The colon cancer chemotherapeutic CPT-11 causes severe diarrhea due gut bacterial beta-glucuronidases that reactivate the drug. To target these enzymes without killing beneficial intestinal bacteria, we identified potent inhibitors by high-throughput screening that have no effect on the endogenous human enzyme, nor do they kill bacteria or harm mammalian cells. Crystal structures established that inhibitor selectivity is based on a loop unique to bacterial beta-glucuronidases (magenta residues in picture). Oral administration of an inhibitor protected mice from CPT-11-induced toxicity. We showed that drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy (Wallace, B.D. et al., Science, 2010).
Phospholipase C isozymes (PLCs) hydrolyze a minor phospholipid whose two products initiate bifurcating signaling cascades required for numerous cellular processes. The crystal structure of Rac1 bound to PLC-b2 and depicted here docked to a model membrane highlights the activation of PLC-b2 by Rac1 (Jezyk, M., Nat. Struct. & Mol. Biol., 2006).
Drugs that target G protein-coupled receptors (GPCRs) represent the largest class of clinically relevant therapeutics. Gbg dimers are obligate heterodimers needed for signaling propagated downstream of GPRCs. The crystal structure of the Gb5 subunit bound to RGS9 (depicted above) highlights an evolutionary distant Gbg-like complex required for signaling downstream of GPCRs in vision and neurotransmission (Cheever, M., Nat. Struct. Mol. Biol., 2008).
Phosphorylated derivatives of phosphatidylinositol (PtdIns), termed phosphoinositides (PIPs) are important components of membrane associated signaling systems in eukaryotes. Crystal structures of Sfh1, a Ptdins/PtdCho-transfer protein, in complex with various phospholipids demonstrate how these ligands are bound and suggest how they are presented/transferred to kinases (Schaaf, G., Mol. Cell, 2008).
This enzyme is critical to breast cancer development as it controls the expression of the aromatase enzyme required for estrogen-dependent tumor growth. Using structural and molecular biology, as well as mass spectrometry, we have shown that human LRH-1 is ligand-dependent and appears to respond to endogenous phospholipids that bind deep within the core of this transcription factor (Ortlund, E.A., Nat. Struct. Mol. Biol., 2005).
PLCs are enzymes involved in cell signaling. Aberrations in PLCs are reported to have an effect on malignant characteristics of tumors, such as motility and invasion capability. It has been suggested that PLC-ß2 constitutes a molecular marker of breast cancer cells able to monitor the progression to invasive cancers, and that it is a target for novel therapeutic approaches for breast cancer. Based on crystallographic and biophysical studies, we have recently proposed a general mechanism for the regulated auto-inhibition and activation of the entire family of phospholipase C (PLC) isozymes. This work is highlighted on the cover of Molecular Cell (Hicks, S., Mol. Cell, 2008).
The Siderovski lab has previously studied GoLoco motif/Galpha interactions by X-ray diffraction crystallography. Their recent structural studies of the KB-752 peptide/Galphai1·GDP dimer represented the first to describe the structural determinants of a Galpha subunit engaging an exchange factor providing strong evidence for a proposed mechanism of GPCR-catalyzed nucleotide exchange. This peptide was one of a collection identified via phase-display screening.
They have also solved the structure of a second peptide from this collection, KB-1753, which recognizes the activated forms of Galphai1. They plan to solve several additional structures from this novel collection of nucleotide-state-selective Galpha binding peptides to illuminate key structural determinants for modulation of Galpha nucleotide cycling.
The SBI Core is helping Dr. Blancafort and researchers in her laboratory design artificial transcription factors. We are involved in designing linkers joining individual transcription factor elements resulting in a new molecule with a unique binding site. We are also aiding design of these transcription factors by analyzing the promotor regions of targeted genes for potential unique binding sites.
We are working with Dr Reader and his laboratory to explore the enzymology, molecular evolution and inhibition of a number of aminoacyl-tRNA synthetases. Using a combination of molecular modeling and biochemical approaches we are characterizing the mode of action of a novel aminoacyl-tRNA synthetase inhibitor.
Dr. Bankaitis and colleagues are using molecular dynamics to study the phosphatidylinositol/ phosphatidylcholine transfer protein Sec14p. Using dynamics they are able to obtain detailed data on crical residues involved in opening and closing of the helical flap. These residues undergo small local conformational changes driving the larger global movements of the helical flap.
: Molecular dynamics studies are also aiding Dr. Redinbo and researchers in his laboratory address the question of why the nuclear receptor PXR is functional as a homodimer. PXR is unique since all other previously studied nuclear receptors are functional as monomers. MD studies have allowed characterization of the dynamical behavior of the monomer and dimer in different liganded states.
: The laboratories of Dr. Chaney and Dr. Dokholyan have joined efforts to study the relationship between structural properties of Pt-DNA adducts and their biological role as anti-cancer drugs. Again, molecular dynamics studies as well as NMR structural studies have elucidated structural differences in oxaliplatinated- and cisplatinated-dna adducts.
Dr. Jones has been working with Dr. Temple of the SBI core to complete an evolutionary analysis of the heterotrimeric G-protein. Their analysis has initially concentrated on the Galpha subunit. They have shown that the plant and fungal Galpha proteins are related to the ancestral Galpha prior to evolution of the 4 major classes and 16 subclasses found in animals.
back to top