Structural and functional determination of peptide versus small molecule ligand binding at the apelin receptor.

We describe a structural and functional study of the G protein-coupled apelin receptor, which binds two endogenous peptide ligands, apelin and Elabela/Toddler (ELA), to regulate cardiovascular development and function. Characterisation of naturally occurring apelin receptor variants from the UK Genomics England 100,000 Genomes Project, and AlphaFold2 modelling, identifies T89 as important in the ELA binding site, and R168 as forming extensive interactions with the C-termini of both peptides. Base editing to introduce an R/H168 variant into human stem cell-derived cardiomyocytes demonstrates that this residue is critical for receptor binding and function.

Comparative Study of Allosteric GPCR Binding Sites and Their Ligandability Potential.

The steadily growing number of experimental G-protein-coupled receptor (GPCR) structures has revealed diverse locations of allosteric modulation, and yet few drugs target them. This gap highlights the need for a deeper understanding of allosteric modulation in GPCR drug discovery. The current work introduces a systematic annotation scheme to structurally classify GPCR binding sites based on receptor class, transmembrane helix contacts, and, for membrane-facing sites, membrane sublocation.

Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound.

Trends in Passive IoT Biomarker Monitoring and Machine Learning for Cardiovascular Disease Management in the U.S. Elderly Population.

It is predicted that the growth in the U.S. elderly population alongside continued growth in chronic disease prevalence will further strain an already overburdened healthcare system and could compromise the delivery of equitable care.

Rosetta's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery.

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in screening.

Automated Urinal-Based Specific Gravity Measurement Device for Real-Time Hydration Monitoring in Male Athletes.

Acute and chronic hydration status is important for athlete safety and performance and is frequently measured by sports scientists and performance staff in team environments via urinalysis. However, the time required for urine collection, staff testing, and reporting often delays immediate reporting and personalized nutrition insight in situations of acute hydration management before training or competition. Furthermore, the burdensome urine collection and testing process often renders chronic hydration monitoring sporadic or non-existent in real-world settings.

Molecular basis for the evolved instability of a human G-protein coupled receptor.

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Although they function at the plasma membrane, mammalian GnRHRs accumulate within the secretory pathway.

Structure, function and pharmacology of human itch GPCRs.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions. MRGPRX2 couples to both G and G in mast cells.

Computational redesign of a fluorogen activating protein with Rosetta.

The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design.

Viewpoints on the First Transatlantic GPCR Symposium for Early-Career Investigators.

In July 2021, we organized a virtual symposium aimed at early-career investigators (ECIs) in G protein-coupled receptor (GPCR) research: the first Transatlantic ECI GPCR Symposium. Here, we discuss the proceedings of this symposium and the unique networking events with GPCR leaders including the Nobel Laureates Dr. Robert Lefkowitz and Dr.

A practical guide to large-scale docking.

Structure-based docking screens of large compound libraries have become common in early drug and probe discovery. As computer efficiency has improved and compound libraries have grown, the ability to screen hundreds of millions, and even billions, of compounds has become feasible for modest-sized computer clusters. This allows the rapid and cost-effective exploration and categorization of vast chemical space into a subset enriched with potential hits for a given target.

Modeling Immunity with Rosetta: Methods for Antibody and Antigen Design.

Structure-based antibody and antigen design has advanced greatly in recent years, due not only to the increasing availability of experimentally determined structures but also to improved computational methods for both prediction and design. Constant improvements in performance within the Rosetta software suite for biomolecular modeling have given rise to a greater breadth of structure prediction, including docking and design application cases for antibody and antigen modeling. Here, we present an overview of current protocols for antibody and antigen modeling using Rosetta and exemplify those by detailed tutorials originally developed for a Rosetta workshop at Vanderbilt University.

Improving homology modeling from low-sequence identity templates in Rosetta: A case study in GPCRs.

As sequencing methodologies continue to advance, the availability of protein sequences far outpaces the ability of structure determination. Homology modeling is used to bridge this gap but relies on high-identity templates for accurate model building. G-protein coupled receptors (GPCRs) represent a significant target class for pharmaceutical therapies in which homology modeling could fill the knowledge gap for structure-based drug design.

Macromolecular modeling and design in Rosetta: recent methods and frameworks.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities.

Comparative modeling and docking of chemokine-receptor interactions with Rosetta.

Chemokine receptors are a subset of G protein-coupled receptors defined by the distinct property of binding small protein ligands in the chemokine family. Chemokine receptors recognize their ligands by a mechanism that is distinct from other class A GPCRs that bind peptides or small molecules. For this reason, structural information on other ligand-GPCR interactions are only indirectly relevant to understanding the chemokine receptor interface.

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor.

The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif.

Modeling the complete chemokine-receptor interaction.

Chemokines are soluble, secreted proteins that induce chemotaxis of leukocytes and other cells. Migratory cells can sense the chemokine concentration gradient following chemokine binding and activation of chemokine receptors, a subset of the G protein-coupled receptor (GPCR) superfamily. Chemokine receptor signaling plays a central role in cell migration during inflammatory responses as well as in cancer and other diseases.

Structural basis of ligand binding modes at the neuropeptide Y Y receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y, Y, Y and Y receptors, with different affinity and selectivity . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y receptor (YR) .

α- and α-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

The poor norepinephrine innervation and high density of Gi/o-coupled α- and α-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D-like receptor ligands, such as the D receptor agonist 7-OH-PIPAT and the D receptor agonist RO-105824, to α-adrenoceptors in cortical and striatal tissue, which express α-adrenoceptors and both α- and α-adrenoceptors, respectively.

Improved Folding of the Y G Protein-Coupled Receptor into Bicelles.

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (YR) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS.

Comparing incidence of emergence delirium between sevoflurane and desflurane in children following routine otolaryngology procedures.

Background: Emergence delirium (ED) is a state of aggressive agitation that can occur temporarily in the process of emerging from anesthesia in children exposed to volatile or intravenous anesthetics. Emergence delirium is typically assessed using the published and validated Pediatric Emergence Delirium (PAED) Scale. Due to some variation in properties between sevoflurane and desflurane for maintenance of anesthesia after standard sevoflurane induction, we designed a prospective study to examine potential differences in emergence behavior and incidence of ED in children undergoing elective ear-nose-throat surgery.

Protocols for Molecular Modeling with Rosetta3 and RosettaScripts.

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al.

Rosetta and the Design of Ligand Binding Sites.

Proteins that bind small molecules (ligands) can be used as biosensors, signal modulators, and sequestering agents. When naturally occurring proteins for a particular target ligand are not available, artificial proteins can be computationally designed. We present a protocol based on RosettaLigand to redesign an existing protein pocket to bind a target ligand.