g - protein 
 coupled receptors ( gpcrs ) are located in the cellular 
 membrane and act as mediators for cell signaling , making them promising 
 therapeutic targets . however , drug design for gpcrs has been challenging 
 due to the paucity of validated 3d structures . although these integral 
 membrane proteins have long been difficult targets for structure determination 
 efforts , recent methodological developments now provide structures 
 for 33 of the 819 human gpcrs . six of these , bovine rhodopsin , human 2 adrenergic receptor , human 
 adenosine a2a receptor , human muscarinic m2 receptor , human neurotensin nts1 receptor , and human p2y12 receptor , have been crystallized in an active or partially active 
 conformation , but only one , human 2 adrenergic receptor , 
 has been cocrystallized with the full heterotrimeric gs protein . the human cannabinoid 
 1 ( cb1 ) is a gpcr with high therapeutic potential 
 as a drug target . this receptor is activated by -tetrahydrocannabinol ( thc ) in marijuana and increases appetite for 
 aids and cancer patients . the cb1-selective inverse agonist / antagonist 
 rimonabant ( also known as sr141716a ) is 
 an antiobesity drug that was available in europe and was in phase 
 iii of us fda clinical trials but had to be withdrawn due to severe 
 depressive effects . there is some indication that these side effects 
 are caused by rimonabant antagonizing the cb1 receptor activity in 
 brain , underscoring the need for biased 
 inverse agonists for cb1 that are devoid of these unacceptable side 
 effects . nonetheless , the intracellular pathways mediating the beneficial 
 and unwanted effects have not yet been elucidated . cb1 has not 
 yet been crystallized ; however our previous studies , 
 using the gensemble ( gpcr ensemble of structures in membrane bilayer 
 environment ) complete conformational sampling method , predicted the 
 ensemble of ten low energy cb1 conformations expected to play a role in binding various ligands and controlling 
 function . briefly , gensemble carries out systematic sampling of trillions 
 of seven - helix bundles by rotating and tilting the transmembrane ( tm ) 
 helices starting with templates from previous calculations or x - ray 
 experiments on other gpcrs . we consider it important to examine this 
 full conformational space for cb1 , because gpcrs are dynamic and flexible , 
 enabling different ligands to bind to and stabilize quite different 
 gpcr conformations . this , in turn , facilitates coupling to multiple 
 types of intracellular proteins . for each of these 
 ten cb1 conformations we used the hierarchical 
 binding site prediction methods , darwindock and gendock , to predict the optimum structures for binding of rimonabant and 
 seven other ligands . receptor complexes 
 are in good agreement with published site - directed mutagenesis data and structure  activity relationship ( sar ) data . we then used these predicted binding sites to design and identify 
 16 new ligands that we expected might act as inverse agonists upon 
 binding to the cb1 receptor . subsequently we tested five of these 
 ligands experimentally and found that two compounds possess inverse 
 agonist properties including reduced g - protein coupling and changes 
 in cellular localization of the receptor . this provides new design 
 strategies for developing more selective and potent cb1 inverse agonists 
 through computational optimization of chemical functionalities of 
 the compounds coupled with further pharmacological characterization . we used our gensemble method to predict an ensemble of ten low energy cb1 receptor structures , 
 described elsewhere . these predictions started with four distinct templates [ bovine rhodopsin , turkey 1 adrenergic receptor , human 2 adrenergic receptor , and human adenosine a2a receptor ] , optimized the helix shapes within the turkey 
 1 adrenergic receptor template , sampled all ( 12 ) = 35 million rotations of each of the seven helices independently , 
 followed by sampling of 13 trillion combinations of helix rotations 
 and tilts to select by energy the best ten conformations of the seven - helix 
 bundles . we then docked various ligands , finding the best gpcr conformation 
 for each ligand . previous papers have analyzed thoroughly this ensemble for the wild - type 
 and several mutant receptors , which were used successfully to predict 
 ( in advance of experiment ) mutations that would bias the ensemble 
 toward the fully inactive , the constitutively active , or the essentially 
 fully active states . to obtain 
 a diverse set of ligand conformations for docking the molecular structure 
 of rimonabant was constructed with maestro software , and a conformational search was performed with macromodel 
