Machine Learning-Enhanced Quantum Chemistry-Assisted Refinement of the Active Site Structure of Metalloproteins.

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Sodium hyaluronate-g-2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide with enhanced affinity towards MMP12 catalytic domain to be used as visco-supplement with increased degradation resistance.

The present paper describes the functionalization of sodium hyaluronate (NaHA) with a small molecule (2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide) (MMPI) having proven inhibitory activity against membrane metalloproteins involved in inflammatory processes (i.e. MMP12). The obtained derivative (HA-MMPI) demonstrated an increased resistance to the in-vitro degradation by hyaluronidase, viscoelastic properties close to those of healthy human synovial fluid, cytocompatibility towards human chondrocytes and nanomolar affinity towards MMP 12. Thus, HA-MMPI can be considered a good candidate as viscosupplement in the treatment of knee osteoarticular disease.

Therapeutic Targeting of MMP-12 for the Treatment of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a lung disorder characterized by progressive airflow obstruction associated with inflammation and emphysema, and it is currently one of the leading causes of death worldwide. Recent studies with genetically engineered mice reported that during pulmonary inflammation, basophil-derived interleukin-4 can act on lung-infiltrating monocytes causing aberrant expression of the matrix metalloproteinase-12 (MMP-12). MMP-12 activity in turn causes the destruction of alveolar walls leading to emphysema, making it potentially a valid target for pharmacological intervention. Using nuclear magnetic resonance (NMR)- and structure-based optimizations, the current study reports on the optimized novel, potent, and selective MMP-12 inhibitors with single-digit nanomolar affinity in vitro and in vivo efficacy. Using a murine model of elastase-induced emphysema we demonstrated that the most potent agents exhibited a significant decrease in emphysema-like pathology compared to vehicle-treated mice, thus suggesting that the reported agents may potentially be translated into novel therapeutics for the treatment of COPD.

Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs.

Matrix metalloproteinase-12 (MMP-12), also known as macrophage elastase, is a potent inflammatory mediator and therefore an important pharmacological target. Clinical trial failures of broad-spectrum compound MMP inhibitors suggested that specificity is the key for a successful therapy. To provide the required selectivity, monoclonal antibody (mAb)-based inhibitors are on the rise. However, poor production of active recombinant human MMP-12 catalytic domain (cdMMP-12) presented a technical hurdle for its inhibitory mAb development. We hypothesized that this problem could be solved by designing an expression-optimized cdMMP-12 mutant without structural disruptions at its reaction cleft and surrounding area, and thus isolated active-site inhibitory mAbs could maintain their binding and inhibition functions toward wild-type MMP-12. We combined three advances in the field-PROSS algorithm for cdMMP-12 mutant design, convex paratope antibody library construction, and functional selection for inhibitory mAbs. As a result, isolated Fab inhibitors showed nanomolar affinity and potency toward cdMMP-12 with high selectivity and high proteolytic stability. Particularly, Fab LH11 targeted the reaction cleft of wild-type cdMMP-12 with 75 nM binding KD and 23 nM inhibition IC50 . We expect that our methods can promote the development of mAbs inhibiting important proteases, many of which are recalcitrant to functional production.

Prediction of matrix metal proteinases-12 inhibitors by machine learning approaches.

Matrix metal proteinases-12 (MMP-12) is a hot pharmaceutical target on the treatment of many human diseases. There's a crying need for designing and finding new MMP-12 inhibitors. In this work, four machine learning approaches, support vector machine, k-nearest neighbor, C4.5 decision tree, and random forest, were employed to derive statistical models from datasets with well distributed biological activities and predict a compound whether it is a MMP-12 inhibitor. The prediction accuracies of the models are in the range of 96.15-98.08% for sensitivity, 87.23-100.00% for specificity, 91.92-98.99% for the overall prediction accuracy and 0.8401-0.9800 for Matthews correlation coefficient, all producing satisfactory results. By means of diverse feature selection methods, several sets of critical descriptors with key information of inhibitory properties were selected by different models, accelerating the classification for MMP-12 inhibitors and non-inhibitors. Communicated by Ramaswamy H. Sarma.

