Sydney P. Craig
University of North Carolina at Chapel Hill
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Featured researches published by Sydney P. Craig.
Antimicrobial Agents and Chemotherapy | 1995
A E Eakin; R Nieves-Alicea; R Tosado-Acevedo; M S Chin; C C Wang; Sydney P. Craig
Expression plasmids encoding the hypoxanthine phosphoribosyltransferases (HPRTs) of Plasmodium falciparum, Schistosoma mansoni, Tritrichomonas foetus, and Homo sapiens were subcloned into genetically deficient Escherichia coli that requires complementation by the activity of a recombinant HPRT for growth on semidefined medium. Fifty-nine purine analogs were screened for their abilities to inhibit the growth of these bacteria. Several compounds that selectively altered the growth of the bacteria complemented by the malarial, schistosomal, or tritrichomonal HPRT compared with the growth of bacteria expressing the human enzyme were identified. These results demonstrate that the recombinant approach to screening compounds by complement selection in a comparative manner provides a rapid and efficient method for the identification of new lead compounds selectively targeted to the purine salvage enzymes of parasites.
Biochimica et Biophysica Acta | 2001
Christian C. Lee; Francisco J. Medrano; Sydney P. Craig; Ann E. Eakin
Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.
Vitamins and Hormones Series | 2000
Sydney P. Craig; Ann E. Eakin
Time and costs associated with the discovery of new drugs have been significantly reduced by enzyme structure-based approaches to the discovery of new chemotherapeutic agents. However, fundamental components of the overall approach continue to rely on technologies which, by their nature, involve relatively random processes (i.e., combinatorial chemistry and high-throughput screening). Thus, the efficiency of the drug discovery process potentially could be further improved through better use of structural information. In this regard, three-dimensional structures of enzymes are now being solved at high resolution and/or in conformations that provide data that should be more useful for inhibitor design or discovery. Scientists are beginning to appreciate the importance of water as a possible competitor of inhibitors for binding to target enzymes. New computational algorithms are improving the efficiency of identifying flexible inhibitors from among the large numbers of compounds in chemical databases. Also, tools of molecular genetics together with structures of target enzymes are likely to be used more frequently in dealing with the development of resistance to novel chemotherapeutic agents. Instead of detailing success stories in structure-based drug discovery, the following article considers how future efforts to discover or design new drugs may increasingly rely on information about molecular targets and less on data acquired via approaches involving random methodologies.
Biochimica et Biophysica Acta | 1998
René Nieves-Alicea; Pamela J. Focia; Sydney P. Craig; Ann E. Eakin
Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.
Biochimica et Biophysica Acta | 1997
Sydney P. Craig; Pamela J. Focia; Robert J. Fletterick
Lysine was substituted for a conserved arginine at position 199 of the schistosomal hypoxanthine phosphoribosyltransferase (HPRT). This resulted in a > or = 35-fold increase in the K(M) for binding phosphoribosyl-pyrophosphate (PRPP). The possible functional role of R199 in tertiary structure, as well as in the binding of PRPP, is interpreted in the context of the reported three dimensional structure for the human HPRT.
Biochemistry | 1998
Pamela J. Focia; Sydney P. Craig; Ann E. Eakin
Biochemistry | 1992
Ling Yuan; Sydney P. Craig; James H. McKerrow; Ching C. Wang
Protein Science | 1999
Ganesaratnam K. Balendiran; José A. Molina; Yiming Xu; Jan Torres-Martinez; Robert D. Stevens; Pamela J. Focia; Ann E. Eakin; James C. Sacchettini; Sydney P. Craig
Biochemistry | 1998
Pamela J. Focia; Sydney P. Craig; René Nieves-Alicea; Robert J. Fletterick; Ann E. Eakin
Chemistry & Biology | 2000
Douglas M. Freymann; Mary Anne Wenck; Juan C Engel; Jun Feng; Pamela J. Focia; Ann E. Eakin; Sydney P. Craig