Opioid receptors (ORs) mediate the actions of endogenous and exogenous opioids for many essential physiological processes including regulation of pain, respiratory drive, mood, and, in the case of κ-opioid receptors (KOR), dysphoria and psychotomimesis. Here we report the crystal structure of the human KOR (hKOR) in complex with the selective antagonist JDTic, arranged in parallel-dimers, at 2.9 angstrom resolution. The structure reveals important features of the ligand binding pocket that contribute to JDTic’s high affinity and subtype-selectivity for hKOR. Modeling of other important KOR-selective ligands, including the morphinan-derived antagonists nor-BNI and GNTI, and the diterpene agonist salvinorin A analog RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for hKOR subtype-selectivity along with insight essential for the design of hKOR compounds with new pharmacological properties.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity and behavior. Here we used a structure-based approach to develop a new Gi coupled DREADD using the kappa-opioid receptor as template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally-expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq coupled M3-DREADD within the same neuronal population facilitated the sequential and bi-directional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators.

Background:The -opioid receptor can be activated by structurally diverse agonists. Results: Four structurally diverse agonists differentially bound to and activated wild type and mutant -opioid receptors. Conclusion:The structural features of the agonists dictate how they interact with and stabilize G i -signaling receptor conformations. Significance: The results provide insights into the structural basis of opioid receptor ligand recognition and activation.

Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

We describe a combinatorial virtual screening approach for predicting high specificity heparin/heparan sulfate sequences using the well-studied antithrombin-heparin interaction as a test case. Heparan sulfate hexasaccharides were simulated in the 'average backbone' conformation, wherein the inter-glycosidic bond angles were held constant at the mean of the known solution values, irrespective of their sequence. Molecular docking utilized GOLD with restrained inter-glycosidic torsions and intra-ring conformations, but flexible substituents at the 2-, 3-, and 6-positions and explicit incorporation of conformational variability of the iduronate residues. The approach reproduces the binding geometry of the sequence-specific heparin pentasaccharide to within 2.5 A. Screening of a combinatorial virtual library of 6,859 heparin hexasaccharides using a dual filter strategy, in which predicted antithrombin affinity was the first filter and self-consistency of docking was the second, resulted in only 10 sequences. Of these, nine were found to bind antithrombin in a manner identical to the natural pentasaccharide, while a novel hexasaccharide bound the inhibitor in a unique but dramatically different geometry and orientation. This work presents the first approach on combinatorial library screening for heparin/heparan sulfate GAGs to determine high specificity sequences and opens up huge opportunities to investigate numerous other physiologically relevant GAG-protein interactions.

Structural plasticity plays a major role in determining differential binding of CXCL8 monomer and dimer to glycosaminoglycans (GAGs) and that dimer is the high-affinity GAG ligand. We propose that these properties play important roles in orchestrating in vivo chemokine-mediated neutrophil function.

Salvinorin A, the most potent naturally occurring hallucinogen, has gained increasing attention since the κ-opioid receptor (KOR) was identified as its principal molecular target by us (Roth et al, PNAS, 2002). Here we report the design, synthesis and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. Based on prior substituted cysteine accessibility and molecular modeling studies, C315 7.38 was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently-bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315 7.38 . 22-thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted based on molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315 7.38 and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence for the presence of a free cysteine in the agonist-bound state of KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates KOR.Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum, is the most potent known naturally-occurring hallucinogen (1,2). In 2002, we discovered that the κ-opioid receptor (KOR) was the molecular target for the actions of salvinorin A in vitro (3). Studies with KOR knock-out mice (4) unequivocally demonstrated that the KOR was also the site of † This research was supported in part by NIH R01DA017204 (to B . NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 28. Published in final edited form as:Biochemistry. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript action of salvinorin A in vivo-a finding which has been widely replicated (see (5) and (6) for reviews). Subsequently, salvinorin A emerged as an attractive lead compound for drug discovery and during the past few years, hundreds of salvinorin A derivates have been synthesized (6). Some of these analogues present interesting pharmacological profiles, from full KOR agonist to partial δ-opioid receptor (DOR) or µ-opioid receptor (MOR) agonists and antagonists (7) (8) (9) (10) (11). However, most of the hundreds of analogues displayed decreased affinity (or even no affinity) to KOR. The present challenge now is to use the knowledge about salvinorin A-KOR interactions (12) (13) to design unique salvinorin A derivatives with novel pharmacological profiles and therapeutic potential. In recent years, covalently-bound ligands emerged as a new class of receptor ligands with unique pharmacologi...