Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

Structural and functional diversity among agonist-bound states of the GLP-1 receptor.

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

Molecular dynamics studies reveal structural and functional features of the SARS-CoV-2 spike protein.

The SARS-CoV-2 virus is responsible for the COVID-19 pandemic the world experience since 2019. The protein responsible for the first steps of cell invasion, the spike protein, has probably received the most attention in light of its central role during infection. Computational approaches are among the tools employed by the scientific community in the enormous effort to study this new affliction. One of these methods, namely molecular dynamics (MD), has been used to characterize the function of the spike protein at the atomic level and unveil its structural features from a dynamic perspective. In this review, we focus on these main findings, including spike protein flexibility, rare S protein conformational changes, cryptic epitopes, the role of glycans, drug repurposing, and the effect of spike protein variants.

Multisite Model of Allosterism for the Adenosine A1 Receptor.

Despite being a target for about one-third of approved drugs, G protein-coupled receptors (GPCRs) still represent a tremendous reservoir for therapeutic strategies against diseases. For example, several cardiovascular and central nervous system conditions could benefit from clinical agents that activate the adenosine 1 receptor (A1R); however, the pursuit of A1R agonists for clinical use is usually impeded by both on- and off-target side effects. One of the possible strategies to overcome this issue is the development of positive allosteric modulators (PAMs) capable of selectively enhancing the effect of a specific receptor subtype and triggering functional selectivity (a phenomenon also referred to as bias). Intriguingly, besides enforcing the effect of agonists upon binding to an allosteric site, most of the A1R PAMs display intrinsic partial agonism and orthosteric competition with antagonists. To rationalize this behavior, we simulated the binding of the prototypical PAMs PD81723 and VCP171, the full-agonist NECA, the antagonist 13B, and the bitopic agonist VCP746. We propose that a single PAM can bind several A1R sites rather than a unique allosteric pocket, reconciling the structure-activity relationship and the mutagenesis results.

Supervised molecular dynamics for exploring the druggability of the SARS-CoV-2 spike protein.

The recent outbreak of the respiratory syndrome-related coronavirus (SARS-CoV-2) is stimulating an unprecedented scientific campaign to alleviate the burden of the coronavirus disease (COVID-19). One line of research has focused on targeting SARS-CoV-2 proteins fundamental for its replication by repurposing drugs approved for other diseases. The first interaction between the virus and the host cell is mediated by the spike protein on the virus surface and the human angiotensin-converting enzyme (ACE2). Small molecules able to bind the receptor-binding domain (RBD) of the spike protein and disrupt the binding to ACE2 would offer an important tool for slowing, or even preventing, the infection. Here, we screened 2421 approved small molecules in silico and validated the docking outcomes through extensive molecular dynamics simulations. Out of six drugs characterized as putative RBD binders, the cephalosporin antibiotic cefsulodin was further assessed for its effect on the binding between the RBD and ACE2, suggesting that it is important to consider the dynamic formation of the heterodimer between RBD and ACE2 when judging any potential candidate.

A Supervised Molecular Dynamics Approach to Unbiased Ligand-Protein Unbinding.

The recent paradigm shift toward the use of the kinetics parameters in place of thermodynamic constants is leading the computational chemistry community to develop methods for studying the mechanisms of drug binding and unbinding. From this standpoint, molecular dynamics (MD) plays an important role in delivering insight at the molecular scale. However, a known limitation of MD is that the time scales are usually far from those involved in ligand-receptor unbinding events. Here, we show that the algorithm behind supervised MD (SuMD) can simulate the dissociation mechanism of druglike small molecules while avoiding the input of any energy bias to facilitate the transition. SuMD was tested on seven different intermolecular complexes, covering four G protein-coupled receptors: the A2A and A1 adenosine receptors, the orexin 2 and the muscarinic 2 receptors, and the soluble globular enzyme epoxide hydrolase. SuMD well-described the multistep nature of ligand-receptor dissociation, rationalized previous experimental data and produced valuable working hypotheses for structure-kinetics relationships.

Addressing free fatty acid receptor 1 (FFAR1) activation using supervised molecular dynamics.

