How exascale computing can shape drug design: A perspective from multiscale QM/MM molecular dynamics simulations and machine learning-aided enhanced sampling algorithms.

Molecular simulations are an essential asset in the first steps of drug design campaigns. However, the requirement of high-throughput limits applications mainly to qualitative approaches with low computational cost, but also low accuracy. Unlocking the potential of more rigorous quantum mechanical/molecular mechanics (QM/MM) models combined with molecular dynamics-based free energy techniques could have a tremendous impact. Indeed, these two relatively old techniques are emerging as promising methods in the field. This has been favored by the exponential growth of computer power and the proliferation of powerful data-driven methods. Here, we briefly review recent advances and applications, and give our perspective on the impact that QM/MM and free-energy methods combined with machine learning-aided algorithms can have on drug design.

Mutational rescue of the activity of high-fidelity Cas9 enzymes.

Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.

Unexpected Single-Ligand Occupancy and Negative Cooperativity in the SARS-CoV-2 Main Protease.

Many homodimeric enzymes tune their functions by exploiting either negative or positive cooperativity between subunits. In the SARS-CoV-2 Main protease (Mpro) homodimer, the latter has been suggested by symmetry in most of the 500 reported protease/ligand complex structures solved by macromolecular crystallography (MX). Here we apply the latter to both covalent and noncovalent ligands in complex with Mpro. Strikingly, our experiments show that the occupation of both active sites of the dimer originates from an excess of ligands. Indeed, cocrystals obtained using a 1:1 ligand/protomer stoichiometry lead to single occupation only. The empty binding site exhibits a catalytically inactive geometry in solution, as suggested by molecular dynamics simulations. Thus, Mpro operates through negative cooperativity with the asymmetric activity of the catalytic sites. This allows it to function with a wide range of substrate concentrations, making it resistant to saturation and potentially difficult to shut down, all properties advantageous for the virus' adaptability and resistance.

Physical Chemistry of Chloroquine Permeation through the Cell Membrane with Atomistic Detail.

We provide a molecular-level description of the thermodynamics and mechanistic aspects of drug permeation through the cell membrane. As a case study, we considered the antimalaria FDA approved drug chloroquine. Molecular dynamics simulations of the molecule (in its neutral and protonated form) were performed in the presence of different lipid bilayers, with the aim of uncovering key aspects of the permeation process, a fundamental step for the drug's action. Free energy values obtained by well-tempered metadynamics simulations suggest that the neutral form is the only permeating protomer, consistent with experimental data. H-bond interactions of the drug with water molecules and membrane headgroups play a crucial role for permeation. The presence of the transmembrane potential, investigated here for the first time in a drug permeation study, does not qualitatively affect these conclusions.

EphrinA5 regulates cell motility by modulating Snhg15/DNA triplex-dependent targeting of DNMT1 to the Ncam1 promoter.

Cell-cell communication is mediated by membrane receptors and their ligands, such as the Eph/ephrin system, orchestrating cell migration during development and in diverse cancer types. Epigenetic mechanisms are key for integrating external "signals", e.g., from neighboring cells, into the transcriptome in health and disease. Previously, we reported ephrinA5 to trigger transcriptional changes of lncRNAs and protein-coding genes in cerebellar granule cells, a cell model for medulloblastoma. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate a lncRNA-mediated targeting of DNMT1 to specific gene loci by the combined power of in silico modeling of RNA/DNA interactions and wet lab approaches, in the context of the clinically relevant use case of ephrinA5-dependent regulation of cellular motility of cerebellar granule cells. We provide evidence that Snhg15, a cancer-related lncRNA, recruits DNMT1 to the Ncam1 promoter through RNA/DNA triplex structure formation and the interaction with DNMT1. This mediates DNA methylation-dependent silencing of Ncam1, being abolished by ephrinA5 stimulation-triggered reduction of Snhg15 expression. Hence, we here propose a triple helix recognition mechanism, underlying cell motility regulation via lncRNA-targeted DNA methylation in a clinically relevant context.

Three-dimensional evaluation of symmetry in facial palsy reanimation using stereophotogrammetric devices: A series of 15 cases.

