Accurate prediction of drug kinetic rates is critical for rational drug design, especially since the drug residence time has been shown to correlate strongly with its efficacy. Molecular dynamics (MD) simulations are widely used to investigate ligand binding and dissociation processes. However, due to the time scale limitation, capturing ligand dissociation events remain a major challenge for MD simulations. Enhanced sampling techniques can provide the potential of mean force (PMF) for the dissociation process, but their modified dynamics often prevent reliable estimation of dissociation rates and mechanistic details. Here, we present a proof-of-principle study that combines replica exchange molecular dynamics (REMD) with Kramers' rate theory, enabling not only the sampling of equilibrium properties but also the extraction of kinetic information on ligand binding kinetics on host-guest systems. The calculated standard binding free energies agreed excellently with experimental data with <0.8 kcal/mol errors for the host-guest systems. Furthermore, the calculated kinetic rate constants compared well with the experimental data within errors less than an order of magnitude. In summary, this work establishes an efficient and reliable approach to characterize both ligand binding thermodynamics and kinetics using well-defined model systems, offering a validated foundation for future extension to complex biomolecular systems.

The single-pass transmembrane receptor guanylyl cyclase A (GC-A), also known as natriuretic peptide receptor A (NPR-A) or NPR1, regulates blood pressure through vasodilation and natriuresis, making it a promising therapeutic target for hypertension and heart failure. We describe two monoclonal antibodies, XX16 and REGN5308, that differentially activate GC-A. Using cryo-electron microscopy and molecular dynamics simulations, we reveal that XX16 stabilizes GC-A in an active conformation even without its ligand ANP, whereas REGN5308 requires ANP to fully promote receptor activation. Both antibodies increase ANP binding affinity to GC-A and enhance GC-A-mediated cGMP signaling, although XX16 exerts a stronger stabilizing influence on ATP and GTP binding. In a mouse model of obesity-induced hypertension, XX16 treatment significantly reduces blood pressure, underscoring its therapeutic potential. These findings outline the structural and functional basis of GC-A activation by antibody positive allosteric modulators, offering strategies for durable antihypertensive therapies and improved management of cardiovascular diseases.

Abstract Positive allosteric modulators (PAMs) of the M 4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M 4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M 4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 Å resolution, revealing the molecular basis for improved M 4 mAChR PAM activity. MK-97 adopts a distinctive ‘boomerang’-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y92 2.64 and a π-π stacking interaction with W435 7.35 . Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M 4 mAChR PAMs with improved pharmacological properties. Graphical Abstract

Amber is a molecular dynamics (MD) software package first conceived by Peter Kollman, his lab and collaborators to simulate biomolecular systems. The pmemd module is available as a serial version for central processing units (CPUs), NVIDIA and Advanced Micro Devices (AMD) graphics processing unit (GPU) versions as well as Message Passing Interface (MPI) parallel versions. Advanced capabilities include thermodynamic integration, replica exchange MD and accelerated MD methods. A brief update to the software and recently added capabilities is described in this Application Note.

The M 5 muscarinic acetylcholine receptor (M 5 mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site poses significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M 5 mAChR and its allosteric binding sites remains limited. Here, we present a 2.8 Å cryo-electron microscopy structure of the M 5 mAChR complexed with heterotrimeric G q protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M 5 -selective PAMs, we implement an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies reveal that selective M 5 PAMs bind outside previously characterized M 5 mAChR allosteric sites. Subsequently, we obtain a 2.1 Å structure of M 5 mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing an allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that is confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify allosteric binding sites.

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via 19F quantitative NMR and molecular dynamics (MD) simulations, we determined the structure of an intermediate GPCR-mini-Gαsβγ complex at 2.6 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we present direct evidence that the complex at this intermediate state initiates a rate-limited nucleotide exchange before transitioning to the fully activated complex. In this state, BODIPY-GDP/GTP based nucleotide exchange assays further indicated the α-helical domain of the Gα is partially open, allowing it to grasp a nucleotide at a non-canonical binding site, distinct from the canonical nucleotide-binding site. These advances bridge a significant gap in our understanding of the complexity of GPCR signaling. Here, the authors employed 19 F NMR and Gaussian accelerated MD simulations to determine the conformational landscape of the adenosine-A2A receptor and mini-Gαsβγ complex, enabling determination of an intermediate structure of the complex with cryoEM.