 software , which generated 83 conformations 
 of the ligand . sytematic and extended torsional sampling options were 
 used where the selected rotatable bonds are rotated 360 in 30 
 increments . ligand conformations that fall within an energy window 
 of 10 kcal / mol and an rmsd diversity of 0.5  were saved for 
 subsequent steps . the conformational search was conducted with the 
 opls 2005 force field and in a dielectric 
 of 80.37 to match water . subsequently , we preformed two rounds of 
 clustering for each ligand ; in the first round we clustered ligands 
 with a 2.0  diversity followed by another round of clustering 
 with a 1.0  diversity . the mulliken populations of each atom 
 were calculated with jaguar software using 
 density functional theory ( dft ) with the b3lyp functional and the 
 6 - 31 g * * basis set . an additional ligand conformation was constructed from existing coordinates 
 from the crystallized rimonabant in methanol solvate deposited in the cambridge structural database . each ligand was minimized using the surface 
 generalize born ( sgb ) solvation model for 100 steps or to a convergence threshold of 0.2 kcal / mol /  
 rms force with the mpsim program . for 
 each of the ten conformations of rimonabant , we used the darwindock 
 and gendock methods to predict the optimum binding site for each of the ten 
 lowest energy protein structures predicted by gensemble for the cb1 
 receptor . the darwindock method 
 aims at generating a complete set of poses for the binding pocket 
 while using rmsd clustering of the poses to dramatically reduce the 
 computational cost . to provide flexibility and space for the ligand 
 to identify favorable binding sites , we replaced the seven bulky hydrophobic 
 residues ( filmyvw ) with alanines . the mutated residues are called 
 alanized residues , and the mutated protein is called alanized protein . for each of the best 100 ligand poses in the alanized protein , we 
 then dealanized the mutated residues back to their original hydrophobic 
 identity and optimized their positions along with those of other residues 
 in the binding site using scream . this 
 leads to a unique set of optimized residue side chains for each of 
 the 100 ligand poses . in this process we did not replace the w5.43 
 residue with alanine because we consider this tryptophan to be critical 
 for ligand interaction based on site - directed mutagenesis data , leaving its side chain in the form predicted 
 by scream for each of the ten protein conformations . in the 
 pose generation step of darwindock , a ligand pose is acceptable if 
 it clashed or bumped the receptor residues at six positions or less . first , we used dock6 to generate 5,000 
 ligand poses ( without evaluating an energy ) and clustered them into 
 families , where every family member is within a 2.00  rmsd of 
 each other . this procedure of adding 5,000 
 poses and reclustering is repeated until the number of new families 
 generated is less than 2% of the total number of families in the preceding 
 iteration . at this point darwindock scores the energies 
 of one representative from each family , the family head , and selects 
 the 10% of family heads with the lowest dreiding energies . all members of these respective families are 
 then scored energetically . from this list of approximately 5000 poses , 
 we select the lowest 50 by each of three criteria : lowest hydrophobic 
 energy , lowest polar energy , and lowest total energy , giving at most 
 150 poses . gendock 
 was used to refine 
 the 150 docked ligand  receptor poses generated by darwindock . 
 in the scream step , residues were replaced with the original hydrophobic residues but 
 using the optimum side chain rotamers to avoid any clashes with the 
 ligand and other protein side chains . then the entire complex was 
 minimized for 10 steps for each case to remove any bad contacts . next , 
 the receptor was neutralized , so that the acidic residues ( aspartic 
 acid and glutamic acid ) each gained a proton , and the basic residues 
 ( lysine and arginine ) each lost a proton . the resulting receptor  ligand 
 complexes were minimized for 60 steps using the dreiding iii ff . then for each of the ten receptor structures 
 and each of the ten ligand conformations ( 100 complexes total ) , we 
 selected the lowest binding energy structure , including strain and 
 ligand solvation , which we expected to best represent the binding 
 affinity . the binding energy with strain and ligand solvation is defined 
 as the energy difference between the complex and the sum of the receptor 
 and ligand energies with ligand strain and ligand solvation included . the selected complex was minimized with the dreiding iii ff with the lj vdw term ( dreiding iii - lj ff ) in 