Facial palsy can severely compromise quality of life, significantly altering the harmony and symmetry of the face, which can be restored by surgical rehabilitation. The aim of the study was the quantification of facial symmetry following facial reanimation. Fifteen consecutive adult patients were surgically treated through triple innervation for reanimation of flaccid unilateral facial paralysis (contralateral facial nerve, masseteric nerve, and hypoglossal nerve) and fascia lata graft for definition of the nasolabial sulcus. In the preoperative stage and at least 11 months after the surgical treatment, three-dimensional facial images were recorded through stereophotogrammetry in a neutral (rest) position, and with Mona Lisa and full-denture (maximum) smiles. Labial commissure inclination relative to the interpupillary axis, and a surface assessment of local facial asymmetry at rest and while smiling were obtained for the upper, middle, and lower facial thirds. The angle between the interpupillary axis and the labial commissure significantly improved in post-surgical acquisitions, regaining symmetry at rest (t-test; p < 0.001). Facial symmetry increased significantly when passing from pre-to postsurgical facial scans, from the lower to the upper facial third, and from the full smile to the rest position (ANOVA; p < 0.001). After treatment, the full smile recovered more symmetry than the other two expressions. In summary, surgical treatment significantly reduced facial asymmetry, but this reduction differed significantly among the various animations and facial thirds. The results of this study confirmed clinical findings of significant static and dynamic improvements in facial symmetry after triple innervation reanimation surgery.

Low Molecular Weight Inhibitors Targeting the RNA-Binding Protein HuR.

The RNA-binding protein human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. This protein has been progressively recognized as a relevant therapeutic target for several pathologies, like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here, we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatic approaches, high-throughput virtual screening, and RNA-protein pulldown assays, we demonstrate that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits a dose-dependent HuR inhibition effect in binding experiments. Importantly, the chemical scaffold is new with respect to the currently known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein.

AI-based identification of therapeutic agents targeting GPCRs: introducing ligand type classifiers and systems biology.

Identifying ligands targeting G protein coupled receptors (GPCRs) with novel chemotypes other than the physiological ligands is a challenge for in silico screening campaigns. Here we present an approach that identifies novel chemotype ligands by combining structural data with a random forest agonist/antagonist classifier and a signal-transduction kinetic model. As a test case, we apply this approach to identify novel antagonists of the human adenosine transmembrane receptor type 2A, an attractive target against Parkinson's disease and cancer. The identified antagonists were tested here in a radio ligand binding assay. Among those, we found a promising ligand whose chemotype differs significantly from all so-far reported antagonists, with a binding affinity of 310 ± 23.4 nM. Thus, our protocol emerges as a powerful approach to identify promising ligand candidates with novel chemotypes while preserving antagonistic potential and affinity in the nanomolar range.

Metadynamics simulations of ligands binding to protein surfaces: a novel tool for rational drug design.

Structure-based drug design protocols may encounter difficulties to investigate poses when the biomolecular targets do not exhibit typical binding pockets. In this study, by providing two concrete examples from our labs, we suggest that the combination of metadynamics free energy methods (validated against affinity measurements), along with experimental structural information (by X-ray crystallography and NMR), can help to identify the poses of ligands on protein surfaces. The simulation workflow proposed here was implemented in a widely used code, namely GROMACS, and it could straightforwardly be applied to various drug-design campaigns targeting ligands' binding to protein surfaces.

SPEADI: Accelerated Analysis of IDP-Ion Interactions from MD-Trajectories.

The disordered nature of Intrinsically Disordered Proteins (IDPs) makes their structural ensembles particularly susceptible to changes in chemical environmental conditions, often leading to an alteration of their normal functions. A Radial Distribution Function (RDF) is considered a standard method for characterizing the chemical environment surrounding particles during atomistic simulations, commonly averaged over an entire or part of a trajectory. Given their high structural variability, such averaged information might not be reliable for IDPs. We introduce the Time-Resolved Radial Distribution Function (TRRDF), implemented in our open-source Python package SPEADI, which is able to characterize dynamic environments around IDPs. We use SPEADI to characterize the dynamic distribution of ions around the IDPs Alpha-Synuclein (AS) and Humanin (HN) from Molecular Dynamics (MD) simulations, and some of their selected mutants, showing that local ion-residue interactions play an important role in the structures and behaviors of IDPs.

Predictions of the Poses and Affinity of a Ligand over the Entire Surface of a NEET Protein: The Case of Human MitoNEET.

