Computational studies to determine the binding site of ORG27569, a negative allosteric modulator, within the cannabinoid receptor 1 (CB1) reveal an intracellular binding domain as the most favorable binding site for ORG27569.https://egrove.olemiss.edu/pharm_facpost/1000/thumbnail.jp

Aderibigbe, AyoOluwa O.

Doerksen, Robert J.

Pandey, Pankaj

English

eGrove (Univ. of Mississippi)

University of Mississippi eGrove Faculty and Student Poster Sessions Pharmacy, School of 10-2019 Determining the binding site of ORG27569 within the CB1 receptor AyoOluwa O. Aderibigbe University of Mississippi, aaderibi@go.olemiss.edu Pankaj Pandey University of Mississippi, ppandey@olemiss.edu Robert J. Doerksen University of Mississippi, rjd@olemiss.edu Follow this and additional works at: https://egrove.olemiss.edu/pharm_facpost  Part of the Natural Products Chemistry and Pharmacognosy Commons Recommended Citation Aderibigbe, AyoOluwa O.; Pandey, Pankaj; and Doerksen, Robert J., "Determining the binding site of ORG27569 within the CB1 receptor" (2019). Faculty and Student Poster Sessions. 1. https://egrove.olemiss.edu/pharm_facpost/1 This Book is brought to you for free and open access by the Pharmacy, School of at eGrove. It has been accepted for inclusion in Faculty and Student Poster Sessions by an authorized administrator of eGrove. For more information, please contact egrove@olemiss.edu. AyoOluwa O. Aderibigbe1, Pankaj Pandey2, and Robert J. Doerksen*1,31 Department of BioMolecular Sciences, Division of Medicinal Chemistry, 1,2 National Center for Natural Products Research, and 3 Research Institute of Pharmaceutical Sciences, School of Pharmacy University of Mississippi, University, MS 38677, USA Determining the binding site of ORG27569 within the CB1 receptorIntroductionØ Functional antagonists of the cannabinoid receptor 1 (CB1)receptor are considered to have great potential in obesitymanagement.ØMost neutral antagonists and inverse agonists bind to the main(orthosteric) site of CB1, and are CNS-active, exhibiting adverseneuropsychiatric effects.Ø In contrast, negative allosteric modulators (NAMs) block CB1signaling by binding at a site that is topologically distinct from theorthosteric site.ØNAMs are highly selective. They exhibit a ceiling effect, probedependence and biased signaling, making them less liable toelicit CNS-mediated adverse effects.ØORG27569, an intrinsic inverse agonist and negative allostericmodulator (NAM) of CB1, increases the binding affinity of agonistCP55940.Ø In this study, we applied computational methods to identify thebinding site of ORG27569, amidst contradictory reports in theliterature (Fig. 1).ØCB1 receptor models were built using each of the four X-raycrystal structures4-6 of CB1 as a template, with the N-terminusadded on through our computational modeling, in order to be ableto include the reported C98–C107 disulfide bridge.ØCP55940 was docked into each of the four CB1 receptor modelsusing the Glide7 Standard Precision (SP) protocol of Schrödinger.ØSiteMap8, Schrödinger was used to identify the top-rankedpotential binding sites in the CB1–CP55940 complexes.ØORG27569 was docked into all the pockets identified usingSiteMap.ØA 50 ns molecular dynamics simulation was performed on the5U09 model of the CB1 receptor bound to CP55940, using theOPLS3 force field.ØA SiteMap analysis was run on frames extracted every 0.5 nsfrom the trajectory.ØMolecular docking studies of ORG27569 were performed on sixpredominant site clusters obtained from the trajectory using theGlide SP protocol, InducedFit docking and Prime9 free energycalculations.1. Shore, D. M.;  Baillie, G. L.;  Hurst, D. H.;  Navas, F.;  Seltzman, H. H.;  Marcu, J. P.;  Abood, M. E.;  Ross, R. A.; Reggio, P. H. Journal of Biological Chemistry 2014, 289 (9), 5828-5845.2. Stornaiuolo, M.;  Bruno, A.;  Botta, L.;  La Regina, G.;  Cosconati, S.;  Silvestri, R.;  Marinelli, L.; Novellino, E. Scientific Reports 2015, 5, 15453.3. Sabatucci, A.;  Tortolani, D.;  Dainese, E.; Maccarrone, M. Biotechnology and Applied Biochemistry 2018, 65 (1), 21-28.4. Hua, T.;  Vemuri, K.;  Pu, M.;  Qu, L.;  Han, G. W.;  Wu, Y.;  Zhao, S.;  Shui, W.;  Li, S.;  Korde, A.;  Laprairie, R.;  Stahl, E.;  Ho, J.-H.;  Zvonok, N.;  Zhou, H.;  Kufareva, I.;  Wu, B.;  Zhao, Q.;  Hanson, M.;  Bohn, L.;  Makriyannis, A.;  Stevens, R.; Liu, Z.-J. Cell 2016, 167 (3), 750-762.5. Shao, Z.;  Yin, J.;  Chapman, K.;  Grzemska, M.;  Clark, L.;  Wang, J.; Rosenbaum, D. M. Nature 2016, 540 (7634), 602-606.6. Hua, T.;  Vemuri, K.;  Nikas, S. P.;  Laprairie, R. B.;  Wu, Y.;  Qu, L.;  Pu, M.;  Korde, A.;  Jiang, S.; Ho, J.-H. Nature 2017, 547, 468-471.7. Glide, Schrödinger Release 2018-1; LLC: New York, NY, 2018. 8. SiteMap, Schrödinger Release 2018-1; LLC: New York, NY, 2018.9. Prime, Schrödinger Release 2018-1; LLC: New York, NY, 2018.10.Liu, X.; Ahn, S.; Kahsai, A. W.; Meng, K.-C.; Latorraca, N. R.;Pani, B.; Venkatakrishnan, A. J.; Masoudi, A.; Weis, W. I.; Dror, R.O.; Chen, X.; Lefkowitz, R. J.; Kobilka, B. K. Nature 2017, 548(7668), 480.Fig. 1. (Left) CB1 model created from the inactive-state X-ray CB1 crystal structure, 5U09, with the orthosteric site highlighted in blue and previously predicted allosteric sites1-3 in orange. (Right) CB1 NAM, ORG27569, and CB1/CB2 agonist, CP55940.