The free fatty acid receptor 1 (FFAR1, formerly GPR40), is a potential G protein-coupled receptor (GPCR) target for the treatment of type 2 diabetes mellitus (T2DM), as it enhances glucose-dependent insulin secretion upon activation by endogenous long-chain free fatty acids. The presence of two allosterically communicating binding sites and the lack of the conserved GPCR structural motifs challenge the general knowledge of its activation mechanism. To date, four X-ray crystal structures are available for computer-aided drug design. In this study, we employed molecular dynamics (MD) and supervised molecular dynamics (SuMD) to deliver insights into the (un)binding mechanism of the agonist MK-8666, and the allosteric communications between the two experimentally determined FFAR1 binding sites. We found that FFAR1 extracellular loop 2 (ECL2) mediates the binding of the partial agonist MK-8666. Moreover, simulations showed that the agonists MK-8666 and AP8 are reciprocally stabilized and that AP8 influences MK-8666 unbinding from FFAR1.

Revisiting the Allosteric Regulation of Sodium Cation on the Binding of Adenosine at the Human A2A Adenosine Receptor: Insights from Supervised Molecular Dynamics (SuMD) Simulations.

One of the most intriguing findings highlighted from G protein-coupled receptor (GPCR) crystallography is the presence, in many members of class A, of a partially hydrated sodium ion in the middle of the seven transmembrane helices (7TM) bundle. In particular, the human adenosine A2A receptor (A2A AR) is the first GPCR in which a monovalent sodium ion was crystallized in a distal site from the canonical orthosteric one, corroborating, from a structural point of view, its role as a negative allosteric modulator. However, the molecular mechanism by which the sodium ion influences the recognition of the A2A AR agonists is not yet fully understood. In this study, the supervised molecular dynamics (SuMD) technique was exploited to analyse the sodium ion recognition mechanism and how its presence influences the binding of the endogenous agonist adenosine. Due to a higher degree of flexibility of the receptor extracellular (EC) vestibule, we propose the sodium-bound A2A AR as less efficient in stabilizing the adenosine during the different steps of binding.

Could the presence of sodium ion influence the accuracy and precision of the ligand-posing in the human A2A adenosine receptor orthosteric binding site using a molecular docking approach? Insights from Dockbench.

The allosteric modulation of G protein-coupled receptors (GPCRs) by sodium ions has received considerable attention as crystal structures of several receptors, in their inactive conformation, show a Na+ ion bound to specific residues which, in the human A2A adenosine receptor (hA2A AR), are Ser913.39, Trp2466.48, Asn2807.45, and Asn2847.49. A cluster of water molecules completes the coordination of the sodium ion in the putative allosteric site. It is absolutely consolidated that the progress made in the field of GPCRs structural determination has increased the adoption of docking-driven approaches for the identification or the optimization of novel potent and selective ligands. Despite the extensive use of docking protocols in virtual screening approaches, to date, almost any of these studies have been carried out without taking into account the presence of the sodium cation and its first solvation shell in the putative allosteric binding site. In this study, we have focused our attention on determining how the presence of sodium ion binding and additionally its first hydration sphere, in hA2AAR could influence the ligand positioning accuracy during molecular docking simulations for most of the available resting and activated hA2A AR crystal structures, using DockBench as a comparative benchmarking tool and implementing a new correlation coefficient (EM). This work provides indications on the evidence that the posing performance (accuracy and/or precision) of the docking protocols in reproducing the crystallographic poses of different hA2A AR antagonists is generally increased in the presence of the sodium cation and its first solvation shell, in agreement with experimental observations. Consequently, the inclusion of sodium ion and its first solvation shell should be considered in order to facilitate the selection of new potential ligands in all molecular docking-based virtual screening protocols that aim to find novel GPCRs antagonists and inverse agonists.

Supporting the Identification of Novel Fragment-Based Positive Allosteric Modulators Using a Supervised Molecular Dynamics Approach: A Retrospective Analysis Considering the Human A2A Adenosine Receptor as a Key Example.

