NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.

Pharmacological evaluation of iodo and nitro analogs of delta 8-THC and delta 9-THC.

One aspect of cannabinoid structure-activity relationships (SARs) that has not been thoroughly investigated is the aromatic (A) ring. Although halogenation of the side chain enhances potency, our recent observation that iodination of the A ring also enhanced activity was surprising. The purpose of this investigation was to establish the steric and electrostatic requirements at these sites of the cannabinoid molecule via molecular modeling, while determining pharmacological activity. Molecular modeling was performed using the Tripos molecular mechanics force field and the semiempirical quantum mechanical package AM1. The Ki values for novel cannabinoids were determined in a [3H]CP-55,940 binding assay and ED50 values generated from four different evaluations in a mouse model. The present studies underscore the increase in potency produced by a dimethylheptyl (DMH) side chain. Trifluoro substitutions on the pentyl side chain, or bromination of the DMH side chain, had little effect on the pharmacological activity. Any substitution at the C4 position of the aryl ring resulted in a loss of activity, which appears to be due to steric hindrances. Nitro, but not iodo, substitution at the C2 position essentially produces an inactive analog, and the drastic alteration of the electrostatic potential appears to be responsible. The altered pharmacological profile of the 2-iodo analog seems to be related to an alteration in the highest occupied molecular orbital because there is no alteration in the electron density map compared to delta 8-tetrahydrocannibinol.

HINT: a new method of empirical hydrophobic field calculation for CoMFA.

An empirical hydrophobic field-like 3D function has been calculated with the program HINT (hydrophobic interactions) and imported into the SYBYL implementation of CoMFA (Comparative Molecular Field Analysis). The addition of hydrophobicity appears to offer increased chemical interpretability of CoMFA models. An example is given using the steroid model reported by Cramer et al. (J. Am. Chem. Soc., 110 (1988) 5959). While addition of the HINT field did not improve statistical parameters in this model, the CoMFA coefficient contours from the hydrophobic field unambiguously define the most active steroid molecules in the chemical terms of hydrophobic and polar substituents.

Modeling the cannabinoid receptor: a three-dimensional quantitative structure-activity analysis.

The structure-activity relationship studies that have been reported for cannabinoids suggest that 1) the conformation of the C-ring at the C9 position, 2) the A-ring phenolic hydroxyl, and 3) the hydrophobic side chain are important determinants for the production of analgesia, as well as other cannabinoid effects. However, either these previous structure-activity studies described for cannabinoid compounds have not been quantitative in nature or the prediction of the activity of known and unknown compounds based on molecular structure has not been tested in a comprehensive manner. In this study we describe a three-dimensional molecular modeling program using comparative molecular field analysis to derive quantitative structure-activity relationships fitting pharmacological potencies and binding affinities of cannabinoids. The analysis has proven to accurately fit the pharmacological activity of cannabinoid analogs, with cross-validated r2 values of greater than 0.3 and final analysis r2 values of greater than 0.88. Additionally, this study has further characterized the steric and electrostatic properties that account for the variations in their potency. The results from this study indicate that steric repulsion behind the C-ring is associated with decreased predicted binding affinity and pharmacological potency. On the other hand, the steric bulk of a side chain that is extended up to seven carbons contributes to predictions of increased binding affinity and potency. The electrostatic fields of cannabinoid analogs also contribute to the predicted in vitro and in vivo potencies. If the biological activities we have investigated are assumed to be the result of interaction with a single binding site, this method indicates the structural and physicochemical properties necessary for binding to the receptor and producing an effect. By defining cannabinoid binding affinity and behavioral activity pharmacophores, this method can be used for designing cannabinoid agonists and it is capable of predicting the activity of unknowns, thereby serving to facilitate rational drug design.

A computergraphic investigation into the pharmacological role of the THC-cannabinoid phenolic moiety.

There has been much debate as to the nature of the cannabinoid-receptor interaction, whether by traditional interpretation or by means of membrane perturbation. Whichever hypothesis is correct, the structural requirements for pharmacological action need determination. Here, we report a computergraphic investigation into the role of the phenolic hydroxyl moiety in such a receptor interaction. It has been determined by molecular mechanics energy calculations and volume map determinations that the proton of the hydroxyl group may be involved in hydrogen bonding at the putative receptor site.

A computergraphic determination of the chemotactic peptide preferred conformation.

Replacement of leucine in the chemotactic peptide For-Met-Leu-Phe by the sterically constrained amino acids alpha-aminoisobutyric acid and aminocyclohexanecarboxylic acid affords compounds of equal or greater activity than the parent. NMR studies indicate that the parent compound is present as a beta-sheet in solution, whereas the analogues prefer a beta-turn. Application of molecular modelling would indicate that the beta-turn conformer is energetically preferable and thus suggests that it is the orientation adopted by the peptides.