Ø To identify the binding pose of CP55940 within the CB1 X-raycrystal structures.Ø To identify potential allosteric sites in the active-state andinactive-state complexes of the CB1 receptor bound to CP55940.Ø To identify the preferred binding site of ORG27569 within aninactive-state CB1–CP55940 complex.Ø To determine the binding site of ORG27569 will aid rationaldesign of novel NAMs of the CB1 receptor.Objectives and SignificanceMethodsConclusionsReferencesFuture workAcknowledgementsResults and DiscussionThis project was supported by R15 GM119061 and P20 GM104932from the National Institutes of Health (NIH) National Institute ofGeneral Medical Sciences (NIGMS). The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of NIGMS or NIH. This investigation was conducted inpart in a facility constructed with support from research facilitiesimprovement program NIH C06 RR-14503-01.ØEnhanced MD simulations (such asmetadynamics) of theORG27569−CP55940−CB1 complex using datafrom previously-reported mutagenesis studies.ØAssessment of the mechanism by whichORG27569 interferes with CP55940-inducedCB1 activation.Ø The orientation of CP55940, which aligned wellwith native poses of the co-crystallized ligandswithin the experimental active-state CB1structures, validated its docking pose within theinactive-state models.ØBinding site analysis and subsequent moleculardocking with ORG27569 on the rigid receptormodels did not provide a consensus bindingsite for ORG27569.ØBinding site analysis performed on the flexiblereceptor model, using a conformationalensemble of the protein, provided moreinformation about the probability of finding aparticular site at any time.Ø The best docking site for ORG27569 was foundat an intracellular site on CB1, near helices 2, 6and 7.Ø The docking orientation of CP55940 in all four models of CB1 was similar (Fig.2).ØDocked poses of CP55940 obtained in the active-state CB1 X-ray structureswere highly similar to the binding poses of the co-crystallized ligands, with anRMSD <1 Å for common heavy atoms.ØSiteMap analysis revealed multiple allosteric sites within the CP55940-boundCB1 complexes (Fig. 3).ØRigid receptor docking within the allosteric sites identified by SiteMap did notprovide a consensus docking site for ORG27569 across all structures (Fig. 4).Fig. 3. Maps of identified allosteric binding sites (each site’s surface in different color) for the CP55940-bound complex of the CB1 receptor, using X-ray structures of the inactive-state (A) 5TGZ and (B) 5U09; and active-state (C) 5XR8 and (D) 5XRA. CP55940 (C magenta; non-polar hydrogen atoms are not shown, for clarity) is shown in the orthosteric site. Fig. 6. ORG27569 (C orange) and CP55940 (C cyan) within the 5U09-based CB1 structure. Hydrogen bonds are shown using black dashed lines. Non-polar hydrogen atoms are not shown for clarity.Fig. 5. Site points of six predominant site clusters (SC) in the 5U09-based CB1–CP55940 complex following a 50 ns MD simulation. ØSince the SiteMap and docking protocolsconsider a rigid receptor conformationthat is most suited for docking of smallmolecules that are very similar to thenative ligand, we performed an MDsimulation of the CB1–CP55940 complexto generate multiple receptorconformations and performed binding siteanalysis on them.ØOf thirty site clusters obtained from theMD simulations, six site clusters werepresent in at least 40 of the framesprocessed (Fig. 5).ØMolecular docking studies of ORG27569within the site clusters revealed that sitecluster 3 was the most favored (Table 1).5TGZ5U095XR85XRAORG27569CP559405XR85XRA 5U09 5TGZFig. 2. Docked CP55940 (C purple) overlaid with the co-crystallized ligand within each of the four X-ray crystal structure-based CB1 models.SC1SC8SC5SC6SC3SC13Site ClusterGlide Gscore a IFD Score a Prime Free Energy a1 −6.190 −549.22 −50.003 −9.740 −556.08 −86.825 −6.542 −489.49 −66.506 −8.439 −495.44 −76.668 −7.862 −495.47 −65.0813 −6.862 −493.61 −64.92Table 1. The docking performance of ORG27569 within the predominant site clusters obtained from the CP55940-bound complex of CB1.R214Y153D403D338Y397TM I5TGZTM I5U095XR85XRAFig. 4. Most favorable docking sites for ORG27569 (C orange) within the rigid CP55940-bound CB1 complexes. CP55940 (C magenta). Non-polar hydrogen atoms are not shown for clarity.ØORG27569 docked best at anintracellular site near helices 2, 6 and 7(SC3) within CB1 (Fig. 6).Ø Two hydrogen bond interactions wereobserved with Y153 and D403,similar to interactions found with theβ2AR intracellular antagonist, Cmpd-15PA10.Ø The close proximity of ORG27569 tothe canonical ionic lock residues,R214 and D338, may account fordisruption of CB1 signaling. a Units: kcal/mol

Determining the binding site of ORG27569 within the CB1 receptor

https://egrove.olemiss.edu/cgi/viewcontent.cgi?article=1000&context=pharm_facpost

Determining the binding site of ORG27569 within the CB1 receptor

Abstract

Similar works

Full text

Available Versions

eGrove (Univ. of Mississippi)