Human NEET proteins contain two [2Fe-2S] iron-sulfur clusters, bound to three Cys residues and one His residue. They exist in two redox states. Recently, these proteins have revealed themselves as attractive drug targets for mitochondrial dysfunction-related diseases, such as type 2 diabetes, Wolfram syndrome 2, and cancers. Unfortunately, the lack of information and mechanistic understanding of ligands binding to the whole functional, cytoplasmatic domain has limited rational drug design approaches. Here, we use an enhanced sampling technique, volume-based metadynamics, recently developed by a team involving some of us, to predict the poses and affinity of the 2-benzamido-4-(1,2,3,4-tetrahydronaphthalen-2-yl)-thiophene-3-carboxylate ligand to the entire surface of the cytoplasmatic domain of the human NEET protein mitoNEET (mNT) in an aqueous solution. The calculations, based on the recently published X-ray structure of the complex, are consistent with the measured affinity. The calculated free energy landscape revealed that the ligand can bind in multiple sites and with poses other than the one found in the X-ray. This difference is likely to be caused by crystal packing effects that allow the ligand to interact with multiple adjacent NEET protein copies. Such extra contacts are of course absent in the solution; therefore, the X-ray pose is only transient in our calculations, where the binding free energy correlates with the number of contacts. We further evaluated how the reduction and protonation of the Fe-bound histidine, as well as temperature, can affect ligand binding. Both such modifications introduce the possibility for the ligand to bind in an area of the protein other than the one observed in the X-ray, with no or little impact on affinity. Overall, our study can provide insights on the molecular recognition mechanisms of ligand binding to mNT in different oxidative conditions, possibly helping rational drug design of NEET ligands.

Molecular Dynamics and Structural Studies of Zinc Chloroquine Complexes.

Chloroquine (CQ) is a first-choice drug against malaria and autoimmune diseases. It has been co-administered with zinc against SARS-CoV-2 and soon dismissed because of safety issues. The structural features of Zn-CQ complexes and the effect of CQ on zinc distribution in cells are poorly known. In this study, state-of-the-art computations combined with experiments were leveraged to solve the structural determinants of zinc-CQ interactions in solution and the solid state. NMR, ESI-MS, and X-ray absorption and diffraction methods were combined with ab initio molecular dynamics calculations to address the kinetic lability of this complex. Within the physiological pH range, CQ binds Zn2+ through the quinoline ring nitrogen, forming [Zn(CQH)Clx(H2O)3-x](3+)-x (x = 0, 1, 2, and 3) tetrahedral complexes. The Zn(CQH)Cl3 species is stable at neutral pH and at high chloride concentrations typical of the extracellular medium, but metal coordination is lost at a moderately low pH as in the lysosomal lumen. The pentacoordinate complex [Zn(CQH)(H2O)4]3+ may exist in the absence of chloride. This in vitro/in silico approach can be extended to other metal-targeting drugs and bioinorganic systems.

Aromaticity at position 39 in α-synuclein: A modulator of amyloid fibril assembly and membrane-bound conformations.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Multiple Poses and Thermodynamics of Ligands Targeting Protein Surfaces: The Case of Furosemide Binding to mitoNEET in Aqueous Solution.

Human NEET proteins, such as NAF-1 and mitoNEET, are homodimeric, redox iron-sulfur proteins characterized by triple cysteine and one histidine-coordinated [2Fe-2S] cluster. They exist in an oxidized and reduced state. Abnormal release of the cluster is implicated in a variety of diseases, including cancer and neurodegeneration. The computer-aided and structure-based design of ligands affecting cluster release is of paramount importance from a pharmaceutical perspective. Unfortunately, experimental structural information so far is limited to only one ligand/protein complex. This is the X-ray structure of furosemide bound to oxidized mitoNEET. Here we employ an enhanced sampling approach, Localized Volume-based Metadynamics, developed by some of us, to identify binding poses of furosemide to human mitoNEET protein in solution. The binding modes show a high variability within the same shallow binding pocket on the protein surface identified in the X-ray structure. Among the different binding conformations, one of them is in agreement with the crystal structure's one. This conformation might have been overstabilized in the latter because of the presence of crystal packing interactions, absent in solution. The calculated binding affinity is compatible with experimental data. Our protocol can be used in a straightforward manner in drug design campaigns targeting this pharmaceutically important family of proteins.