 vacuum for 50 steps or to an rms force threshold of 0.5 kcal / mol /  
 using the mpsim program . we took the most stable predicted rimonabant - cb1 
 receptor complex shown in figure 1 and extracted the rimonabant . then , we modified it 
 with maestro software to look like the new derivative , calculated 
 the mulliken charges at the b3lyp dft level ( with jaguar software ) , 
 minimized this configuration with 100 steps of conjugate gradients 
 ( or to a threshold of 0.2 kcal / mol /  ) using the mpsim program , 
 and predicted the binding site using darwindock as described above . however , instead of docking the new ligands to all ten cb1 conformations , 
 we docked only to the alanized cb1 receptor conformation wild - type 
 6 ( wt6 ) because it produced the lowest energy ( most stable ) complex 
 with rimonabant shown in figure 1 . we repeated these steps for seven 
 derivatives and for the original rimonabant ligand as a control . for 
 rimonabant , we did not alter the ligand structure but rather docked 
 the optimized ligand conformation . the final complexes were chosen 
 according to the lowest binding energy including strain and ligand 
 solvation . for comparison of the experimental binding affinities with 
 the computational energies , we used the binding energy , that is , the 
 difference in energy between the receptor  ligand complex and 
 the receptor and the ligand structures calculated separately . the 
 ligand strain and ligand solvation were ignored since we were comparing 
 energetics across different ligands based on the same rimonabant conformation . the experimental change in free energy upon ligand binding to the 
 receptor , gbindexp , is obtained 
 from the pki in eq 1:1 the pki is the experimental binding equilibrium constant of 
 the inhibitory 
 ligand . since the pki describes the strength 
 of the interaction between the receptor and ligand , we use it to derive 
 gbindexp . the g , or the difference in the change in experimental 
 free energy upon ligand binding for a given inverse agonist ( gbindexp ) with respect to the corresponding 
 value of rimonabant , is determined by eq 2:2 this expression 
 was used to compare experimental changes in binding 
 energy with our predicted changes in binding energy for the series 
 of ligands . our predicted rimonabant - cb1 binding site was used to identify 
 new potential cb1 inverse agonists . the predicted pharmacophore ( shown 
 in figure 1b ) , was 
 used in a preliminary search over the 2 million compounds in pubchem for ligands that are similar to rimonabant according 
 to the tanimoto coefficient , an indicator of how 
 similar two 2d ligands structures are to one another . the tanimoto 
 coefficient ranges from 0 to 1 , with 0 being no resemblance between 
 the molecules and 1 being identical molecules . we wanted ligands that 
 are similar to rimonabant , but which could exploit nearby underused 
 polar and aromatic residues in the predicted binding site . for example , 
 k7.32 is located near rimonabant but does not form a hydrogen bond 
 or salt bridge with it ; thus we searched for ligands similar to rimonabant , 
 with an appropriately placed functional group to create the new polar 
 interaction . we also wanted a ligand that is commercially available 
 but has not been previously tested with cb1 , so that our prediction 
 can be tested easily . once we had identified a ligand ( msc1 , described 
 later ) from pubchem that met the above criteria , we performed a search 
 in pubchem to identify ligands similar to msc1 ( scoring > 0.90 on 
 the 
 tanimoto coefficient ) or that have a 2d structure that is 90% similar 
 to the new pubchem ligand msc1 and satisfy specific constraints coming 
 from the cb1 binding site . the 16 ligands found by using pubchem in 
 this protocol are denoted by the acronym hek293 cells were maintained in 
 dulbecco s modified eagle s 
 medium supplemented with 10% fetal bovine serum and 3.5% mg / ml glucose 
 at 37 c in 5% co2 . one day prior to transfection , 
 cells were seeded at approximately 1 million cells/100 mm dishes . the cells were transiently transfected by the calcium phosphate precipitation 
 method . at 24 h post - transfection , the 
 cells were harvested in phosphate buffered saline ( pbs ) containing 
 mammalian protease inhibitor cocktail ( ( 42-aminoethyl)benzene - sulfonyl 
 fluoride , pepstatin a , e-64 , bestatin , leupeptin , and aprotinin ) ( sigma - aldrich , 
 st . louis , mo ) and lysed by nitrogen cavitation at 750 psi for 5 min . the lysate was spun at 500 g for 10 min at 4 c , and the supernatant 
 was subsequently spun at 100,000 g for 45 min at 4 c . the membrane - containing 
 pellet was resuspended in tme buffer ( 25 mm tris - hcl , 5 mm mgcl2 , 