Development of Thioaryl-Based Matrix Metalloproteinase-12 Inhibitors with Alternative Zinc-Binding Groups: Synthesis, Potentiometric, NMR, and Crystallographic Studies.

Matrix metalloproteinase-12 (MMP-12) selective inhibitors could play a role in the treatment of lung inflammatory and cardiovascular diseases. In the present study, the previously reported 4-methoxybiphenylsulfonyl hydroxamate and carboxylate based inhibitors (1b and 2b) were modified to enhance their selectivity for MMP-12. In the newly synthesized thioaryl derivatives, the nature of the zinc binding group (ZBG) and the sulfur oxidation state were changed. Biological assays carried out in vitro on human MMPs with the resulting compounds led to identification of a sulfide, 4a, bearing an N-1-hydroxypiperidine-2,6-dione (HPD) group as new ZBG. Compound 4a is a promising hit compound since it displayed a nanomolar affinity for MMP-12 with a marked selectivity over MMP-9, MMP-1, and MMP-14. Solution complexation studies with Zn2+ were performed to characterize the chelating abilities of the new compounds and confirmed the bidentate binding mode of HPD derivatives. X-ray crystallography studies using MMP-12 and MMP-9 catalytic domains were carried out to rationalize the biological results.

An integrated structure- and pharmacophore-based MMP-12 virtual screening.

MMP-12 belongs to a large family of proteases called matrix metalloproteinases (MMPs) that degrades elastin. The main pathologic role of MMP-12 overexpression was suggested to be associated with pathogenesis mechanism of inflammatory respiratory diseases and atherosclerosis. An integrated ligand- and structure-based virtual screening was employed in hope of finding inhibitors with new scaffolds and selectivity for MMP-12. Seven compounds among 18 experimentally tested compounds had a measurable effect on the inhibition of MMP-12 enzyme. Our results demonstrated the applicability of the developed pharmacophore model and selected crystal structure (PDB code: 3F17) to discover new MMP-12 inhibitors. The receptor structure was selected based on cross-docking results. Here, we report the discovery of new class of MMP-12 inhibitors that could be used for lead optimization. For the inhibition of MMP-12, the significance of its interactions with the catalytic residues Glu219 and Ala182 was emphasized through the inspection of the docking poses.

Peripheral membrane associations of matrix metalloproteinases.

Water soluble matrix metalloproteinases (MMPs) have been regarded as diffusing freely in the extracellular matrix. Yet multiple MMPs are also observed at cell surfaces. Their membrane-proximal activities include sheddase activities, collagenolysis, bacterial killing, and intracellular trafficking reaching as far as the nucleus. The catalytic domains of MMP-7 and MMP-12 bind bilayers peripherally, each in two different orientations, by presenting positive charges and a few hydrophobic groups to the surface. Related peripheral membrane associations are predicted for other soluble MMPs. The peripheral membrane associations may support pericellular proteolysis and endocytosis. The isolated soluble domains of MT1-MMP can also associate with membranes. NMR assays suggest transient association of the hemopexin-like domains of MT1-MMP and MMP-12 with lipid bilayers. Peripheral association of soluble MMP domains with bilayers or heparin sulfate proteoglycans probably concentrates them near the membrane. This could increase the probability of forming complexes with membrane-associated proteins, such as those targeted for proteolysis. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Synthesis and in Vitro and in Vivo Evaluation of MMP-12 Selective Optical Probes.

