De Novo Design of κ-Opioid Receptor Antagonists Using a Generative Deep-Learning Framework.

Likely effective pharmacological interventions for the treatment of opioid addiction include attempts to attenuate brain reward deficits during periods of abstinence. Pharmacological blockade of the κ-opioid receptor (KOR) has been shown to abolish brain reward deficits in rodents during withdrawal, as well as to reduce the escalation of opioid use in rats with extended access to opioids. Although KOR antagonists represent promising candidates for the treatment of opioid addiction, very few potent selective KOR antagonists are known to date and most of them exhibit significant safety concerns. Here, we used a generative deep-learning framework for the de novo design of chemotypes with putative KOR antagonistic activity. Molecules generated by models trained with this framework were prioritized for chemical synthesis based on their predicted optimal interactions with the receptor. Our models and proposed training protocol were experimentally validated by binding and functional assays.

Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin αVß3.

Inhibitors of integrin αVß3 have therapeutic promise for a variety of diseases. Most αVß3-targeting small molecules patterned after the RGD motif are partial agonists because they induce a high-affinity, ligand-binding conformation and prime the receptor to bind the ligand without an activating stimulus, in part via a charge-charge interaction between their aspartic acid carboxyl group and the metal ion in the metal-ion-dependent adhesion site (MIDAS). Building upon our previous studies on the related integrin αIIbß3, we searched for pure αVß3 antagonists that lack this typical aspartic acid carboxyl group and instead engage through direct binding to one of the coordinating residues of the MIDAS metal ion, specifically ß3 E220. By in silico screening of two large chemical libraries for compounds interacting with ß3 E220, we indeed discovered a novel molecule that does not contain an acidic carboxyl group and does not induce the high-affinity, ligand-binding state of the receptor. Functional and structural characterization of a chemically optimized version of this compound led to the discovery of a novel small-molecule pure αVß3 antagonist that (i) does not prime the receptor to bind the ligand and does not induce hybrid domain swing-out or receptor extension as judged by antibody binding and negative-stain electron microscopy, (ii) binds at the RGD-binding site as predicted by metadynamics rescoring of induced-fit docking poses and confirmed by a cryo-electron microscopy structure of the compound-bound integrin, and (iii) coordinates the MIDAS metal ion via a quinoline moiety instead of an acidic carboxyl group.

Lipids Alter Rhodopsin Function via Ligand-like and Solvent-like Interactions.

Rhodopsin, a prototypical G protein-coupled receptor, is a membrane protein that can sense dim light. This highly effective photoreceptor is known to be sensitive to the composition of its lipidic environment, but the molecular mechanisms underlying this fine-tuned modulation of the receptor's function and structural stability are not fully understood. There are two competing hypotheses to explain how this occurs: 1) lipid modulation occurs via solvent-like interactions, where lipid composition controls membrane properties like hydrophobic thickness, which in turn modulate the protein's conformational equilibrium; or 2) protein-lipid interactions are ligand-like, with specific hot spots and long-lived binding events. By analyzing an ensemble of all-atom molecular dynamics simulations of five different states of rhodopsin, we show that a local ordering effect takes place in the membrane upon receptor activation. Likewise, docosahexaenoic acid acyl tails and phosphatidylethanolamine headgroups behave like weak ligands, preferentially binding to the receptor in inactive-like conformations and inducing subtle but significant structural changes.

Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation.

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase called M.BceJIV with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates N6 of the adenine at the fifth base position. Here, we present crystal structures of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structures show not one, but two DNA substrates bound to the M.BceJIV dimer, with each monomer contributing to the recognition of two recognition sequences. We also show that methylation at the two recognition sequences occurs independently, and that the GTWWAC motifs are enriched in intergenic regions in the genomes of B. cenocepacia strains. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation.

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase (M.BceJIV) with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates the N6 of the adenine at the fifth base position (GTWWAC). Here, we present a high-resolution crystal structure of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structure shows not one, but two DNA substrates bound to the M.BceJIV dimer, wherein each monomer contributes to the recognition of two recognition sequences. This unexpected mode of DNA binding and methylation has not been observed previously and sets a new precedent for a DNA methyltransferase. We also show that methylation at two recognition sequences occurs independently, and that GTWWAC motifs are enriched in intergenic regions of a strain of B. cenocepacia's genome. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

Structural basis for partial agonism in 5-HT3A receptors.

Hyperactivity of serotonin 3 receptors (5-HT3R) underlies pathologies associated with irritable bowel syndrome and chemotherapy-induced nausea and vomiting. Setrons, a class of high-affinity competitive antagonists, are used in the treatment of these conditions. Although generally effective for chemotherapy-induced nausea and vomiting, the use of setrons for treating irritable bowel syndrome has been impaired by adverse side effects. Partial agonists are now being considered as an alternative strategy, with potentially less severe side effects than full antagonists. However, a structural understanding of how these ligands work is lacking. Here, we present high-resolution cryogenic electron microscopy structures of the mouse 5-HT3AR in complex with partial agonists (SMP-100 and ALB-148471) captured in pre-activated and open-like conformational states. Molecular dynamics simulations were used to assess the stability of drug-binding poses and interactions with the receptor over time. Together, these studies reveal mechanisms for the functional differences between orthosteric partial agonists, full agonists and antagonists of the 5-HT3AR.

