Abstract: (-)-4-Amino-2-thiabicyclo-[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-3) is a highly potent and selective agonist of metabotropic glutamate receptors 2 (mGlu2) and 3 (mGlu3). As part of our ongoing research program, we have prepared S-oxidized variants of (-)-3, compounds (-)-10, (+)-11 (LY404040), and (-)-12 (LY404039). Each of these chiral heterobicyclic amino acids displaced specific binding of the mGlu2/3 receptor antagonist 3H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid (3H-LY341495) from membranes expressing recombinant human mGlu2 or mGlu3 and acted as potent agonists in cells expressing these receptor subtypes. Docking of the most potent of these derivatives, (+)-11, to mGlu2 revealed the possibility of an additional H-bond interaction between the sulfoxide oxygen of (+)-11 with tyrosine residue Y236. Pharmacokinetic analysis of mGlu active enantiomers (+)-11 and (-)-12 in rats showed each to be well absorbed following oral administration. Consistent with their mGlu2/3 agonist potency and pharmacokinetic properties, both (+)-11 and (-)-12 blocked phencyclidine-evoked ambulations in a dose-dependent manner, indicating their potential as nonclassical antipsychotic agents.
Abstract: The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.
Abstract: Anxiety and stress disorders are the most commonly occurring of all mental illnesses, and current treatments are less than satisfactory. So, the discovery of novel approaches to treat anxiety disorders remains an important area of neuroscience research. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, and G-protein-coupled metabotropic glutamate (mGlu) receptors function to regulate excitability via pre- and postsynaptic mechanisms. Various mGlu receptor subtypes, including group I (mGlu(1) and mGlu(5)), group II (mGlu(2) and mGlu(3)), and group III (mGlu(4), mGlu(7) and mGlu(8)) receptors, specifically modulate excitability within crucial brain structures involved in anxiety states. In addition, agonists for group II (mGlu(2/3)) receptors and antagonists for group I (in particular mGlu(5)) receptors have shown activity in animal and/or human conditions of fear, anxiety or stress. These studies indicate that metabotropic glutamate receptors are interesting new targets to treat anxiety disorders in humans.
Abstract: LY354740 (1) is a highly potent and selective agonist of metabotropic glutamate (mGlu) receptors 2 and 3. In the present study, we have prepared C3- and C4-methyl-substituted variants of rac-1, compounds 5, 9, and 13. Each of these racemic methyl-substituted analogues displaced specific binding of the mGlu2/3 receptor antagonist (3)H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid ((3)H-LY341495) from membranes expressing mGlu2 or mGlu3 receptor subtypes. Evaluation of the functional effects of this series on second messenger responses in cells expressing human mGlu2 or mGlu3 receptors revealed C3beta-methyl analogue 5 to possess antagonist properties at both mGlu2 and mGlu3 receptors while C4beta-methyl analogue 9 acts as a full agonist at each of these targets. Unexpectedly, we found that incorporation of a methyl substituent at the C4alpha-position as in analogue 13 results in a mixed mGlu2 agonist/mGlu3 antagonist pharmacological profile. All of the mGlu2 agonist and mGlu3 antagonist activity of rac-13 was found to reside in its resolved (+)-isomer.
Abstract: Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA-DrrA histidine kinase-response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis-Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5'-beta,gamma-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.
Abstract: A detailed chemometric analysis of ligand binding to domain-3A of human serum albumin is described. NMR and fluorescence data on a set of 889 chemically diverse compounds were used to develop a group contribution model based on 74 chemical fragments that is in good agreement with the experimental data (R2 = 0.94, Q2 = 0.90). The structural descriptors used in this analysis comprise a convenient look-up table for quantitatively estimating the effect that a particular group will have on albumin binding. This information can be valuable for optimizing a particular series of compounds for drug development.
Abstract: A statistical analysis of NMR-derived binding data on 11 protein targets was performed to identify molecular motifs that are preferred for protein binding. The analysis indicates that compounds which contain a biphenyl substructure preferentially bind to a wide range of proteins and that high levels of specificity (>250-fold) can be achieved even for these small molecules. These results suggest that high-throughput screening libraries that are enriched with biphenyl-containing compounds can be expected to have increased chances of yielding high-affinity ligands for proteins, and they suggest that the biphenyl can be utilized as a template for the discovery and design of therapeutics with high affinity and specificity for a broad range of protein targets.
Abstract: Molecular diversity, combinatorial chemistry and automated synthesis are helping usher in a new age in medicinal chemistry. The tools and practices of computational chemistry and molecular modeling are rising to the challenges and opportunities presented by the current trends in drug discovery and design. Recent advances include a number of new and meaningful measures of molecular diversity and the use of genetic algorithms to help design diverse libraries.
