A new 4-point pharmacophore method for molecular similarity and diversity that rapidly calculates all potential pharmacophores/pharmacophoric shapes for a molecule or a protein site is described. The method, an extension to the ChemDiverse/Chem-X software (Oxford Molecular, Oxford, England), has also been customized to enable a new internally referenced measure of pharmacophore diversity. The "privileged" substructure concept for the design of high-affinity ligands is presented, and an example of this new method is described for the design of combinatorial libraries for 7-transmembrane G-protein-coupled receptor targets, where "privileged" substructures are used as special features to internally reference the pharmacophoric shapes. Up to 7 features and 15 distance ranges are considered, giving up to 350 million potential 4-point 3D pharmacophores/molecule. The resultant pharmacophore "key" ("fingerprint") serves as a powerful measure for diversity or similarity, calculable for both a ligand and a protein site, and provides a consistent frame of reference for comparing molecules, sets of molecules, and protein sites. Explicit "on-the-fly" conformational sampling is performed for a molecule to enable the calculation of all geometries accessible for all combinations of four features (i.e., 4-point pharmacophores) at any desired sampling resolution. For a protein site, complementary site points to groups displayed in the site are generated and all combinations of four site points are considered. In this paper we report (i) the details of our customized implementation of the method and its modification to systematically measure 4-point pharmacophores relative to a "special" substructure of interest present in the molecules under study; (ii) comparisons of 3- and 4-point pharmacophore methods, highlighting the much increased resolution of the 4-point method; (iii) applications of the 4-point potential pharmacophore descriptors as a new measure of molecular similarity and diversity and for the design of focused/biased combinatorial libraries.

A series of omega-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3beta-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C).oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.

Prostanoids play important physiological roles in the cardiovascular and immune systems and in pain sensation in peripheral systems through their interactions with eight G-Protein Coupled Receptors. These receptors are important drug targets, but development of subtype specific agonists and antagonists has been hampered by the lack of 3D structures for these receptors. We report here the 3D structure for the human DP G-Protein Coupled Receptor (GPCR) predicted by the MembStruk computational method. To validate this structure we use the HierDock computational method to predict the binding mode for the endogenous agonist (PGD2) to DP. Based on our structure, we predicted the binding of a new family of antagonists which has been confirmed experimentally.We find that PGD2 binds vertically to DP in the TM1237 region with the α chain toward the extracellular (EC) region and the ω chain toward the middle of the membrane. This structure explains the selectivity of the DP receptor and the residues involved in the predicted binding site correlate very well with available mutation experiments on DP, IP, TP, FP, and EP subtypes. We report molecular dynamics of DP in explicit lipid and water and find that the binding of the PGD2 agonist leads to correlated rotations of helices of TM3 and TM7, whereas binding of antagonist leads to no such rotations. Thus these motions may be related to the mechanism of activation.

Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporated into the shortest fragment of hPTH with recognized efficacy in the OVX rat model of osteoporosis, hPTH-(1-31)NH 2 (1). Cyclo(Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ]hPTH(1-31)NH 2 (2) is a potent agonist of the PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulate adenylyl cyclase activity (EC 50 ) 0.29 nM). A second analogue, which constrains the entire C-terminal receptor binding domain, bicyclo(Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ] hPTH(1-31)NH 2 ( 6), is also shown to be a potent agonist (EC 50 ) 0.13 nM), thus providing further evidence for an extended helix as the relevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated into the analogue bicyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (7) to evaluate the receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is also a highly potent agonist (EC 50 ) 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactive form of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporation of all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported, tricyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (9). Peptide 9 (EC 50 ) 0.14 nM) forces residues 13-30 into an extended helical conformation and is a more potent PTH receptor agonist than the parent linear hPTH(1-31)NH 2 (1, EC 50 ) 4.7 nM). Comparative circular dichroism studies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1-12 are also likely to be helical in the bioactive conformation. Taken together, these results provide strong structural evidence that hPTH binds to its receptor in an extended helical conformation.