Structural basis of positive allosteric modulation of metabotropic glutamate receptor activation and internalization.

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export.

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator.

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the ß2-adrenergic receptor (ß2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from ß2AR and Gßγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

Structural basis of allosteric modulation of metabotropic glutamate receptor activation and desensitization.

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted either at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric TMD-targeting compounds as therapeutics, an understanding of the functional and structural basis of their effects on mGluRs is limited. Here we use a battery of approaches to dissect the distinct functional and structural effects of orthosteric versus allosteric ligands. We find using electrophysiological and live cell imaging assays that both agonists and positive allosteric modulators (PAMs) can drive activation and desensitization of mGluRs. The effects of PAMs are pleiotropic, including both the ability to boost the maximal response to orthosteric agonists and to serve independently as desensitization-biased agonists across mGluR subtypes. Conformational sensors reveal PAM-driven inter-subunit re-arrangements at both the LBD and TMD. Motivated by this, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling. Highlights: -Agonists and PAMs drive mGluR activation, desensitization, and endocytosis-PAMs are desensitization-biased and synergistic with agonists-Four combinatorial ligand conditions reveal an ensemble of full-length mGluR structures with novel interfaces-Activation and desensitization involve rolling TMD interfaces which are re-shaped by PAM.

De Novo Design of κ-Opioid Receptor Antagonists Using a Generative Deep Learning Framework.

Likely effective pharmacological interventions for the treatment of opioid addiction include attempts to attenuate brain reward deficits during periods of abstinence. Pharmacological blockade of the κ-opioid receptor (KOR) has been shown to abolish brain reward deficits in rodents during withdrawal, as well as to reduce the escalation of opioid use in rats with extended access to opioids. Although KOR antagonists represent promising candidates for the treatment of opioid addiction, very few potent selective KOR antagonists are known to date and most of them exhibit significant safety concerns. Here, we used a generative deep learning framework for the de novo design of chemotypes with putative KOR antagonistic activity. Molecules generated by models trained with this framework were prioritized for chemical synthesis based on their predicted optimal interactions with the receptor. Our models and proposed training protocol were experimentally validated by binding and functional assays.

Metadynamics simulations leveraged by statistical analyses and artificial intelligence-based tools to inform the discovery of G protein-coupled receptor ligands.

G Protein-Coupled Receptors (GPCRs) are a large family of membrane proteins with pluridimensional signaling profiles. They undergo ligand-specific conformational changes, which in turn lead to the differential activation of intracellular signaling proteins and the consequent triggering of a variety of biological responses. This conformational plasticity directly impacts our understanding of GPCR signaling and therapeutic implications, as do ligand-specific kinetic differences in GPCR-induced transducer activation/coupling or GPCR-transducer complex stability. High-resolution experimental structures of ligand-bound GPCRs in the presence or absence of interacting transducers provide important, yet limited, insights into the highly dynamic process of ligand-induced activation or inhibition of these receptors. We and others have complemented these studies with computational strategies aimed at characterizing increasingly accurate metastable conformations of GPCRs using a combination of metadynamics simulations, state-of-the-art algorithms for statistical analyses of simulation data, and artificial intelligence-based tools. This minireview provides an overview of these approaches as well as lessons learned from them towards the identification of conformational states that may be difficult or even impossible to characterize experimentally and yet important to discover new GPCR ligands.

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors.

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G-protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. Although numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here, we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric, and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study shows that inter-TMD assembly and dynamic rearrangement drive mGluR function with distinct properties between subtypes.

Chemically Accurate Relative Folding Stability of RNA Hairpins from Molecular Simulations.

To benchmark RNA force fields, we compared the folding stabilities of three 12-nucleotide hairpin stem loops estimated by simulation to stabilities determined by experiment. We used umbrella sampling and a reaction coordinate of end-to-end (5' to 3' hydroxyl oxygen) distance to estimate the free energy change of the transition from the native conformation to a fully extended conformation with no hydrogen bonds between non-neighboring bases. Each simulation was performed four times using the AMBER FF99+bsc0+χOL3 force field, and each window, spaced at 1 Å intervals, was sampled for 1 µs, for a total of 552 µs of simulation. We compared differences in the simulated free energy changes to analogous differences in free energies from optical melting experiments using thermodynamic cycles where the free energy change between stretched and random coil sequences is assumed to be sequence-independent. The differences between experimental and simulated ΔΔ G° are, on average, 0.98 ± 0.66 kcal/mol, which is chemically accurate and suggests that analogous simulations could be used predictively. We also report a novel method to identify where replica free energies diverge along a reaction coordinate, thus indicating where additional sampling would most improve convergence. We conclude by discussing methods to more economically perform these simulations.