The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

The furan route to tropolones: probing the antiproliferative effects of ß-thujaplicin analogs.

A direct route to analogs of the naturally occurring tropolone ß-thujaplicin has been developed in just four steps from furan. Using this method, a series of derivatives were synthesized and evaluated. Several of these compounds demonstrated very high levels of potency against bacterial and fungal pathogens with good selectivity over mammalian cells.

ZINC: a free tool to discover chemistry for biology.

ZINC is a free public resource for ligand discovery. The database contains over twenty million commercially available molecules in biologically relevant representations that may be downloaded in popular ready-to-dock formats and subsets. The Web site also enables searches by structure, biological activity, physical property, vendor, catalog number, name, and CAS number. Small custom subsets may be created, edited, shared, docked, downloaded, and conveyed to a vendor for purchase. The database is maintained and curated for a high purchasing success rate and is freely available at zinc.docking.org.

Towards new antifolates targeting eukaryotic opportunistic infections.

Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

In pursuit of virtual lead optimization: pruning ensembles of receptor structures for increased efficiency and accuracy during docking.

Representing receptors as ensembles of protein conformations during docking is a powerful method to approximate protein flexibility and increase the accuracy of the resulting ranked list of compounds. Unfortunately, docking compounds against a large number of ensemble members can increase computational cost and time investment. In this article, we present an efficient method to evaluate and select the most contributive ensemble members prior to docking for targets with a conserved core of residues that bind a ligand moiety. We observed that ensemble members that preserve the geometry of the active site core are most likely to place ligands in the active site with a conserved orientation, generally rank ligands correctly and increase interactions with the receptor. A relative distance approach is used to quantify the preservation of the three-dimensional interatomic distances of the conserved ligand-binding atoms and prune large ensembles quickly. In this study, we investigate dihydrofolate reductase as an example of a protein with a conserved core; however, this method for accurately selecting relevant ensemble members a priori can be applied to any system with a conserved ligand-binding core, including HIV-1 protease, kinases, and acetylcholinesterase. Representing a drug target as a pruned ensemble during in silico screening should increase the accuracy and efficiency of high-throughput analyses of lead analogs.

Structure-based approach to the development of potent and selective inhibitors of dihydrofolate reductase from cryptosporidium.

Cryptosporidiosis is an emerging infectious disease that can be life-threatening in an immune-compromised individual and causes gastrointestinal distress lasting up to 2 weeks in an immune-competent individual. There are few therapeutics available for effectively treating this disease. We have been exploring dihydrofolate reductase (DHFR) as a potential target in Cryptosporidium. On the basis of the structure of the DHFR enzyme from C. hominis, we have developed a novel scaffold that led to the discovery of potent (38 nM) and efficient inhibitors of this enzyme. Recently, we have advanced these inhibitors to the next stage of development. Using the structures of both the protozoal and human enzymes, we have developed inhibitors with nanomolar potency (1.1 nM) against the pathogenic enzyme and high levels (1273-fold) of selectivity over the human enzyme.

Protein quaternary structure and expression levels contribute to peroxisomal-targeting-sequence-1-mediated peroxisomal import of human soluble epoxide hydrolase.

The peroxisomal targeting sequence 1 (PTS1) is a consensus tripeptide 1 (S/C/A)(K/R/H)(L/M) that is found at the C-terminus of most peroxisomal proteins. However, the only known mammalian protein containing a terminal methionine PTS1 (SKM), human soluble epoxide hydrolase (hsEH), shows both peroxisomal and cytosolic localizations in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized green fluorescent protein-hsEH fusion constructs to study the peroxisomal targeting of hsEH in transiently and stably transfected Chinese hamster ovary cells. Our results suggest that the peroxisomal import of hsEH is regulated by three factors. First, we show that SKM is required, but not sufficient, for peroxisomal import. Second, by manipulating protein expression levels, we show that SKM mediates peroxisomal import of wild-type hsEH only when expression levels are high. Third, we show that amino acid modifications that decrease subunit oligomerization and presumably enhance accessibility of the SKM motif confer peroxisomal targeting even at low protein expression levels. We conclude that, in hsEH, SKM is a necessary but inefficient and context-dependent PTS1. Peroxisomal import occurs when expression levels are high or when the SKM motif is accessible. These results provide a mechanistic basis for understanding the cell-specific and tissue-specific localization of hsEH in vivo.

In pursuit of virtual lead optimization: the role of the receptor structure and ensembles in accurate docking.

Accurate ranking during in silico lead optimization is critical to drive the generation of new ligands with higher affinity, yet it is especially difficult because of the subtle changes between analogs. In order to assess the role of the structure of the receptor in delivering accurate lead ranking results, we docked a set of forty related inhibitors to structures of one species of dihydrofolate reductase (DHFR) derived from crystallographic, NMR solution data, and homology models. In this study, the crystal structures yielded the superior results: the compounds were placed in the active site in the conserved orientation and the docking scores for 80% percent of the compounds clustered into the same bins as the measured affinity. Single receptor structures derived from NMR data or homology models did not serve as accurate docking receptors. To our knowledge, these are the first experiments that assess ranking of homologous lead compounds using a variety of receptor structures. We then extended the study to investigate whether ensembles, either computationally or experimentally derived, of all of the single starting structures aid, hinder or have no effect on the performance of the starting template. Impressively, when ensembles of receptor structures derived from NMR data or homology models were employed, docking accuracy improved to a level equal to that of the high resolution crystal structures. The same experiments using a second species of DHFR and set of ligands confirm the results. A comparison of the structures of the individual ensemble members to the starting structures shows that the effect of the ensembles can be ascribed to protein flexibility in addition to absorption of computational error.

Substrate specificity of prostate-specific membrane antigen.

A series of putative dipeptide substrates of prostate-specific membrane antigen (PSMA) was prepared that explored alpha- and beta/gamma-linked acidic residues at the P1 position and various chromophores at the P2 position, while keeping the P1' residue constant as L-Glu. Four chromophores were examined, including 4-phenylazobenzoyl, 1-pyrenebutyryl, 9-anthracenylcarboxyl-gamma-aminobutyryl, and 4-nitrophenylbutyryl. When evaluating these chromophores, it was found that a substrate containing 4-phenylazobenzoyl at the P2 position was consumed most efficiently. Substitution at the P1 position with acidic residues showed that only gamma-linked L-Glu and D-Glu were recognized by the enzyme, with the former being more readily proteolyzed. Lastly, binding modes of endogenous substrates and our best synthetic substrate (4-phenylazobenzoyl-Glu-gamma-Glu) were proposed by computational docking studies into an X-ray crystal structure of the PSMA extracellular domain.

Highly efficient ligands for dihydrofolate reductase from Cryptosporidium hominis and Toxoplasma gondii inspired by structural analysis.

The search for effective therapeutics for cryptosporidiosis and toxoplasmosis has led to the discovery of novel inhibitors of dihydrofolate reductase (DHFR) that possess high ligand efficiency: compounds with high potency and low molecular weight. Detailed analysis of the crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis and a homology model of DHFR from Toxoplasma gondii inspired the synthesis of a new series of compounds with a propargyl-based linker between a substituted 2,4-diaminopyrimidine and a trimethoxyphenyl ring. An enantiomerically pure compound in this series exhibits IC50 values of 38 and 1 nM against C. hominis and T. gondii DHFR, respectively. Improvements of 368-fold or 5714-fold (C. hominis and T. gondii) relative to trimethoprim were generated by synthesizing just 14 new analogues and by adding only a total of 52 Da to the mass of the parent compound, creating an efficient ligand as an excellent candidate for further study.