Developing a computational model of asparagine synthetase-B towards rational inhibitor design.

摘要

Although Acute Lymphoblastic Leukemia (ALL) has an initial treatment rate of nearly 95%, it claims the lives of over 1,000 people in the US each year. These patients often develop resistance to L-asparaginase, a main chemotherapeutic agent used in the treatment of ALL. There has been a long withstanding, inverse correlation to patients' resistance to L-asparaginase and up-regulation of the enzyme, asparagine synthetase. Therefore, selective potent inhibitors of asparagine synthetase (AS) may be useful for the clinical treatment of ALL.;This work focuses on the development of a structural model of E. coli asparagine synthetase-B (AS-B); the glutamine-dependent bacterial form that is similar to human AS. Novel parameters were developed in CHARMM for incorporation of the AS reaction intermediate, beta-aspartyl-AMP, into the AS-B model. This intermediate has been successfully used as a model for inhibitor design. Therefore, modeling this intermediate will provide understanding of the protein structure and active site interactions, which will aid in the development of future inhibitors.;Simulated annealing (SA) was used to arrive at an optimized model of AS-B with glutamine bound in the glutaminase active site and beta-aspartyl-AMP, pyrophosphate and Mg2+ bound in the synthetase active site. The final model was heated from 0 K--600 K; cooled back down to 300 K; then equilibrated at 300 K before a final minimization. The optimized model had a very interesting side chain flip occur for Glu-348. The repositioned side chain would make the translocation of NH3 from the glutaminase site to the synthetase site more probable than the translocation is in the current AS-B crystal structure.;The availability of a structural model for AS sets the stage for the application of in silico screening of virtual libraries to identify novel dugs for the treatment of asparaginase-resistant ALL. The validation of algorithms and potentials for molecular docking is a necessary prelude to such efforts. Therefore, an enhanced Potential of Mean Force (PMF) scoring function was developed for in silico screening studies.;The sum of the work presented here sets the stage for in silico screening or the rational design of inhibitors for AS.