Biology subjects

Kobilka, B.

Publications and source records attributed to Kobilka, B..

7 recordsLinked to original sources

bioRxiv2026

Discovery and dynamic pharmacology of μ-opioid receptor positive allosteric modulators

Opioid agonists such as morphine and fentanyl exert analgesic effects by binding and activating the {micro}-opioid receptor ({micro}OR), yet agonism of the {micro}OR causes a slate of serious side effects. {micro}OR-mediated addiction and respiratory depression are the major causes of the current opioid overdose crisis, largely driven by the explosion in illicit use of fentanyl, a potent opioid receptor full agonist. Given these serious side effects (and high resulting societal cost), molecules that act as analgesics with distinct mechanisms of action are of great interest. Positive allosteric modulators (PAMs) of the {micro}OR have the potential to avoid many off-target side effects of conventional opioid orthosteric agonists by enhancing the signaling properties of natural opioid peptide systems. We used a DNA-encoded chemical library screening approach to selectively discover active-state-specific {micro}OR PAMs. Two out of 3 selected prospective PAMs displayed the anticipated enhancement in agonist activity. The most effective of these compounds enhanced the activity of all orthosteric opioid agonists tested, including the native opioid peptide met-enkephalin. Little is known about the underlying dynamic basis of allosteric modulation of Family A GPCRs like the {micro}OR. To that end, we used single-molecule fluorescence resonance energy transfer experiments to detail the impact that our novel {micro}OR PAM has on the dynamic activation behavior of a key region on the intracellular face of the receptor. Our results here provide both a new chemical scaffold that acts as a {micro}OR PAM and detailed pharmacological and dynamic insights into its mechanism of action.

biophysics↗

bioRxiv2025

The role of intrinsically disordered domains in regulating G protein coupled receptor signaling

The 2A adrenergic receptor (2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of 2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of 2AAR. Our study reveals unique TM6 dynamics in 2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into 2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.

biophysics↗

bioRxiv2025

Ligand-dependent G protein dynamics underlying opioid signaling efficacy

Activation of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) requires large-scale opening of the G -helical domain (AHD) to expose the nucleotide-binding site and facilitate GDP-GTP exchange. While orthosteric ligands are known to modulate GPCR conformation and signaling efficacy, how these effects propagate to the G protein itself remains unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we monitored AHD motions in Gi proteins coupled to the -opioid receptor (OR) across a spectrum of ligand- and nucleotide-bound states. We find that receptor ligands differentially modulate these dynamics from over 70 [A] away, with higher-efficacy agonists more effectively promoting transitions to an open, low-nucleotide-affinity conformation. These data also capture transient OR-Gi intermediates during nucleotide binding and suggest that -opioid ligand efficacy arises in part from allosteric control over G protein conformational equilibria that kinetically gate activation.

biophysics↗

bioRxiv2023

Conformational dynamics of the μ-opioid receptor determine ligand intrinsic efficacy

The -opioid receptor (OR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the OR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of {beta}-arrestin-1 with the OR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi. One-Sentence SummaryLigand-dependent conformational dynamics of the -opioid receptor determine downstream signaling efficacy.

biochemistry↗

bioRxiv2022

Structure-based Evolution of G protein-biased μ-opioid Receptor Agonists

The -opioid receptor (OR) is the major target for opioid analgesics. Activation of OR initiates signaling through G protein pathways as well as through {beta}-arrestin recruitment. OR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased OR agonist. Here we report the cryoEM structure of PZM21 bound OR in complex with Gi protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced Gi protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to OR in complex with Gi protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce {beta}-arrestin recruitment by the OR, and hold promise for developing next-generation analgesics with fewer adverse effects. Table of Contents Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/485330v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@125808corg.highwire.dtl.DTLVardef@11aa15corg.highwire.dtl.DTLVardef@157caaaorg.highwire.dtl.DTLVardef@ba837d_HPS_FORMAT_FIGEXP M_FIG C_FIG We obtained cryoEM structures of the -opioid receptor (OR) bound to the lead compound PZM21 and the newly developed agonist FH210 to understand the mechanism of their biased signaling and to guide the evolution of next-generation analgesics with fewer adverse effects.

pharmacology and toxicology↗

bioRxiv2021

An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

Allosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

biophysics↗

bioRxiv2018

Stable networks of water-mediated interactions are conserved in activation of diverse GPCRs

G protein-coupled receptors (GPCRs) have evolved to recognize incredibly diverse extracellular ligands while sharing a common architecture and structurally conserved intracellular signaling partners. It remains unclear how binding of diverse ligands brings about GPCR activation, the common structural change that enables intracellular signaling. Here, we identify highly conserved networks of water-mediated interactions that play a central role in activation. Using atomic-level simulations of diverse GPCRs, we show that most of the water molecules in GPCR crystal structures are highly mobile. Several water molecules near the G protein-coupling interface, however, are stable. These water molecules form two kinds of polar networks that are conserved across diverse GPCRs: (i) a network that is maintained across the inactive and the active states and (ii) a network that rearranges upon activation. Comparative analysis of GPCR crystal structures independently confirms the striking conservation of water-mediated interaction networks. These conserved water-mediated interactions near the G protein-coupling region, along with diverse water-mediated interactions with extracellular ligands, have direct implications for structure-based drug design and GPCR engineering.

biophysics↗