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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.

The Berkeley Pit, an acid mine waste lake, is a source of extremophilic microorganisms that produce interesting bioactive compounds. We have previously reported the isolation of berkeleydione 1, berkeleytrione 2, the berkeleyacetals and the berkeleyamides from the Pit Lake fungus Penicillium rubrum. In this paper we report the isolation and characterization of berkeleyones A-C (4, 5 and 7) as well as previously described preaustinoid A (3) and A1(6) from this same fungus. These compounds were evaluated as inhibitors of the signaling enzyme caspase-1 and as potential inhibitors of interleukin 1-β production by inflammasomes in induced THP-1 cell line assays.

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.

The Peroxisomal Targeting Sequence 1 (PTS1) is a consensus tripeptide, (S/C/A)(K/R/H)(L/M), found on 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 localization in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized GFP-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 which 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-and tissue-specific localization of hsEH in vivo.