 and 1 mm edta , ph 7.4 ) containing 7% w / v sucrose . competition 
 binding assays were performed as described previously to determine the binding affinity of the test compounds 
 to the receptor . briefly , 6 g of membrane preparation was incubated 
 for 60 min in tme buffer containing 0.1% fatty acid - free bsa with 
 a fixed concentration of tracer [ h]sr141716a ( 43 ci / mmol , 
 perkinelmer life sciences ( boston , ma ) ) typically at its kd which was determined from a saturation binding isotherm . at least nine concentrations of the unlabeled test compound ( ranging 
 between 100 pm and 32 m ) were used for the binding assays . nonspecific binding was determined in the presence of unlabeled sr141716a 
 ( 1 m ) . the reaction was terminated by filtration with a brandell 
 cell harvester through whatman gf / c filter paper , and the radioactivity 
 was measured . briefly , 6 g of membranes was incubated 
 for 60 min at 30 c in gtps binding assay buffer ( 50 mm 
 tris - hcl , ph 7.4 , 3 mm mgcl2 , 0.2 mm egta , and 100 mm nacl ) 
 with the unlabeled 2-arachidonoylglycerol ( 2-ag ) ( at least nine different 
 concentrations were used ranging between 100 pm and 1 m ) , 0.1 
 nm [ s]gtps ( 1250 ci / mmol ; perkinelmer life sciences , 
 boston , ma ) , 10 m gdp ( sigma , st . louis , mo ) , and 0.1% ( w / v ) 
 bsa in the absence and presence of 10 m test compounds . the 
 effect of the compound on inhibiting the level of basal gtps 
 binding was evaluated in the absence of agonist . nonspecific binding 
 was determined with 10 m unlabeled gtps ( sigma , st . after rapid filtration through whatman gf / c filters the 
 radioactivity trapped in the filters was determined by liquid scintillation 
 counting . data are presented as the mean  se or 
 the mean with the corresponding 95% confidence limits from at least 
 three independent experiments . the ki values 
 of the test compounds were calculated by nonlinear regression using 
 prism 6.0 ( graphpad software inc . , san diego , ca ) . hek293 cells 
 expressing the cb1 receptor c - terminally fused to gfp were seeded 
 onto 35 mm glass - bottomed dishes ( mattek , ma ) precoated with poly - d - lysine . cells were treated with 10 m msc compounds 
 or 1 m rimonabant for various lengths of time and then washed 
 three times with pbs , followed by fixation with 4% paraformaldehyde 
 for 10 min at room temperature . cells were mounted in vectashield 
 mounting medium ( vector laboratories , ca ) and visualized using a leica 
 tcs sp2 confocal microscope ( leica microsystems , wetzler , germany ) . images were collected from at least three independently transfected 
 cell dishes and processed for presentation in figures using adobe 
 photoshop 6.0 ( adobe systems , san jose , ca ) . the predicted lowest energy pose 
 for the cb1-rimonabant complex is shown in figure 1a , and the corresponding pharmacophore is 
 shown in figure 1b . we predict that rimonabant is anchored by hydrogen bonds to w5.43 
 and k3.28 [ residues are numbered according to the ballesteros weinstein 
 scheme . ] , and indeed previous site - directed 
 mutagenesis data indicate that mutations of w5.43 and k3.28 to alanine 
 have the largest effect in decreasing experimental binding affinity 
 upon mutation to alanine with > 1000-fold and 17.2-fold , respectively . the components of the predicted binding energies for this complex 
 are shown in table 1 , showing contributions from each residue in the binding site . the 
 most important residue is w5.43 with a predicted binding contribution 
 to rimonabant of 9.36 kcal / mol , consistent with the decrease 
 by a factor of > 1000 upon mutation to alanine . indeed our predicted pharmacophore for rimonabant ( figure 1b ) has the significant 
 polar and hydrophobic contacts with the cb1 receptor expected for 
 a strongly bonding ligand . according to the cavity analysis 
 in table 1 , k3.28 has 
 the third largest binding interaction energy ( 5.57 kcal / mol ) 
 with rimonabant due to its hydrogen bond with the amide carbonyl of 
 the ligand . the same experiments with the rimonabant derivative 
 vchsr , an analogue of rimonabant with hydrocarbons replacing the amide 
 and piperidine groups , showed that the binding affinity remained unaffected 
 upon mutation of k3.28 to alanine . our study shows that k3.28 interacts 
 with the polar atoms of the amide group or piperidine ring , including 
 the carbonyl , which agrees with the mutagenesis study on this ligand 
 that lacks these polar atoms . in 
 our docked pose of rimonabant with the wt receptor , the f3.36 
 residue has a significant interaction with the ligand , including a 
 van der waals component of 3.90 kcal / mol . in binding assays , the f3.36a mutation decreased the binding affinity of this inverse 
