by Xiaoxuan Zhang & Christopher J. Danpure, Grant holder: Christopher J. Danpure

Second year report (May, 2002 - April, 2003)

Research Objectives

The main aims of this project are to design small molecules that might stabilise mutant forms of AGT and develop relevant biossays in which to test them. Such drugs have enormous potential for the treatment of PH1.

Achievements

Our achievements during the second year of this research project can be divided into two sections: 1) De novo drug design and virtual screening, 2) cell-based bioassay development.

De novo drug design and virtual screening.

Following on from our first year's work, we have continued to develop our in silico approach to the structure-based design and screening of small molecules predicted to bind to AGT and possibly act as stabilising agents. We have installed more sophisticated computer packages that allow us to take advantage of the subtleties of normal and mutant AGT crystal structure (see previous reports). To the structural data already obtained, we have collected new X-ray diffraction data from polymorphic AGT (containing the Pro11Leu replacement) to 3.1 Å. We have identified regions of AGT predicted to be involved in its stability and dimerization, some of which bind the non-specific proteins stabilising agent glycerol. Our progress based on this strategy was presented by Dr Zhang and the NIDDK/OHF meeting in Annapolis in November 2003.

Cell-based bioassay development.

In collaboration with another member of my laboratory Dr Joseph Behnam, whose work is supported by the Jules Thorn Charitable Trust (co-PIs Chris Danpure & Gill Rumsby), we have designed a novel cell-based system with considerable potential for screening drugs that might be of benefit in the treatment of primary hyperoxaluria types 1 and 2. This system is based on the use of stably-transformed Chinese hamster ovary (CHO) cells expressing normal glycolate oxidase (GO), and normal and mutant alanine:glyoxylate aminotransferase (AGT, deficient in PH1) and glyoxylate/hydroxypyruvate reductase (GRHPR, deficient in PH2). The system is based on the fact that the cytotoxicity of glyoxylate is much greater than that of oxalate and related metabolites, such as glycolate and glycine. We have shown that although glycolate is not toxic to untransformed CHO cells, it kills CHO cells expressing GO because it is converted to cytotoxic glyoxylate. The indirect toxicity of glycolate in such a system is directly related to the level of GO expression. Low GO expressers survive much better than high expressers presumably because they produce less glyoxylate. Co-expression of either normal AGT or normal GRHPR, in addition to GO, protects the cells from indirect glycolate toxicity, so that even high GO expressers survive. It is predicted that mutant AGT (e.g. AGT containing Pro11Leu and Gly170Arg which mistargets from the peroxisomes to the mitochondria) or mutant GRHPR would not provide any protection, except in the presence of chemical chaperones that stabilise the enzymes and, in the case of AGT, correct its targeting. Thus this novel cell-based system could be used to screen for such stabilising drugs, the endpoint assay being either correct targeting, cell survival or GO expression.

Future

During the third and final year of this grant, we plan to develop our cell-based bioassay system by making CHO cell stable transformants expressing normal GO and various mutant forms of AGT and GRHPR to test our prediction that they would not protect the cells from indirect glycolate toxicity (see above). We then plan to use these cells to test the efficacy of drugs designed in silico based on the crystal structures of normal and mutant AGTs (see above). In addition, the cell based bioassay will be developed for high-throughput screening using multiwell plate-reader immunofluorescence and fluorescence activated cell sorting (FACS) analyses. Data from these studies will be fed back into the in silico drug screening programme in an iterative manner to design additional, hopefully more-efficacious drugs.

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