Solving an Old Puzzle: Elucidation and Evaluation of the Binding Mode of Salvinorin A at the Kappa Opioid Receptor

Abstract

The natural product Salvinorin A (SalA) was the first nitrogen-lacking agonist discovered for the opioid receptors and exhibits high selectivity for the kappa opioid receptor (KOR) turning SalA into a promising analgesic to overcome the current opioid crisis. Since SalA’s suffers from poor pharmacokinetic properties, particularly the absence of gastrointestinal bioavailability, fast metabolic inactivation, and subsequent short duration of action, the rational design of new tailored analogs with improved clinical usability is highly desired. Despite being known for decades, the binding mode of SalA within the KOR remains elusive as several conflicting binding modes of SalA were proposed hindering the rational design of new analgesics. In this study, we rationally determined the binding mode of SalA to the active state KOR by in silico experiments (docking, molecular dynamics simulations, dynophores) in the context of all available mutagenesis studies and structure-activity relationship (SAR) data. To the best of our knowledge, this is the first comprehensive evaluation of SalA’s binding mode since the determination of the active state KOR crystal structure. SalA binds above the morphinan binding site with its furan pointing toward the intracellular core while the C2-acetoxy group is oriented toward the extracellular loop 2 (ECL2). SalA is solely stabilized within the binding pocket by hydrogen bonds (C210ECL2, Y3127.35, Y3137.36) and hydrophobic contacts (V1182.63, I1393.33, I2946.55, I3167.39). With the disruption of this interaction pattern or the establishment of additional interactions within the binding site, we were able to rationalize the experimental data for selected analogs. We surmise the C2-substituent interactions as important for SalA and its analogs to be experimentally active, albeit with moderate frequency within MD simulations of SalA. We further identified the non-conserved residues 2.63, 7.35, and 7.36 responsible for the KOR subtype selectivity of SalA. We are confident that the elucidation of the SalA binding mode will promote the understanding of KOR activation and facilitate the development of novel analgesics that are urgently needed.

Discovery of Novel, Selective, and Nonbasic Agonists for the Kappa-Opioid Receptor Determined by Salvinorin A-Based Virtual Screening

“…The first workflow was based on structure-based 3D pharmacophores developed with LigandScout, while the second workflow was based on threedimensional shape and chemical feature similarity using ROCS. Due to the absence of an experimentally determined structure of the SalA-KOR complex at the start of the study, we utilized our previously published binding hypothesis of SalA at the KOR 52 to generate both of our virtual screening queries (Figure 2). Further details about the query generation can be found in the Experimental Section.…”

“…The data set only includes nonbasic KOR agonists, primarily SalA analogues, due to the limited number of different nonbasic KOR ligand scaffolds known. 52 of SalA at the KOR used for query generation is shown on the left side. At the top, the 3D pharmacophore information used as query for the screening in LigandScout 53,100 is depicted.…”

“…49−51 In this study, we conducted a 3D pharmacophore-based virtual screening campaign aiming to discover novel, nonbasic, and selective KOR agonists as potential therapeutics. We employed our recently published binding mode of SalA at the KOR 52 as a starting point for the 3D pharmacophore generation. Selected virtual screening hits were subjected to pharmacological in vitro evaluation resulting in the discovery of two new nonbasic KOR agonists with nanomolar affinity and potency, as well as very good selectivity for the KOR over the other opioid receptor subtypes, MOR and DOR.…”

Discovery of an Ortho-Substituted N-Cyclopropylmethyl-7α-phenyl-6,14-endoethano-tetrahydronorthebaine Derivative as a Selective and Potent Kappa Opioid Receptor Agonist with Subsided Sedative Effect

“…In fact, the role of V118 2.63 or its allelic residues (N127 in MOR and K108 in DOR) in determining the ligand subtype selectivity toward opioid receptors has been demonstrated using numerous site-directed mutagenesis and independent molecular modeling studies. − In particular, V118 2.63 was claimed to be critical for the selectivity of salvinorin A, evidenced by a substantial 36-fold decrease in affinity to KOR with V118 2.63 K mutation . Contrary to the importance attributed to V118 2.63 in determining selectivity, X-ray diffraction structures of both 4,5-epoxymorphinan-based KOR agonists (MP1104, PDB entry, 6B73; nalfurafine, PDB entry, 7YIT) and other nonmorphinan-based KOR agonists (GR89,696, PDB entry, 8DZR; momSalB, PDB entry, 8DZQ; Dynorphin, PDB entry, 8F7W) displayed negligible interactions with V118 2.63 in their KOR-bound states.…”

Involvement of the Opioid Peptide Family in Cancer Progression

“…The analysis of the opioid binding sites in MOP and KOP has given clues regarding the importance of the region defined by ECL2 and TM5 for controlling β-arrestin recruitment [78]. The docking manner of the natural compound salvinorin, a diterpenoid furanelactone, to KOP has also generated valuable structural details for designing and evaluating KOP selective and effective drugs [79].…”