Computationally designed hyperactive Cas9 enzymes.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.

An anti-diabetic drug targets NEET (CISD) proteins through destabilization of their [2Fe-2S] clusters.

Elevated levels of mitochondrial iron and reactive oxygen species (ROS) accompany the progression of diabetes, negatively impacting insulin production and secretion from pancreatic cells. In search for a tool to reduce mitochondrial iron and ROS levels, we arrived at a molecule that destabilizes the [2Fe-2S] clusters of NEET proteins (M1). Treatment of db/db diabetic mice with M1 improved hyperglycemia, without the weight gain observed with alternative treatments such as rosiglitazone. The molecular interactions of M1 with the NEET proteins mNT and NAF-1 were determined by X-crystallography. The possibility of controlling diabetes by molecules that destabilize the [2Fe-2S] clusters of NEET proteins, thereby reducing iron-mediated oxidative stress, opens a new route for managing metabolic aberration such as in diabetes.

Molecular Dynamics-Assisted Interpretation of Experimentally Determined Intrinsically Disordered Protein Conformational Components: The Case of Human α-Synuclein.

Mass spectrometry and single molecule force microscopy are two experimental approaches able to provide structural information on intrinsically disordered proteins (IDPs). These techniques allow the dissection of conformational ensembles in their main components, although at a low-resolution level. In this work, we interpret the results emerging from these experimental approaches on human alpha synuclein (AS) by analyzing a previously published 73 µs-long molecular-dynamics (MD) simulation of the protein in explicit solvent. We further compare MD-based predictions of single molecule Förster resonance energy transfer (smFRET) data of AS in solution with experimental data. The combined theoretical and experimental data provide a description of AS main conformational ensemble, shedding light into its intramolecular interactions and overall structural compactness. This analysis could be easily transferred to other IDPs.

Sodium Channels and Local Anesthetics-Old Friends With New Perspectives.

The long history of local anesthetics (LAs) starts out in the late 19th century when the content of coca plant leaves was discovered to alleviate pain. Soon after, cocaine was established and headed off to an infamous career as a substance causing addiction. Today, LAs and related substances-in modified form-are indispensable in our clinical everyday life for pain relief during and after minor and major surgery, and dental practices. In this review, we elucidate on the interaction of modern LAs with their main target, the voltage-gated sodium channel (Navs), in the light of the recently published channel structures. Knowledge of the 3D interaction sites of the drug with the protein will allow to mechanistically substantiate the comprehensive data available on LA gating modification. In the 1970s it was suggested that LAs can enter the channel pore from the lipid phase, which was quite prospective at that time. Today we know from cryo-electron microscopy structures and mutagenesis experiments, that indeed Navs have side fenestrations facing the membrane, which are likely the entrance for LAs to induce tonic block. In this review, we will focus on the effects of LA binding on fast inactivation and use-dependent inhibition in the light of the proposed new allosteric mechanism of fast inactivation. We will elaborate on subtype and species specificity and provide insights into modelling approaches that will help identify the exact molecular binding orientation, access pathways and pharmacokinetics. With this comprehensive overview, we will provide new perspectives in the use of the drug, both clinically and as a tool for basic ion channel research.

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

Towards design of drugs and delivery systems with the Martini coarse-grained model.

Coarse-grained (CG) modelling with the Martini force field has come of age. By combining a variety of bead types and sizes with a new mapping approach, the newest version of the model is able to accurately simulate large biomolecular complexes at millisecond timescales. In this perspective, we discuss possible applications of the Martini 3 model in drug discovery and development pipelines and highlight areas for future development. Owing to its high simulation efficiency and extended chemical space, Martini 3 has great potential in the area of drug design and delivery. However, several aspects of the model should be improved before Martini 3 CG simulations can be routinely employed in academic and industrial settings. These include the development of automatic parameterisation protocols for a variety of molecule types, the improvement of backmapping procedures, the description of protein flexibility and the development of methodologies enabling efficient sampling. We illustrate our view with examples on key areas where Martini could give important contributions such as drugs targeting membrane proteins, cryptic pockets and protein-protein interactions and the development of soft drug delivery systems.