G protein–coupled receptors (GPCRs), the largest superfamily of human membrane proteins with >800 members, are primary targets for ~1/3 of all marketed drugs. Recent fluorescence experiments underscored the pivotal role of GPCR–G protein complex lifetime in their coupling efficiency and selectivity. However, these experiments are often expensive, time-consuming, and limited to a small number of GPCR–G protein systems. On the other hand, it is challenging to simulate GPCR–G protein dissociation using molecular dynamics (MD) methods. Here, we have employed Protein–Protein Interaction Gaussian accelerated MD (PPI-GaMD) simulations and experiments to probe the kinetics and pathways of G protein dissociation from GPCRs. For five systems with published experimental kinetic data, PPI-GaMD simulations successfully captured G protein dissociation from the GPCRs, including the adrenergic, adenosine, and muscarinic receptors. The simulations allowed identification of two distinct dissociation pathways and calculation of the G protein dissociation rates, which were in good agreement with experimental data. Additionally, we simulated the effect of positive allosteric modulators (PAMs) of the adenosine A 1 receptor (A 1 R) in Gi protein dissociation and supported simulation findings with bioluminescence resonance energy transfer biosensor experiments evaluating G βγ kinetics following A 1 R activation. A 1 R PAMs were found to strengthen the agonist–receptor and receptor–G protein interactions and significantly reduce dissociation rates of the Gi protein. In summary, complementary PPI-GaMD simulations and kinetic assays have enabled detailed characterization of the kinetics and pathways of G protein dissociation, a critical event in the GPCR signaling cascade, and the effects of GPCR allosteric modulators.

The M5 muscarinic acetylcholine receptor (M5 mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site has posed significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M5 mAChR and its allosteric binding sites has remained limited. Here, we present a 2.8 Å cryo-electron microscopy structure of the M5 mAChR complexed with heterotrimeric Gq protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M5-selective PAMs, we implemented an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies revealed that selective M5 PAMs bind outside previously characterized M5 mAChR allosteric sites. Subsequently, we obtained a 2.1 Å structure of M5 mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing a novel allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that was confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify novel allosteric binding sites.

Accurate prediction of ligand binding thermodynamics and kinetics is crucial in drug design. However, it remains challenging for conventional molecular dynamics (MD) simulations due to sampling issues. Gaussian accelerated MD (GaMD) is an enhanced sampling method that adds a harmonic boost to overcome energy barriers, which has demonstrated significant benefits in exploring protein-ligand interactions. Especially, the ligand GaMD (LiGaMD) applies a selective boost potential to the ligand nonbonded potential energy, significantly improving sampling for ligand binding and dissociation. Furthermore, a selective boost potential is applied to the potential of both ligand and protein residues around binding pocket in LiGaMD2 to further increase the sampling of protein-ligand interaction. LiGaMD and LiGaMD2 simulations could capture repetitive ligand binding and unbinding events within microsecond simulations, allowing to simultaneously characterize ligand binding thermodynamics and kinetics, which is expected to greatly facilitate drug design. In this chapter, we provide a brief review of the status of LiGaMD in drug discovery and outline its usage.

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via 19F quantitative NMR and molecular dynamics (MD) simulation, we determined the structure of an intermediate GPCR-mini-Gαsβγ complex at 2.8 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we presented direct evidence that the intermediate complex initiates a rate-limited nucleotide exchange without progressing to the fully activated complex, in which the α-helical domain (AHD) of the Gα is partially open engaged by a second nucleotide. Our MD simulation supported the pose of the AHD domain. These advances bridge a significant gap in our understanding the complexity of GPCR signaling.