Structure-driven fragment-based (SDFB) approaches have provided efficient methods for the identification of novel drug candidates. This strategy has been largely applied in discovering several pharmacological ligand classes, including enzyme inhibitors, receptor antagonists and, more recently, also allosteric (positive and negative) modulators. Recently, Siegal and collaborators reported an interesting study, performed on a detergent-solubilized StaR adenosine A2A receptor, describing the existence of both fragment-like negative allosteric modulators (NAMs), and fragment-like positive allosteric modulators (PAMs). From this retrospective study, our results suggest that Supervised Molecular Dynamics (SuMD) simulations can support, on a reasonable time scale, the identification of fragment-like PAMs following their receptor recognition pathways and characterizing the possible allosteric binding sites.

Deciphering the Complexity of Ligand-Protein Recognition Pathways Using Supervised Molecular Dynamics (SuMD) Simulations.

Molecular recognition is a crucial issue when aiming to interpret the mechanism of known active substances as well as to develop novel active candidates. Unfortunately, simulating the binding process is still a challenging task because it requires classical MD experiments in a long microsecond time scale that are affordable only with a high-level computational capacity. In order to overcome this limiting factor, we have recently implemented an alternative MD approach, named supervised molecular dynamics (SuMD), and successfully applied it to G protein-coupled receptors (GPCRs). SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand, and also from its receptor binding affinity. In this article, we present an extension of the SuMD application domain including different types of proteins in comparison with GPCRs. In particular, we have deeply analyzed the ligand-protein recognition pathways of six different case studies that we grouped into two different classes: globular and membrane proteins. Moreover, we introduce the SuMD-Analyzer tool that we have specifically implemented to help the user in the analysis of the SuMD trajectories. Finally, we emphasize the limit of the SuMD applicability domain as well as its strengths in analyzing the complexity of ligand-protein recognition pathways.

Understanding allosteric interactions in G protein-coupled receptors using Supervised Molecular Dynamics: A prototype study analysing the human A3 adenosine receptor positive allosteric modulator LUF6000.

The search for G protein-coupled receptors (GPCRs) allosteric modulators represents an active research field in medicinal chemistry. Allosteric modulators usually exert their activity only in the presence of the orthosteric ligand by binding to protein sites topographically different from the orthosteric cleft. They therefore offer potentially therapeutic advantages by selectively influencing tissue responses only when the endogenous agonist is present. The prediction of putative allosteric site location, however, is a challenging task. In facts, they are usually located in regions showing more structural variation among the family members. In the present work, we applied the recently developed Supervised Molecular Dynamics (SuMD) methodology to interpret at the molecular level the positive allosteric modulation mediated by LUF6000 toward the human adenosine A3 receptor (hA3 AR). Our data suggest at least two possible mechanisms to explain the experimental data available. This study represent, to the best of our knowledge, the first case reported of an allosteric recognition mechanism depicted by means of molecular dynamics simulations.

Structural dynamics of therapeutic nucleic acids with phosphorothioate backbone modifications.

Antisense oligonucleotides (ASOs) offer ground-breaking possibilities for selective pharmacological intervention for any gene product-related disease. Therapeutic ASOs contain extensive chemical modifications that improve stability to enzymatic cleavage and modulate binding affinity relative to natural RNA/DNA. Molecular dynamics (MD) simulation can provide valuable insights into how such modifications affect ASO conformational sampling and target binding. However, force field parameters for chemically modified nucleic acids (NAs) are still underdeveloped. To bridge this gap, we developed parameters to allow simulations of ASOs with the widely applied phosphorothioate (PS) backbone modification, and validated these in extensive all-atom MD simulations of relevant PS-modified NA systems representing B-DNA, RNA, and DNA/RNA hybrid duplex structures. Compared to the corresponding natural NAs, single PS substitutions had marginal effects on the ordered DNA/RNA duplex, whereas substantial effects of phosphorothioation were observed in single-stranded RNA and B-DNA, corroborated by the experimentally derived structure data. We find that PS-modified NAs shift between high and low twist states, which could affect target recognition and protein interactions for phosphorothioated oligonucleotides. Furthermore, conformational sampling was markedly altered in the PS-modified ssRNA system compared to that of the natural oligonucleotide, indicating sequence-dependent effects on conformational preference that may in turn influence duplex formation.

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction.

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions.

