Complications of diabetes are responsible for approximately 15% of blindness in the United States. There is a body of evidence that both the retinopathy and the cataracts found in diabetic patients are caused by sorbitol from the aldose reductase(AR)-catalyzed reduction of glucose. Sorbitol accumulation in tissues causes cataracts, retinopathy, peripheral neuropathy, and nephropathy by disturbing either the osmotic or redox balance. Inhibitors of AR provide a therapeutically rational means to delay the onset of diabetic cataracts or microaneurysms in the retina. Although some AR inhibitors successfully prevent cataracts in animal models, the inhibitors developed to date are either ineffective or toxic in diabetic patients. It is presumed that the toxicity of these drugs is due to inhibition of other enzymes which are members of the aldo-keto reductase super family. X-ray crystallography, site-directed mutagenesis, and mechanistic enzymology have been used to propose a likely chemical mechanism for the reduction reaction catalyzed by AR. Based on the structural and mechanistic information currently available, the PI will take three different crystallographic approaches to determine which features are required to enhance inhibitor specificity: 1) The recently determined structure of the AR-Alrestatin complex will be used as a scaffold for designing ferrocene acetic acid derivatives to act as probes of the """"""""specificity pocket."""""""" 2) The more general approach of """"""""solvent mapping"""""""" will be applied to crystals of AR. Using organic solvents as probe molecules, the location of potential binding sites will be mapped to the surface of the enzyme. 3) The binding modes of a set of small molecules designed to block a potentially important """"""""proton wire"""""""" leading to the active site will be studied complexed to AR by X-ray crystallography. 4) To increase our knowledge of the catalytic mechanism, the last structurally unknown, catalytically important state of AR, the reduced cofactor bound to enzyme, will be determined using the non-oxidizable NADPH analogue, NADPH4. A NADP-glyceraldehyde adduct will be examined to understand the substrate binding geometry. The resolution of the current X-ray structure will be extended to beyond 1.2 A to locate hydrogen atoms associated with NADP. This will be accomplished by developing a suitable cryo-protectant and using synchrotron radiation to obtain ultra high resolution data. These structures will directly lead to a better understanding of how AR recognizes both substrates and inhibitors and how it promotes the reduction of aldehydes by NADPH.