 agonist by 20-fold , or 1.78 kcal / mol , 
 for the cb1 receptor . interestingly , the f3.36l mutation decreased 
 the binding affinity by only 2-fold , indicating the importance of 
 the bulky hydrophobic residue for receptor  ligand interaction . ( a ) the predicted 
 structures for inverse agonist rimonabant to 
 wt6 , the sixth best energetic conformation of the cb1 receptor . ( b ) our predicted 
 pharmacophore shows that in our gensemble - derived cb1 structure rimonabant 
 forms two hydrogen bonds , including one with w5.43 , and have strong 
 aromatic interactions with the receptor . only residues with energies with 
 contributions stronger than 1.0 kcal / mol are shown . the nonbonding 
 ( nonbond ) energy 
 in the far right column is the sum of the van der waals ( vdw ) energy , 
 the electrostatic ( coulomb ) energy , and hydrogen bond ( h - bond ) energy . we predicted that m6.55 has the second strongest interaction ( 6.8 
 kcal / mol ) with cb1 , which is almost entirely hydrophobic ( table 1 ) . however , experiments 
 show that mutating m6.55 to alanine decreases the binding affinity 
 to cb1 by only 3-fold or by 0.65 kcal / mol 
 in energy . one possible explanation is that the m6.55a mutation alters 
 the binding site and pose for rimonabant in a way that other residues 
 become available to compensate for the lost interaction with methionine . a very important advantage of the gensemble 
 approach is that we have an ensemble of ten low energy structures 
 to which rimonabant is allowed to bind . we showed for the adenosine 
 a3 receptor that neither the selective agonists nor the selective 
 antagonists prefer the lowest energy apoprotein structures . also , for ccr5 we showed that the antagonists 
 all prefer to bind to structures that are not the lowest energy for 
 the apoprotein . similarly for cb1 , the 
 most favorable binding is with wt6 , the sixth best conformation for 
 the apo - cb1 structure , not with the lowest energy apo - cb1 conformation 
 ( wt1 ) ( table 2 ) . since 
 the ligands do not preferentially bind to the lowest energy conformation , 
 these docking results support the concept that gpcrs are highly dynamic 
 structures that can sample many conformations , providing an opportunity 
 for different ligands that can selectively bind to different conformations , 
 and perhaps lead to different function . the ten cb1 conformations 
 used in this study range in backbone rmsd from 0.42.1  . 
 of course the protein conformation may change when complexed with 
 the ligand . surprisingly , the wt6 cb1 structure , to which the 
 inverse agonist rimonabant preferentially binds , lacks the r3.50 and 
 d6.30 ionic lock , which is generally believed to be important for 
 preventing activation . experimental data have shown that rimonabant 
 inactivates the cb1 receptor , while our calculations 
 find that the inverse agonist has the best binding energies with activated 
 cb1 conformations . indeed of the top four rimonabant - cb1 complexes , 
 we find that three conformations ( wt6 , wt1 , and wt5 ) do not form an 
 ionic lock to the conserved r3.50 and d6.30 . the complexes are 
 ranked according 
 to best binding energy with strain and ligand solvation included . receptor conformation numbers 
 that 
 do not contain the conserved r3.50 and d6.30 salt bridge are in bold . on the other hand , several 
 studies have suggested that a r3.50 
 to d6.30 salt bridge is not necessary for maintaining the inactive 
 conformation . for example , audet and bouvier showed that the r3.50 and d6.30 salt bridge is broken in all but 
 three of the 36 crystallized gpcr structures bound to antagonists 
 and inverse agonists analyzed . it should be noted that many crystallized 
 gpcr structures , such as the human adenosine a2a receptor , 
 contain an inserted t4 lysozyme for stabilization , which may impact 
 the conformation of the cytocellular ends of the helices and thus 
 prevent the formation of the ionic lock . in our previous work with the human cb1 receptor , we showed 
 that 
 a different residue , r2.37 , plays an important role in preventing 
 receptor activation . we found that this residue forms an  ionic lock  via 
 a salt bridge interaction with d6.30 to stabilize the inactive conformation . indeed our calculations found that the rimonabant inverse agonist 
 did not prefer to bind to the conformations that contained this signature 
 contact as shown in table 2 . the rimonabant - bound cb1 conformation wt6 resembles the 
 predicted structure of a constitutively active mutant , the two structures have a c-rmsd of 1.2  , which is 
 smaller than most crystal structure resolutions suggesting that the two conformations are very similar . 
