Suzanne Tomlinson, PhD
Department of Biochemistry and Molecular Biology
University of Texas Medical Branch
Aldose reductase (ALR2) has recently been shown to be an important mediator of colon cancer signaling pathways. The reduction of glutathione (GS-) conjugated aldehydes by ALR2 produces intermediates that are involved in inflammation and cancer-signaling pathways. Although ALR2 inhibitors have been shown to arrest tumor growth in mice, the majority have failed in clinical trials due to undesirable side effects. It is likely that a portion of the observed side effects were due to cellular toxicity resulting from indiscriminate inhibition of GS-aldehyde reduction as well as non-GS-conjugated aldehyde reduction. Analysis of our crystal structure of ALR2 with a bound GS-aldehyde analog revealed a GS-binding site separate from the catalytic site implying that one could develop selective inhibitors of the GS-binding site, which would alleviate GS-aldehyde reduction and subsequent cancer signaling, while still allowing aliphatic aldehyde reduction and thus reducing associated side effects.
Clustering analyses performed on the ~ 100 ALR2 structures in the Protein Data Bank (PDB) revealed three primary "conformations" in the GS-binding site. Docking studies utilizing the AUTODOCK Vina program and the Directory of Useful Decoys (DUD) ALR2 database of known binders/decoys revealed that by docking to representatives from each of the conformations represented in the PDB, and developing a composite ranking of combined energy scores, early discrimination of known ALR2 binders was near ideal. Additionally, testing of the top compounds from the composite ranking revealed two previously unidentified ALR2 inhibitors.
Predoctoral Fellow at Baylor College of Medicine
Cyclic guanosine monophosphate (cGMP) is a key secondary messenger that is produced in response to nitric oxide. One of the key mediators of cGMP signaling, cGMP-dependent protein kinase (PKG), is activated upon binding to cGMP and phosphorylates downstream substrates in a process required for important physiological processes such as vasodilation, nociception, and memory formation. PKGs are also known to mediate most effect of drugs that increase cellular cGMP levels, including nitric oxide-releasing agents and phosphodiesterase inhibitors, which are used for the treatment of angina pectoris and erectile dysfunction, respectively. We have investigated the mechanism of cyclic nucleotide selectivity by PKG by determining crystal structures of the cGMP-selective carboxyl-terminal cyclic nucleotide-binding domain (CNB-B) of human PKG I bound to cGMP and in the apo form. Our crystal structure of CNB-B with bound cGMP reveals that cGMP adopts the syn configuration in the binding pocket and is coordinated by a previously unidentified arginine residue. Furthermore, comparison of the cGMP-bound crystal structure of the apo structure suggests a role for a C-terminal tyrosine residue in capping the nucleotide into the binding pocket. The interaction of this tyrosine residue with cGMP appears to result in a large conformational change in CNB-B, suggesting a mechanism for kinase activation by cGMP.