3D-QSAR CoMFA study of benzoxazepine derivatives as mGluR5 positive allosteric modulators

Metabotropic glutamate receptor subtype 5 (mGluR5) belongs to a family of G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes 1-3. Prevailing cellular studies increasingly demonstrate a positive association between N-methyl D-aspartate (NMDA) receptors and mGluR5, suggesting that activation of mGluR5 potentiates NMDA receptor function4-6. Given evidence which suggests the role of hypofunction of NMDA receptors in the manifestation of the positive, negative, and cognitive symptoms of schizophrenia, potentiation of mGluR5, specifically through its allosteric binding site, is considered a possible avenue for ameliorating the symptoms of this debilitating disease5, 7. HEK293 cells expressing rat mGluR5 were cultured and plated as previously described8. The cells were loaded with a Ca2+-sensitive fluorescent dye and the plates were washed and placed in the Functional Drug Screening System (Hamamatsu). Test compound was applied to cells 3 s after baseline readings were taken. Cells were incubated with the test compounds for 140 s then stimulated with an EC20 concentration of glutamate and readings taken for an additional 60 s. Allosteric modulation by the compounds was measured by comparing the amplitude of the responses at the time of glutamate addition plus and minus test compound. A comparative molecular field analysis (CoMFA)9, 10 was conducted on 118 benzoxazepine derivatives which act as positive allosteric modulators for mGluR5. Briefly, CoMFA correlates sterics and electrostatics to the bioactivity data establishing a three-dimensional quantitative structural activity relationship (3D QSAR). The biological data for the 118 benzoxazepine derivatives investigated in the present study resulted from validated hits of a high-throughput screen (HTS)88. Corresponding pEC50 values were derived from the reported bioactivity values (pEC50=-log(EC50)) and used as the dependent variables in the CoMFA analysis. Geometries for the benzoxazepine derivatives were generated using the Corina software package11, 12. Prior to CoMFA analysis, molecular alignment was achieved by the SYBYL routine database align method, whereby the superimposition of molecules was based on minimizing root-means-squares (rms) differences in the fitting of selected atoms with those of a template molecule. All molecules were aligned based on the assumption that they bind to the allosteric site of mGluR5 in the same manner, wherein, all atoms of the benzoxazepine moiety were used as the structural element for superimposition. Partial atomic charges were calculated using the Gasteiger-Marsilli method13. Steric and electrostatic interactions were calculated using the Tripos force field14 using a 2 A grid spacing, while a sp3 carbon atom and +1 charge were used as the steric and electrostatic probes, respectively. All CoMFA calculations were performed using electrostatic fields with Lennard-Jones and Coulombtype potentials, a dielectric constant of 1/r, and a cutoff of 30 kcal mol-1. Regression analysis was executed using the leave one out (LOO) cross-validated partial least squares (PLS) method15 with a minimum sigma of 2.0 kcal/mol to improve the signal to noise ratio. LOO-PLS was thus used to ascertain the predictive ability of the models and to determine the optimal number of components to be used in the final QSAR models, which were derived from the non cross-validated, conventional analysis. All calculations were performed using Sybyl 7.316 on a Dell Precision 380 workstation. Initially, alignment of the compound set afforded a median q2 of 0.45. Outliers based on residual value (molecules 45 and 78 in supp. mat.) were omitted and region focusing using Determinant Power as a weight was implemented to yield an improved q2 of 0.51. Region focusing corresponds to rotating the model components through a high-order space, and thus refining the model by increasing the weight for the lattice points which are most pertinent to the model. This enhances the resolution and predictive power of a corresponding PLS analysis17. Thereby the cross-correlated “brown” noise in the data matrix is reduced. This procedure was accomplished through SYBYL PLS region focusing which selectively re-weights the grid points in a region to create a new CoMFA model. Two flavors of region focusing were tested: 1) Weights were derived for each lattice point according to the fraction of the variation of the model's components attributable to each lattice point (menu option “Discriminate Power”). 2) Weights were assigned to each lattice point according to the product of the variation at each lattice point and the lattice point's regression coefficient from the model (menu option “Stdev*Coefficients”).The accuracy of prediction of CoMFA models has been shown to depend strongly on the geometry and structural alignment of molecules18. To test influence of conformation and superimposition on the present model, the 118 benzoxazepine structures were geometry optimized at the HF 6-31G* level of theory using Firefly19 Usage of the resulting conformations yields an improved q2 of 0.53 with no observed outliers (Table 1). Ultimately, the model with the highest predictive power was obtained using the CoMFA standard model with region focusing. Variance×coefficients were assigned as weight values, through which significant cross-validated correlation coefficients, a q2 of 0.58 and r2 of 0.81, were retained. The graph of the actual pEC50 versus the predicted pEC50 values for this model demonstrates that these values are in good agreement, and compared to the model without region focusing, shows an improvement. Table 1 Summary of PLS analyses of 3 CoMFA sets Most of the variation in the data set resides at the R1 position (Figure 2). Thus, a model was constructed with the R1 groups aligned in an attempt to reduce noise in the model. The best model with this alignment, q2 of 0.37 and r2 of 0.78, was achieved with region focusing with weight by “Discriminant Power.” Figure 2 A plot of the 118 benzoxazepine derivatives after geometry optimization, overlapped for the CoMFA analysis to illustrate the variability at R1. Oxygen is shown in red, carbon in silver, hydrogen in cyan, nitrogen in blue, chlorine in green, and sulfur ... The CoMFA contours of the best model (Figure 4) reveal that primarily bulky substituents are required at the meta position of the 1,2,4-triazine ring (R3). With most analogs containing a ring-substitution at the R1 position, the contour maps reflect the preference for electropositive character proximal to the acyl carbonyl oxygen at the R2 position of the oxazepine moiety. The thiophene group at the R1 position of the most potent mGluR5 potentiator (Figure 1b) reflects the ambivalent electrostatic character with electropositivity found along the aromatic carbons and electronegativity due to the sulfur on the opposite side of the ring. Figure 1 (a) Base structure of the benzoxazepine analogs examined. (R1) predicted to prefer ambivalent electronegative and electropositive character with low steric bulk, (R2) no signal, and (R3) predicted to prefer high steric bulk Figure 4 Electrostatic (a) and steric (b) contour plots of the best CoMFA model, respectively. Areas favoring steric bulk are indicated by green polyhedra (contribution level 80%), while areas where steric bulk is unfavorable are represented by yellow polyhedra ... To better assess the predictive power of the models constructed, an independent data set was created containing 18 molecules from the original training set. A new model was constructed using the parameters of the best model (StDev*Coefficient) with the remaining 100 molecules as a training set. The non cross-validated QSAR model was then used to predict the activities of the independent test set. The model produced a good predictive r2 value of 0.88 for this validation test set. In conclusion, we present here the first example in which a comparative molecular field analysis (CoMFA) based on 3D-QSAR was conducted on 118 benzoxazepine derivatives shown to positively modulate mGluR5 allosterically. The final CoMFA model, derived with region focusing, where variance×coefficients were assigned as weight values, suggests the potential, statistical significance of this class of compounds. The resulting contour maps afford insight into the features vital for the design of efficacious benzoxazepine-based analogs as positive allosteric modulators for mGluR5. Specifically, the analysis suggests bulky substituents at the meta position of the 1,2,4-triazine ring (R3), and substituents at the 2 position of the oxazepine moiety (R1) with both an electropositive and an electronegative functional group are preferred.