is “closed” in the absence and “open” in the presence of bound cations; a notable exception is the unusual Ca2+-bound closed domain in the essential light chain of the Ca2+-activated scallop muscle myosin. Here we have reported the 1.8 Å resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca2+-induced change in structural dynamics of RD is a major factor in Ca2+-mediated regulation of Physarum myosin II activity.

Design, selection, and evaluation of a general kinase-focused library

The human kinome is a highly conserved target family composed of more than 500 different proteins. Kinases play fundamental roles in many intracellular pathways such as cytokinesis, cell proliferation, differentiation, and apoptosis, and are therefore implicated in various diseases. Currently there are significant efforts in therapeutic areas such as cancer and inflammation to identify novel kinase inhibitors. Random screening of a discovery compound library often results in a hit rate of 0.1 %, whereas focused library screening could improve this rate to 1 %. Consequently, libraries focused toward kinases have become starting points in screening campaigns and are complementary to conventional high-throughput screening (HTS) of discovery libraries. Compound collections catered to target families such as kinases present a unique opportunity to explore discrete chemical, biological, and property spaces. In contrast, HTS libraries are built to represent maximum diversity in the chemical and biological properties of compounds. Focused libraries are also appealing due to decreased synthesis, repository management, and screening costs. The present work describes a rapid computational process to select a collection of compounds targeted as kinase inhibitors, combining the advantages of 2D and 3D virtual screening methods for use in kinase-focused screening campaigns. In designing a general kinase screening library, the challenge lies in defining the chemical and biological space that identifies compounds with utility against any of the many possible kinase targets. Whereas some inhibitors such as staurosporine are known to be active against many kinases, others show a specific inhibitory profile. 7] The limited selectivity of many inhibitors is due to the fact that the catalytic ATP binding domain targeted is highly conserved. In recognition of the difficulty of designing selective kinase inhibitors, dualand multitarget kinase inhibitors were developed. Such “dirty drugs” (i.e. , sorefenib, sunitinib) have gained much attention in recent years and show potential to be advantageous in cancer therapy. These clinical developments have also contributed to the increased interest in general kinase inhibitor libraries. In this work, our goal was to develop a focused library selection procedure based on a 2D similarity search combined with 3D target-based filtering. A recent review argues that “2D fingerprints are surprisingly effective in many search situations in comparison with more complex 3D designs”. Indeed, 2D approaches allow a rapid analogue search from various databases. We find the combination of 2D and 3D methods has distinct advantages; it can decrease the number of false negatives, and 2D methods can represent a pre-filtering tool that enables real-time 3D virtual screening using traditional docking algorithms tailored to the evaluation of large numbers of molecules. Herein we describe our methods and characterize the general kinase-focused library.

Combination of 2D/3D Ligand-Based Similarity Search in Rapid Virtual Screening from Multimillion Compound Repositories. Selection and Biological Evaluation of Potential PDE4 and PDE5 Inhibitors

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.

Combining 2D and 3D in silico methods for rapid selection of potential PDE5 inhibitors from multimillion compounds’ repositories: biological evaluation

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach when starting new drug discovery projects. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compounds’ databases. This in silico approach can be combined with physico-chemical parameter filtering based on the property space of the active compounds and 3D virtual screening if the structure of the target protein is available. A multi-step virtual screening procedure was developed and applied to select potential phosphodiesterase 5 (PDE5) inhibitors in real time. The combined 2D/3D in silico method resulted in the identification of 14 novel PDE5 inhibitors with <1 μMIC50 values and the hit rate in the second in silico selection and in vitro screening round exceeded the 20%.

Structural plasticity of the Salmonella FliS flagellar export chaperone

The Salmonella FliS flagellar export chaperone is a highly α‐helical protein. Proteolytic experiments suggest that FliS has a compact core. However, the calorimetric melting profile of FliS does not show any melting transition in the 25–110 °C temperature range. Circular dichroism measurements reveal that FliS is losing its helical structure over a broad temperature range upon heating. These observations indicate that FliS unfolds in a noncooperative way and its native state shows features reminiscent of the molten globule state of proteins possessing substantial structural plasticity. As FliS has several binding partners within the cell, conformational adaptability seems to be an essential requirement to fulfill its multiple roles.

Combination of Pharmacophore Matching, 2D Similarity Search, and In Vitro Biological Assays in the Selection of Potential 5-HT6 Antagonists from Large Commercial Repositories

Rapid in silico selection of target‐focused libraries from commercial repositories is an attractive and cost‐effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5‐HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second‐round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first‐round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed.

Target Identification and Polypharmacology of Nutraceuticals

Abstract Nutraceuticals are mostly multi-target agents and affect the cellular signaling systems in various stages. The multi-target feature of the herbal components allows interactions with several targets that might cooperate in a synergistic way. To elucidate the exact molecular mechanism that the nutraceuticals exhibit, identification of their beneficial effects at the direct protein target is extremely important. The completion of the human genome project has accelerated the development of the target identification methods and many of the traditional techniques were revisited and upgraded. In this chapter, the multi-target or polypharmacology aspects of the nutraceuticals are discussed, which is followed by a detailed overview of the contemporary procedures for the identification of the direct molecular targets of the most important active nutraceutical components.

Monoclonal antibody proteomics: Use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

Development of matrix metalloproteinase-2 inhibitors for cardioprotection

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 μM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.