Abstract: Ofloxacin, a potent quinolone antibacterial agent, has a tricyclic ring structure with a methyl group attached to the asymmetric carbon at the C-3 position on the oxazine ring. The S isomer (DR-3355) of ofloxacin has antibacterial activity up to 2 orders of magnitude greater than that of the R isomer (DR-3354). This differential antibacterial activity was not due to different drug transport mechanisms of the two isomers but was found to be derived from the inhibitory activity against the target enzyme, DNA gyrase. Previous mechanistic studies have suggested that the bactericidal effect of the drug is mediated through the stabilization of a cleavable complex via a cooperative drug binding process to a partially denatured DNA pocket created by DNA gyrase. The drug binds to supercoiled DNA in a manner similar to that to which it binds to the enzyme-DNA complex. In the present studies, we first examined the binding of the two radiolabeled ofloxacin enantiomers to supercoiled pUC9 plasmid DNA. Surprisingly, the two enantiomers possessed similar apparent binding affinities and binding cooperatives. The major difference in binding between the two stereoisomers was the molar binding ratio: 4 for the more active S isomer versus 2 for the less active R isomer. We next examined the relative binding potencies of the stereoisomers to the DNA-DNA gyrase complex. The results of a competition assay showed that (S)-ofloxacin binds 12-fold better to the complex than (R)-ofloxacin. The binding potencies of the two enantiomers and two other quinolones correlated well with their respective concentrations causing 50% inhibition against DNA gyrase. The results are interpreted by a stacking model by using the concept of the cooperative drug-DNA binding mechanism, indicating that the potencies of quinolones cannot be determined solely by the DNA binding affinity and cooperativity but can also be determined by their capability in maximally saturating the binding site. The capability of the drug in saturating the binding pocket manifests itself in an increased efficacy at inhibiting the enzyme through a direct interaction between the drug and the enzyme. The results augment the previous suggestion that the binding pocket in the enzyme-DNA complex involves multiple receptor groups including not only DNA bases but also a gyrase subunit. The higher level of potency of (S)-ofloxacin is proposed to derive from the fact that a greater number of molecules are assembled in the pocket. This greater number of molecules optimizes the interaction between the drug and the enzyme, possibly through a contact between the C-7 substituent and the quinolone pocket on the B subunit of DNA gyrase.
Abstract: (2RS,4R)-3-(2-(3-Pyridinyl)thiazolidin-4-oyl)indoles represent a new class of potent, orally active antagonists of platelet activating factor (PAF). The compounds were prepared by acylation of the magnesium or zinc salts of substituted indoles with (2RS,4R)-2-(3-pyridinyl)-3-(tert-butoxycarbonyl)thiazolidin-4-oyl chloride. The 3-acylindole moiety functions as a hydrolytically stabilized and conformationally restricted anilide replacement, which imparts a considerable boost in potency to the series. Structure-activity relationships observed for substitution on the indole ring system are discussed. Members of the series compare favorably with other reported PAF antagonists.
Abstract: In the absence of a 3D structure of the target biomolecule, to propose the 3D requirements for a small molecule to exhibit a particular bioactivity, one must supply both a bioactive conformation and a superposition rule for every active compound. Our strategy identifies both simultaneously. We first generate and optimize all low-energy conformations by any suitable method. For each conformation we then use ALADDIN to calculate the location of points to be considered as part of the superposition. These points include atoms in the molecule and projections from the molecule to hydrogen-bond donors and acceptors or charged groups in the binding site. These positions and the relative energy of each conformation are the input to our new program DISCO. It uses a clique-detection method to find superpositions that contain at least one conformation of each molecule and user-specified numbers of point types and chirality. DISCO is fast; for example, it takes about 1 min CPU to propose pharmacophores from 21 conformations of seven molecules. We typically run DISCO several times to compare alternative pharmacophore maps. For D2 dopamine agonists DISCO shows that the newer 2-aminothiazoles fit the traditional pharmacophore. Using site points correctly identifies the bioactive enantiomers of indoles to compare with catechols whereas using only ligand points leads to selecting the inactive enantiomer for the pharmacophore map. In addition, DISCO reproduces pharmacophore maps of benzodiazepines in the literature and proposes subtle improvements. Our experience suggests that clique-detection methods will find many applications in computational chemistry and computer-assisted molecular design.
Abstract: Molecular modeling techniques and three-dimensional (3D) pattern analysis have been used to investigate the chemical and steric properties of compounds that inhibit transport of the plant hormone auxin. These compounds bind to a specific site on the plant plasma membrane characterized by its affinity for the herbicide N-1-naphthylphthalamic acid (NPA). A 3D model was derived from critical features of a set of ligands for the NPA receptor, a suggested binding conformation is proposed, and implications for the topographical features of the NPA receptor are discussed. This model, along with 3D structural analysis techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the criteria of the model, 77 representative molecules were evaluated for their ability to compete for the binding of [3H]NPA to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, representing chemical classes not included in the original compound set, were also found to inhibit polar auxin transport through corn coleoptile sections. Thus, this study demonstrates that 3D analysis techniques can identify active, novel ligands for biochemical target sites with concomitant physiological activity.