In designing new tracers consisting of a small peptide conjugated to a reporter of comparable size, particular attention needs to be paid to the selection of the reporter group, which can dictate both the in vitro and the in vivo performances of the whole conjugate. In the case of fluorescent tracers, this is particularly true given the large numbers of available dye moieties differing in their structures and properties. Here, we have investigated the in vitro and in vivo properties of a novel series of MMP-12 selective probes composed of cyanine dyes varying in their structure, net charge, and hydrophilic character, tethered through a linker to a potent and specific MMP-12 phosphinic pseudopeptide inhibitor. The impact of linker length has been also explored. The crystallographic structure of one tracer in complex with MMP-12 has been obtained, providing the first crystal structure of a Cy5.5-derived probe and confirming that the binding of the targeting moiety is unaffected. MMP-12 remains the tracers' privileged target, as attested by their affinity selectivity profile evaluated in solution toward a panel of 12 metalloproteases. In vivo assessment of four selected probes has highlighted not only the impact of the dye structure but also that of the linker length on the probes' blood clearance rates and their biodistributions. These experiments have also provided valuable data on the stability of the dye moieties in vivo. This has permitted the identification of one probe, which combines favorable binding to MMP-12 in solution and on cells with optimized in vivo performance including blood clearance rate suitable for short-time imaging. Through this series of tracers, we have identified various critical factors modulating the tracers' in vivo behavior, which is both useful for the development and optimization of MMP-12 selective radiolabeled tracers and informative for the design of fluorescent probes in general.

Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors.

Matrix metalloproteinase-12 (MMP-12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a ß-N-acetyl-d-glucosamine moiety, were designed and synthesized as MMP-12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP-12 inhibitor with improved water solubility, compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-ß-d-glucopyranosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid], was identified.

Path to Collagenolysis: COLLAGEN V TRIPLE-HELIX MODEL BOUND PRODUCTIVELY AND IN ENCOUNTERS BY MATRIX METALLOPROTEINASE-12.

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å(2)of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling.

New insights into the substrate specificity of macrophage elastase MMP-12.

Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

Receptor-based 3D-QSAR approach to find selectivity features of flexible similar binding sites: case study on MMP-12/MMP-13.

Design of selective matrix metalloproteinases (MMPs) inhibitors is still a challenging task because of binding pocket similarities and flexibility among MMPs family. To overcome this issue we try to generate a (three-dimensional quantitative structure activity relationship) 3D-QSAR model that might reflect, at least in part, the differential properties of MMP-12 and MMP-13 active sites compared to each other. The different alignment rules were applied for CoMFA/CoMSIA model development. In an approach the best docked poses were followed by alignment based on their zinc binding group. As it was suggested by comparison of CoMSIA contour maps of MMP-12 with MMP-13, the ligand based approach can find more detailed features of specificity for MMPs that have similar highly flexible active sites, than solely analysis of available crystal structures. The residues Val(194), Leu(214) and Thr(220) of MMP-13 were suggested to be investigated for flexibility upon binding of different ligands.

The impact of binding thermodynamics on medicinal chemistry optimizations.

Ligand binding thermodynamics has been attracted considerable interest in the past decade owing to the recognized relation between binding thermodynamic profile and the physicochemical and druglike properties of compounds. In this review, the relation between optimization strategies and ligand properties is presented based on the structural and thermodynamic analysis of ligand-protein complex formation. The control of the binding thermodynamic profile is beneficial for the balanced affinity and physicochemical properties of drug candidates, and early phase optimization gives more opportunity to this control.

Identification of matrix metalloproteinase inhibitors by chemical arrays.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.

A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form.

