1. Structure-Activity Correlations for Dihydrofolate Reductase Inhibition
In order to understand the role of key active site residues among fungal, mammalian, and bacterial species of dihydrofolate reductase (DHFR), mutational studies are being carried out to measure the influence on binding specificity and selectivity. Structural studies have of inhibitor complexes with various DHFR species (human, mouse, rat, beef, Pneumocystis carinii, Pneumocystis jirovecii and E. coli DHFR) are under investigation. These data have revealed novel modes of binding for potent inhibitors and show that the dynamics of binding can control the preferred conformation for binding.
The focus is on the design of inhibitors that are selective for the inhibition of DHFR from the opportunistic pathogen Pneunocystis jirovecii, the causative agent for AIDS-related pneumonia. Mutation of key active site residues have been carried out to measure the inhibitory and kinetic effect of inhibitors on selectivity and specificity of DHFR inhibition. Currently mutations in Pneumocystis jirovecii DHFR observed in clinical isolates of patients who are trimethoprim resistant are under investigation to understand the mechanism of drug-resistance.
In a second study, homodimers of E. coli DHFR in complex with bifunctional inhibitors is being carried out to develop new methods of drug delivery.
2. Histidine Triad Nucleotide Binding Proteins (Hint)
The histidine triad nucleotide proteins (Hints) from human and E. coli have been established as purine nucleoside phosphoramidases. Human Hint1 possesses tumor suppressor activity whereas E. coli HinT is involved in high salt tolerance for E. coli. The homodimeric Hint proteins are characterized by a His-X-His-X-His-XX motif where X is a hydrophobic residue. Additionally, human HinT1 and its bacterial homolog have different substrate specificities. To understand the mechanism of substrate specificity, kinetic and structural studies of mutants that replace the active site histidines were carried out. Current studies will focus on the interactions of ecHint with LysRS.
3. E. coli Enterotoxin LT-IIb-B5 pentamer mutants
Structural studies of mutants of the heat-labile enterotoxin LT-IIb-B5 pentamer are being carried out to understand the effect of these mutations on the activation of toll-like receptor signaling. Biochemical data reveal that the B pentamer lacks enterotoxicity and has acquired toll-like receptor 2 (TLR2) binding capacity. A second aspect of this work involves use of small-angle X-ray solution scattering (SAXS) experiments to map the molecular shape profile of the interactions of the B pentamer with toll-like receptor 2. The crystal structure of the complex of LT-IIb-B5 with TLR2 will also be carried out.
4. Human Papollomavirus (HPV)
Interactions between HPV E1 DNA helicase (E1) and human Topoisomerase I can be disrupted by small peptiometics of E1. Such molecules will inhibit HPV DNA replication, thereby preventing HPV proliferation. Recent data have shown that E1 interactions with, and stimulates the enzymatic activity of topoisomerase I. Further, a mutation in E1 compromises this interaction and is also compromised for HPV DNA replication. Stuctural studies of the E1- TopoI interaction will provide insight into the specific residues in E1 that can be modified to prevent HPV proliferation. Small angle solution X-ray scattering (SAXS) data will be carried out to determine the molecular shape of this complex.
5. Thyroid Hormone Integrin Interactions
Integrins are ubiquitous heterodimeric structural proteins of the cell membrane that convey signals to and from the cell interior to the extracellular matrix. A novel cell surface receptor for thyroid hormone has been identified on the extracellular domain of integrin alphaVbeta3 that leads in a variety of human cell lines to activation by the hormone of the mitogen-activated protein kinase (MAPK) signal transduction cascade within the cell. The arginine-glycine-aspartate (RGD) recognition site on integrin is essential to the binding of a variety of extra cellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding (T4ac inhibits T4-induced MAPK activity) suggesting that the hormone interaction site is located near the RGD recognition site on integrin alphaVbeta3. Structural data show that an RGD cyclic peptide binds at the interface of the propeller of the alphaV and the B domains on the integrin head (Arnaout, et. al, Science, 2002). To model potential interactions of thyroid (T4, T4ac, T3, GC-1) and steroid (E2, resveratrol, stilene) hormone analogues with integrin, molecular docking combined with quantum chemical calculations were carried out. These results are compared to previous modeling studies (Cody, et al, Steroids, 2007) that indicated the thyroid hormones had preferential binding at the surface of the B domain whereas the more planar molecules could bind in an alternate pocket. These computational results showed preferential binding to the alternate site and showed there was a strong electronic contribution to binding energies by the presence of Mg near the active site that impacts ligand binding. These data show that CG-1, T4ac and c-resveratrol have the strongest stabilization energies of the compounds tested. These calculations also showed that there can be significant difference in the binding orientation of similar ligands.