Protein Structure Inspired Drug Discovery.

Drug discovery starts with known function, either of a compound or a protein, in-turn prompting investigations to probe 3D structure of the compound-protein interface. As protein structure determines function, we hypothesized that unique 3D structural motifs represent primary information denoting unique function that can drive discovery of novel agents. Using a physics-based protein structure analysis platform developed by us, designed to conduct computationally intensive analysis at supercomputing speeds, we probed a high-resolution protein x-ray crystallographic library developed by us. We selected 3D structural motifs whose function was not otherwise established, that offered environments supporting binding of drug-like chemicals and were present on proteins that were not established therapeutic targets. For each of eight potential binding pockets on six different proteins we accessed a 60 million compound library and used our analysis platform to evaluate binding. Using eight-day colony formation assays acquired compounds were screened for efficacy against human breast, prostate, colon and lung cancer cells and toxicity against human bone marrow stem cells. Compounds selectively inhibiting cancer growth segregated to two pockets on separate proteins. The compound, Dxr2-017, exhibited selective activity against human melanoma cells in the NCI-60 cell line screen, had an IC50 of 19 nM against human melanoma M14 cells in our eight-day assay, while over 2100-fold higher concentrations inhibited stem cells by less than 30%. We show that Dxr2-017 induces anoikis, a unique form of programmed cell death in need of targeted therapeutics. The predicted target protein for Dxr2-017 is expressed in bacteria, not in humans. This supports our strategy of focusing on unique 3D structural motifs. It is known that functionally important 3D structures are evolutionarily conserved. Here we demonstrate proof-of-concept that protein structure represents high value primary data to support discovery of novel therapeutics. This approach is widely applicable.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.