 for comparison , the difference between the wt receptor conformations 
 wt2 ( which contains the r3.50+d6.30 salt bridge ) and wt6 ( without 
 that salt bridge ) is 1.8  . to validate our proposed 
 binding site , we docked seven known derivatives of rimonabant ( figure 2a ) to the cb1 conformation 
 and plotted the calculated binding energies against the experimental 
 binding affinity , pki , to find the correlation 
 between the two data sets ( figure 3 ) . for each ligand , we found the same binding site 
 as for rimonabant ( figure 2b ) . figure 3a shows a comparison between the pki of 
 the inverse agonists from hurst et al . and our calculated binding energies . we observe a very high correlation 
 of 93.4% , indicating good agreement between the trends observed in 
 experimental binding affinities and our own calculated binding energies . the portions of the ligand 
 that are different from rimonabant are colored in red . the derivatives 
 in the first two rows were used in the hurst et al . , 2006 study , and the derivatives in the third row were used 
 in the dantona et al . comparison of experimental binding affinities and computationally 
 calculated energies between receptor and inverse agonists . ( a ) plot 
 showing the correlation of the experimental binding affinity , pki , for the six inverse agonists in hurst et 
 al . this leads to r = 0.93 , 
 indicating that the error bar for our calculations is 2 kcal / mol , 
 while the range is 18 kcal / mol . ( b ) plot showing the correlation of 
 the g for seven previously 
 published inverse agonists with respect to that of rimonabant versus the calculated binding energy . r = 0.89 , indicating that the error bar for our calculations is 2 
 kcal / mol , while the range is 14 kcal / mol . for the inverse agonists from dantona et al . the gbindexp values are within 1 kcal / mol of each other since 
 the energies are 9.33 kcal / mol for am-281 , 10.0 kcal / mol 
 for am-251 , and 9.70 kcal / mol for rimonabant . similarly , both 
 the calculated cavity and binding energies for the three ligands are 
 approximately within 1 kcal / mol . the binding energies are 67.4 
 to 69.0 kcal / mol , which are very similar and agree with the 
 experimentally observed negligible changes in free energy upon binding . this excellent correlation relies on the predicted energies that are 
 based purely on enthalpy and do not include entropy . however , the 
 entropy change upon binding of all the ligands should be similar since 
 they all have the same number of rotatable bonds . figure 3b combines 
 the results from figure 3a with those regarding the inverse agonists from dantona 
 et al . for a single comparison of our 
 calculated binding energies and the binding affinities from the two 
 experiments . the respective g values from eq 2 are 
 plotted against the calculated binding energies for all eight ligands , 
 leading to an 89.4% correlation . the excellent agreement between our 
 predicted energies and the experimental binding affinity shown in figure 3b provides confidence 
 that our predicted binding site for rimonabant is reasonable . with 
 a reliable rimonabant binding pharmacophore for cb1 , which 
 is supported by site - directed mutagenesis and sar data , we aimed to 
 design a new , more selective cb1 inverse agonist . after the informed 
 search in pubchem , described in the methods section , we selected zinc08587042 , which 
 we refer to as msc1 ( figure 4 ) , a ligand with a 68% similarity to rimonabant according 
 to the 2d tanimoto coefficient . rather 
 than relying exclusively on pubchem s search algorithm , which 
 looks for molecules based on a 0.90 tanimoto similarity score with 
 respect to rimonabant , we used the predicted cb1 binding site and 
 searched for a ligand that maintained key structural aspects of rimonabant 
 and additionally took advantage of underused residues in the binding 
 site . for example , we wanted to find a ligand having the same amide 
 and pyrazole functional groups to maintain the hydrogen bonds with 
 k3.28 and w5.43 , respectively . yet , we wanted to replace the rimonabant 
 piperidine ring with a phenyl ring and a substituted polar group to 
 improve the interactions with the aromatic residues that we found 
 nearby and to gain an additional hydrogen bond with k7.32 , which is 
 within the rimonabant binding site . furthermore , we wanted to replace 
 the methyl group of the pyrazole group with a polar substitute to 
 form a hydrogen bond with s7.39 . msc1 is attractive because it contains 
 an acetylphenyl group , which we predicted would reach into the aromatic 
 pocket of the extracellular end , and a triazole ring , which would 
 replace a methyl group with a nitrogen atom . we also wanted a small 
 molecule that was commercially available and that had not been previously 
 tested with cb1 in bioactivity assays . portions of msc1 highlighted in red 
 are different from the corresponding groups in rimonabant . pubchem also identified 15 other small molecule 
 ligands that have 
 a 2d structural similarity tanimoto score of 0.90 with msc1 or have 
 2d structures that are 90% similar to that of msc1 ( figure 5 ) . we docked these 16 ligands 
 to the ensemble of predicted cb1 structures , found their corresponding 
 binding energies , and predicted their respective pki values based on figure 3 . of the 
 16 msc small molecules , 14 compounds , msc1 msc6 , msc8msc11 , 
 and msc13msc16 were commercially available . pubchem identified 
 15 ligands that have 2d structures similar to msc1 . from the 16 compounds 
 identified from the computational analyses , we chose five compounds 
 based on the 2d structural similarity , tanimoto score , and commercial 
 availability . to evaluate 
 experimentally the binding affinities of the compounds , we performed 
 competition binding experiments using [ h ] rimonabant as 
 a tracer . msc1 , msc3 , and msc9 bound the receptor with ki values of 502 nm , 495 nm , and 4619 nm , respectively 
 ( table 3 ) . these data 
 indicate that while removal of the chloro group from the chlorophenyl 
 ring of msc1 does not impact receptor binding ( msc1 versus msc3 ) , 
 the position of the methyl group in one of the biphenyl rings and 
 the acetyl group in the amide - linked phenyl ring is critical for receptor 
 binding ( msc3 versus msc9 ) . msc5 and msc8 failed to bind cb1 up to 
 32 m ( table 3 ) . unexpectedly , our experimental ki values 
 suggested a weaker affinity than rimonabant compared to the predictions . 