BACKGROUND: Mild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies. RESULTS: Here we presented an efficient method for mild solubilization of inclusion bodies by using a freeze-thawing process in the presence of low concentration of urea. We used two different proteins to demonstrate the advantage of this method over the traditional urea-denatured method: enhanced green fluorescent protein (EGFP) and the catalytic domain of human macrophage metalloelastase (MMP-12_CAT). Firstly, PBS buffer at pH 8 containing different molar concentration of urea (0-8 M) were used to solubilize EGFP and MMP-12-CAT inclusion bodies and the solubility achieved in 2 M urea in PBS buffer by freeze-thawing method was comparable to that of PBS buffer containing 8 M urea by traditional urea-denatured method. Secondly, different solvents were used to solubilize EGFP and MMP-12_CAT from inclusion bodies and the results indicated that a wide range of buffers containing 2 M urea could efficiently solubilize EGFP and MMP-12_CAT inclusion bodies by freeze-thawing method. Thirdly, the effect of pH and freezing temperature on the solubility of EGFP and MMP-12_CAT inclusion bodies were studied, revealing that solubilization of inclusion bodies by freeze-thawing method is pH dependent and the optimal freezing temperature indicated here is -20°C. Forth, the solubilized EGFP and MMP-12_CAT from inclusion bodies were refolded by rapid dilution and dialysis, respectively. The results showed that the refolded efficiency is much higher (more than twice) from freeze-thawing method than the traditional urea-denatured method. The freeze-thawing method containing 2 M urea also effectively solubilized a number of proteins as inclusion bodies in E.coli. CONCLUSIONS: Mild solubilization of inclusion body proteins using the freeze-thawing method is simple, highly efficient and generally applicable. The method can be utilized to prepare large quantities of bioactive soluble proteins from inclusion bodies for basic research and industrial purpose.

A QSAR study on the inhibition mechanism of matrix metalloproteinase-12 by arylsulfone analogs based on molecular orbital calculations.

A binding mechanism between human matrix metalloproteinase-12 (MMP-12) and eight arylsulfone analogs having two types of carboxylic and hydroxamic acids as the most representative zinc binding group is investigated using a quantitative structure-activity relationship (QSAR) analysis based on a linear expression by representative energy terms (LERE). The LERE-QSAR analysis quantitatively reveals that the variation in the observed (experimental) inhibitory potency among the arylsulfone analogs is decisively governed by those in the intrinsic binding and dispersion interaction energies. The results show that the LERE-QSAR analysis not only can excellently reproduce the observed overall free-energy change but also can determine the contributions of representative free-energy changes. An inter-fragment interaction energy difference (IFIED) analysis based on the fragment molecular orbital (FMO) method (FMO-IFIED) leads to the identification of key residues governing the variation in the inhibitory potency as well as to the understanding of the difference between the interactions of the carboxylic and hydroxamic acid zinc binding groups. The current results that have led to the optimization of the inhibitory potency of arylsulfone analogs toward MMP-12 to be used in the treatment of chronic obstructive pulmonary disease may be useful for the development of a new potent MMP-12 inhibitor.

Designing lead optimisation of MMP-12 inhibitors.

The design of new molecules with desired properties is in general a very difficult problem, involving heavy experimentation with high investment of resources and possible negative impact on the environment. The standard approach consists of iteration among formulation, synthesis, and testing cycles, which is a very long and laborious process. In this paper we address the so-called lead optimisation process by developing a new strategy to design experiments and modelling data, namely, the evolutionary model-based design for optimisation (EDO). This approach is developed on a very small set of experimental points, which change in relation to the response of the experimentation according to the principle of evolution and insights gained through statistical models. This new procedure is validated on a data set provided as test environment by Pickett et al. (2011), and the results are analysed and compared to the genetic algorithm optimisation (GAO) as a benchmark. The very good performance of the EDO approach is shown in its capacity to uncover the optimum value using a very limited set of experimental points, avoiding unnecessary experimentation.

SSNMR of biosilica-entrapped enzymes permits an easy assessment of preservation of native conformation in atomic detail.

Bioinspired silica coprecipitates of enzymes find a wide range of applications for analysis and catalysis in industrial and academic research. Here we used SSNMR as a proxy for the 3D structure of enzymes trapped in bioinspired silica. We show that it is easy to assess whether the enzymes retain their native conformation in atomic detail. We thus propose SSNMR as a rapid and reliable tool to analyze this kind of samples at high resolution.

Crystallization of bi-functional ligand protein complexes.

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.