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New Inhibitors of the Peripheral Site in Acetylcholinesterase that Specifically Block Organophosphorylation

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Final rept.

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Examination of the enzyme structure for acetylcholinesterase AChE reveals two sites of ligand interaction The peripheral site P-site located at the entrance of the gorge, and the acylation site A-site at the base of the gorge. Our goal is to develop high affinity cyclic peptide ligands specific for the P-site that would block the access of organophosphate agents while allowing the passage of acetylcholine to the A-site for use by personnel at risk for nerve gas exposure. Our immediate strategy involves the covalent tethering of cyclic inhibitors via methanethiosulfonate MTS linkage to a cyteine on the AChE mutant, H287C. The modified AChEs linked to candidate peptides that inhibit P-site access are selected by affinity chromatography and tested for P-site and A-site affinity by measuring competitive inhibition constants K12 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. We are using a combinatorial approach to identify tethered cyclic peptides with high affinity for the P-site. A cyclic octapeptide library with 3000 compounds was synthesized in 30 subsets. The initial subset of 100 compounds has been screened by covalent tethering, affinity chromatography selection, and analysis of competitive inhibition by ligands that bind the site. Candidate cyclic peptides were released from modified AChEs with substantial P-site blockade by reduction with dithiothreitol, and mass spectrometry MS techniques were used to characterized the peptides. MALDI TOF MS revealed a mixture of peptides by detecting a series of predicted peptide masses. Peptide sequences were obtained on an ESI ion trap mass spectrometer with MSn capabilities by following peptide fragments through several stages of consecutive collisionally activated decomposition CAD mass spectra. Two peptide masses were selected from this first screen, corresponding to sixteen individual peptides because of several D- and L-amino acid combinations.

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  • Biochemistry
  • Medicine and Medical Research
  • Organic Chemistry

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