 this may be due to the limitations of force fields to reliably predict 
 relative binding affinities . data are the median and corresponding 95% 
 confidence limits of three independent experiments performed in duplicate . since the experimental binding affinities to the cb1 receptor were 
 higher for msc1 and msc3 than for msc5 , msc8 , and msc9 , we further 
 tested msc1 and msc3 using [ s]gtps binding assays 
 to evaluate changes in g - protein coupling . we assessed the effect 
 of the compounds on the basal and the 2-ag - induced gtps binding . we chose 2-ag , the endogenous eicosanoid cb1 agonist for the assay 
 since it showed binding affinity comparable to msc1 and msc3 . the addition 
 of 10 m of msc1 and msc3 resulted in substantial reduction 
 in emax values with 90 fmol / mg and 81 
 fmol / mg , respectively , compared with that in the absence of these 
 msc compounds ( emax = 126 fmol / mg ) ( figure 6a ) . these compounds 
 also exhibited small but not statistically significant shifts in ec50 values ( 785 nm for msc1 , 770 nm for msc3 , compared to 521 
 nm for the absence of any msc compound ) . thus , the extent of g protein 
 coupling ( emax values ) changes , though 
 the amount of agonist needed to achieve this level of gtps 
 binding ( ec50 values ) does not . furthermore , figure 6b showed that both msc1 and 
 msc3 substantially decreased the basal [ s]gtps 
 binding ( 67 fmol / mg ) with specific binding of 50 fmol / mg and 47 fmol / mg , 
 respectively . whereas prolonged agonist treatment can remove 
 receptors from the 
 cell surface by endocytosis , inverse agonists have been shown to enhance 
 the cell surface localization , consistent with receptor inactivation . cb1 exhibits some constitutive activity , and the majority of cb1 
 receptors are localized on intracellular vesicles even in the absence 
 of agonist . we tested if the msc compounds can affect cellular localization 
 of the receptor using confocal microscopy of cells expressing gfp - tagged 
 cb1 . similarly to rimonabant treatment , upon treatment with 10 m 
 msc1 or msc3 the level of cell surface localization of the receptor 
 was enhanced though the extent for msc3 is less than msc1 ( figure 6c ) . collectively , 
 although msc1 and msc3 exhibited somewhat lower potency and efficacy 
 compared to rimonabant , these data suggest that they possess inverse 
 agonist properties as evidenced by reduction in g - protein coupling 
 and enhancement of cb1 cell surface expression . we took the msc1-bound 
 cb1 structure and relaxed it in an explicit lipid bilayer environment 
 using 50 ns of molecular dynamics ( md ) simulations . isobaric conditions ( npt ) at 
 310 k and 1 atm , using periodic boundary conditions with particle - mesh 
 ewald summation method for calculating long - range coulomb interactions . the temperature is controlled using langevin dynamics , and the pressure 
 is maintained using a langevin - hoover barostat . these simulations 
 showed that binding of msc1 to the cb1 receptor constrains tm helix 
 6 more through additional intracellular couplings preventing the g - protein 
 from engaging the receptor , which is consistent with experimental 
 findings of msc1 ligand s inverse agonism . these results offer 
 opportunities for developing new cb1 inverse agonists through the 
 optimization of chemical functionalities of the compounds and further 
 pharmacological characterization . effect of msc1 and msc3 on the [ s]gtps binding 
 to hek293 cell membranes expressing the cb1 wt receptor . 2-ag - induced [ s]gtps binding in the 
 absence (  ) or presence of 10 m (  ) msc1 and 
 msc3 . the levels of untransfected cells ( no cb1 ) and rimonabant 
 treated samples are shown for comparison . all data are the mean  se of at least three independent experiments performed in 
 duplicate . ( c ) cellular distribution of the wt receptor upon treatment 
 with msc compounds . hek293 cells expressing the cb1 wt - gfp receptor 
 were incubated with vehicle alone ( 0.03% dmso ) , msc1 ( 10 m ) 
 or msc3 ( 10 m ) for 6 h. rimonabant - treated ( 1 m ) cell 
 is shown for comparison . our predicted rimonabant binding site 
 in cb1 is different from previous computational studies that used 
 the bovine rhodopsin template to create a cb1 homology structure for 
 docking rimonabant.supporting information figure s1 compares the rimonabant pharmacophore published by lange 
 and kruse ( figure s1a ) with the pharmacophore from our current study ( figure s1b ) . both agree that rimonabant ( figure s1a ) spans the width of the binding site 
 with the chlorophenyl rings near tm5 and the piperidine ring near 