Targeting hPKM2 in cancer: A bio isosteric approach for ligand design.

The term cancer refers to a plethora of diseases characterized by the development of abnormal cells that divide uncontrollably and can infiltrate further proximal or distal body tissues. Each type of cancer can be defined by aggressiveness, localization, metabolism, and response to available treatments. Among the most common hallmarks of cancer is a more acidic intracellular microenvironment. Offset pH values are due to an excess of lactate and an increased hypoxia-inducible factor (HIF) expression, which leads to a hypoxic state and a metabolic shift towards glycolysis to produce adenosine-5'-triphosphate (ATP) necessary for cellular metabolism. Warburg's hypothesis underpins this concept, making glycolysis and its central enzyme pyruvate kinase (hPKM2), an ideal target for drug development. Using molecular docking and extensive molecular dynamics (MD) simulations we investigated the binding mode of phosphoenolpyruvate (PEP) inside the hPKM2 active site, and then evaluated a set of known bio-isosteric inhibitors to understand the differences caused by their substitutions on their binding mode. Ultimately, we propose a new molecular entity to hamper hPKM2, unbalance cellular energy, and possibly trigger autophagic mechanisms.

Computer-aided de novo design and optimization of novel potential inhibitors of HIV-1 Nef protein.

Nef is a small accessory protein pivotal in the HIV-1 viral replication cycle. It is a multifunctional protein and its interactions with kinases in host cells have been well characterized through many in vitro and structural studies. Nef forms a homodimer to activate the kinases and subsequently the phosphorylation pathways. The disruption of its homodimerization represents a valuable approach in the search for novel classes of antiretroviral. However, this research avenue is still underdeveloped as just a few Nef inhibitors have been reported so far, with very limited structural information about their mechanism of action. To address this issue, we have employed an in silico structure-based drug design strategy that combines de novo ligand design with molecular docking and extensive molecular dynamics simulations. Since the Nef pocket involved in homodimerization has high lipophilicity, the initial de novo-designed structures displayed poor drug-likeness and solubility. Taking information from the hydration sites within the homodimerization pocket, structural modifications in the initial lead compound have been introduced to improve the solubility and drug-likeness, without affecting the binding profile. We propose lead compounds that can be the starting point for further optimizations to deliver long-awaited, rationally designed Nef inhibitors.

Is the Stalk of the SARS-CoV-2 Spike Protein Druggable?

The spike protein is key to SARS-CoV-2 high infectivity because it facilitates the receptor binding domain (RBD) encounter with ACE2. As targeting subunit S1 has not yet delivered an ACE2-binding inhibitor, we have assessed the druggability of the conserved segment of the spike protein stalk within subunit S2 by means of an integrated computational approach that combines the molecular docking of an optimized library of fragments with high-throughput molecular dynamics simulations. The high propensity of the spike protein to mutate in key regions that are responsible for the recognition of the human angiotensin-converting enzyme 2 (hACE2) or for the recognition of antibodies, has made subunit S1 of the spike protein difficult to target. Despite the inherent flexibility of the stalk region, our results suggest two hidden interhelical binding sites, whose accessibility is only partially hampered by glycan residues.

A Pathway Model to Understand the Evolution of Spike Protein Binding to ACE2 in SARS-CoV-2 Variants.

After the SARS-CoV-2 Wuhan variant that gave rise to the pandemic, other variants named Delta, Omicron, and Omicron-2 sequentially became prevalent, with mutations spread around the viral genome, including on the spike (S) protein; in order to understand the resultant in gains in infectivity, we interrogated in silico both the equilibrium binding and the binding pathway of the virus' receptor-binding domain (RBD) to the angiotensin-converting enzyme 2 (ACE2) receptor. We interrogated the molecular recognition between the RBD of different variants and ACE2 through supervised molecular dynamics (SuMD) and classic molecular dynamics (MD) simulations to address the effect of mutations on the possible S protein binding pathways. Our results indicate that compensation between binding pathway efficiency and stability of the complex exists for the Omicron BA.1 receptor binding domain, while Omicron BA.2's mutations putatively improved the dynamic recognition of the ACE2 receptor, suggesting an evolutionary advantage over the previous strains.