Successes and challenges in multiscale modelling of artificial metalloenzymes: the case study of POP-Rh2 cyclopropanase.

Molecular modelling applications in metalloenzyme design are still scarce due to a series of challenges. On top of that, the simulations of metal-mediated binding and the identification of catalytic competent geometries require both large conformational exploration and simulation of fine electronic properties. Here, we demonstrate how the incorporation of new tools in multiscale strategies, namely substrate diffusion exploration, allows taking a step further. As a showcase, the enantioselective profiles of the most outstanding variants of an artificial Rh2-based cyclopropanase (GSH, HFF and RFY) developed by Lewis and co-workers (Nat. Commun., 2015, 6, 7789 and Nat. Chem., 2018, 10, 318-324) have been rationalized. DFT calculations on the free-cofactor-mediated process identify the carbene insertion and the cyclopropanoid formation as crucial events, the latter being the enantiodetermining step, which displays up to 8 competitive orientations easily altered by the protein environment. The key intermediates of the reaction were docked into the protein scaffold showing that some mutated residues have direct interaction with the cofactor and/or the co-substrate. These interactions take the form of a direct coordination of Rh in GSH and HFF and a strong hydrophobic patch with the carbene moiety in RFY. Posterior molecular dynamics sustain that the cofactor induces global re-arrangements of the protein. Finally, massive exploration of substrate diffusion, based on the GPathFinder approach, defines this event as the origin of the enantioselectivity in GSH and RFY. For HFF, fine molecular dockings suggest that it is likely related to local interactions upon diffusion. This work shows how modelling of long-range mutations on the catalytic profiles of metalloenzymes may be unavoidable and software simulating substrate diffusion should be applied.

Influence of Association on Binding of Disaccharides to YKL-39 and hHyal-1 Enzymes.

Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.

BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization.

With a large amount of research dedicated to decoding how metallic species bind to proteins, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal-binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parameterization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 90 metal-binding X-ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems whose structures (either experimental or theoretical) are not optimal for metal-binding sites. We report here its application on three different challenging cases: (i) the modulation of metal-binding sites during conformational transition in human serum albumin, (ii) the identification of possible routes of metal migration in hemocyanins, and (iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding. BioMetAll is an open-source application available at https://github.com/insilichem/biometall.

Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability.

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.

GARLEEK: Adding an extra flavor to ONIOM.

The ONIOM method, developed in the group of Keiji Morokuma, is one of the most successful examples of quantum mechanics/molecular mechanics (QM/MM) treatments, and of multilayer methods in general. Its implementation in the Gaussian program package is in particular widely used. This implementation has access to the wide variety of QM methods available in Gaussian, but is limited to only three specific force fields. The current article presents the GARLEEK interface, which expands the availability of molecular mechanics methods to the wide variety of force fields available in MM packages. The focus is in the simple installation and use. Two examples of the performance of the interface with selected systems are provided. GARLEEK is MIT-licensed and freely available at https://github.com/insilichem/garleek. © 2018 Wiley Periodicals, Inc.

GPathFinder: Identification of Ligand-Binding Pathways by a Multi-Objective Genetic Algorithm.

Protein-ligand docking is a widely used method to generate solutions for the binding of a small molecule with its target in a short amount of time. However, these methods provide identification of physically sound protein-ligand complexes without a complete view of the binding process dynamics, which has been recognized to be a major discriminant in binding affinity and ligand selectivity. In this paper, a novel piece of open-source software to approach this problem is presented, called GPathFinder. It is built as an extension of the modular GaudiMM platform and is able to simulate ligand diffusion pathways at atomistic level. The method has been benchmarked on a set of 20 systems whose ligand-binding routes were studied by other computational tools or suggested from experimental "snapshots". In all of this set, GPathFinder identifies those channels that were already reported in the literature. Interestingly, the low-energy pathways in some cases indicate novel possible binding routes. To show the usefulness of GPathFinder, the analysis of three case systems is reported. We believe that GPathFinder is a software solution with a good balance between accuracy and computational cost, and represents a step forward in extending protein-ligand docking capacities, with implications in several fields such as drug or enzyme design.

Investigating Potential Inhibitory Effect of Uncaria tomentosa (Cat's Claw) against the Main Protease 3CLpro of SARS-CoV-2 by Molecular Modeling.

COVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2. Presently, there is no effective treatment for COVID-19. As part of the worldwide efforts to find efficient therapies and preventions, it has been reported the crystalline structure of the SARS-CoV-2 main protease Mpro (also called 3CLpro) bound to a synthetic inhibitor, which represents a major druggable target. The druggability of Mpro could be used for discovering drugs to treat COVID-19. A multilevel computational study was carried out to evaluate the potential antiviral properties of the components of the medicinal herb Uncaria tomentosa (Cat's claw), focusing on the inhibition of Mpro. The in silico approach starts with protein-ligand docking of 26 Cat's claw key components, followed by ligand pathway calculations, molecular dynamics simulations, and MM-GBSA calculation of the free energy of binding for the best docked candidates. The structural bioinformatics approaches led to identification of three bioactive compounds of Uncaria tomentosa (speciophylline, cadambine, and proanthocyanidin B2) with potential therapeutic effects by strong interaction with 3CLpro. Additionally, in silico drug-likeness indices for these components were calculated and showed good predicted therapeutic profiles of these phytochemicals. Our findings suggest the potential effectiveness of Cat's claw as complementary and/or alternative medicine for COVID-19 treatment.

Computational insight into the interaction of oxaliplatin with insulin.

In an organism, cisplatin and its derivatives are known to interact with proteins besides their principal DNA target. These off-target interactions have major therapeutic consequences including undesired side effects, loss of bioavailability and emergence of resistance. Insulin is one of the prototypical protein targets of platinum drugs as it has been seen to be involved in bioavailability reduction and might also determine resistance in certain cancer lines. However, despite the interest in understanding the nature of the oxaliplatin-insulin adducts, no 3D models have been achieved so far. In this study, we apply our recent computational multiscale protocol optimized for bioinorganic interactions to provide structural insights into these systems. To do so, the initial structures are predicted by blind protein-metalloligand docking calculations optimized to account for a metal-containing species, and then refined using a Molecular Dynamics (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) integrated protocol. The results are consistent with experimental information obtained from fragment analysis, and also provide novel structural information like conformational changes occurring upon binding and potential effects on the biological functions of the protein. This study opens an avenue towards applying similar strategies to a wide ensemble of metallodrug-protein/peptide systems for which no structural data are available.

Synthesis of polyfluoroalkyl sp2-iminosugar glycolipids and evaluation of their immunomodulatory properties towards anti-tumor, anti-leishmanial and anti-inflammatory therapies.

Immunomodulatory glycolipids, among which α-galactosylceramide (KRN7000) is an iconic example, have shown strong therapeutic potential in a variety of conditions ranging from cancer and infection to autoimmune or neurodegenerative diseases. A main difficulty for those channels is that they often provoke a cytokine storm comprising both pro- and anti-inflammatory mediators that antagonize each other and negatively affect the immune response. The synthesis of analogues with narrower cytokine secretion-inducing capabilities is hampered by the intrinsic difficulty at controlling the stereochemical outcome in glycosidation reactions, particularly if targeting the α-anomer, which seriously hampers drug optimization strategies. Here we show that replacing the monosaccharide glycone by a sp2-iminosugar glycomimetic moiety allows accessing N-linked sp2-iminosugar glycolipids (sp2-IGLs) with total α-stereocontrol in a single step with no need of protecting groups or glycosidation promotors. The lipid tail has been then readily tailored by incorporating polyfluoroalkyl segments of varied lengths in view of favouring binding to the lipid binding site of the master p38 mitogen activated protein kinase (p38 MAPK), thereby polarizing the immune response in a cell-context dependent manner. The compounds have been evaluated for their antiproliferative, anti-leishmanial and anti-inflammatory activities in different cell assays. The size of the fluorous segment was found to be critical for the biological activity, probably by regulating the aggregation and membrane-crossing properties, whereas the hydroxylation profile (gluco or galacto-like) was less relevant. Biochemical and computational data further support a mechanism of action implying binding to the allosteric lipid binding site of p38 MAPK and subsequent activation of the noncanonical autophosphorylation route. The ensemble of results provide a proof of concept of the potential of sp2-IGLs as immunoregulators.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.