 tm3 . however , we predict that rimonabant ( figure s1b ) lies parallel to the z - axis passing from 
 the extracellular to intracellular side of the membrane , which is 
 perpendicular to the previous models ( figure s1a ) . although both agree that a hydrogen bond forms between k3.28 and 
 the amide carbonyl of rimonabant , the earlier models suggest that 
 k3.28 also participates in a salt bridge with d6.58 , which we do not observe . in addition , 
 the previous models have the w5.43 residue sandwiched by the two chlorophenyl 
 groups of rimonabant , leading to strong aromatic interactions . furthermore , 
 multiple other aromatic residues in the binding pocket ( f3.25 , f3.36 , 
 w4.64 , y5.39 , w6.48 ) were found to participate in stacking with the 
 ligand , whereas in 
 our predicted binding site , it is w5.43 that has the biggest impact 
 on binding affinity , from the hydrogen bond with the pyrazole ring 
 of rimonabant . in our predicted structure , we find that the f3.36 
 residue is sandwiched by the chlorophenyl groups rather than w5.43 . in addition , a paper from shim et al . based its cb1 model on the human 
 2 adrenergic receptor to identify a binding site similar to the one from lange and krause but with the ligand lying perpendicular to our 
 predicted structure , with the piperidine ring pointing to the tm127 
 binding pocket and the chlorophenyl rings pointing toward tm5 , where 
 they form aromatic stacks with w5.43 and w6.48 . one would expect 
 our predicted rimonabant binding site to be different from the previously 
 published ones since our predicted receptor structures are quite different . specifically , we sample all rotations of all helices about their axes 
 and helix tilt and sweep angles , a total of nearly 13 trillion receptor 
 conformations . in contrast , the previous studies used homology models 
 based on the bovine rhodopsin or human 2 adrenergic 
 receptor structure templates , so that no rotations or tilting of the 
 helices were conducted . these three gpcrs have very similar global 
 conformations , but we performed considerable sampling to obtain cb1 
 receptor conformations that were significantly different and more 
 stable than the starting template . the method we used to identify our final receptor - inverse 
 agonist complex is also different . the study from shim et al . used the selection criterion that one of the 
 two chlorophenyl rings should be near the tm356 hydrophobic 
 site , so their final pose was chosen based on the interactions of 
 rimonabant with residues for which there was site - directed mutagenesis 
 data . however , the structures in our study were based on the binding 
 energy of the complexes , with no extra conditions imposed . the previously 
 suggested rimonabant binding pose most similar to ours came from another 
 study in which the ligand was docked to a bovine rhodopsin structure - based 
 homology model . this predicted binding 
 site is similar to the current model , with rimonabant sitting vertically 
 in the binding pocket the piperidine ring points toward the 
 extracellular end and the chlorophenyl rings point toward the intracellular 
 end . however , it does not lead to any hydrogen bonds with w5.43 and 
 finds a hydrogen bond with y5.39 , that we did not observe . since many antiobesity drugs including 
 rimonabant have been suspended 
 from the market , there remains an enormous unmet need for compounds 
 that reduce food intake . in an effort to develop novel inverse agonists 
 for cb1 , we predicted the binding sites and energies for rimonabant 
 and structurally related molecules to our previously predicted and 
 validated cb1 receptor conformations . our calculated binding energies 
 for the compounds are in good agreement with the previously reported 
 mutagenesis and sar data . based on the predicted binding site of these inverse agonists , we 
 identified 16 new potential cb1 inverse agonists . we tested five of 
 these experimentally and found that two of them ( msc1 and msc3 ) antagonized 
 g - protein coupling and enhanced surface localization of the receptor 
 suggesting they are inverse agonists . the md simulations of msc1:cb1 
 complex in explicit lipid bilayer environment show that the relaxed 
 receptor exhibits additional intracellular couplings that will prevent 
 g - protein coupling , which is consistent with the characterization 
 of msc1 as an inverse agonist . although msc1 and msc3 are less potent 
 than rimonabant , they provide new starting points for developing new 
 candidates for antiobesity drugs that target cb1 .