Heterocylic diamidines have been proven effective in the treatment of diseases caused by parasitic microorganisms. Previous studies suggest that these compounds target specific sequences on DNA. DB293 and DB75 are heterocyclic diamidines that have shown to bind to the minor grove of DNA at AT rich sequences. DB293 has also been observed by the laboratory of W.D. Wilson, Georgia State University, to bind cooperatively, forming dimers in the minor groove of ATGA sites of DNA. This research focuses on the effect of DNA sequences on both DB293 and DB75’s binding properties as well as how the drugs affect the activity of enzymes acting on DNA. DB75 and DB293 were provided by D.W. Boykin and W.D. Wilson, Georgia State University. Restriction enzyme kinetics assays using three restriction enzymes, [Bssh II (cleavage site GCGCGC), Mlu I (ACGCGT) and Stu I (AGGCCT)], with different reaction sites and differing flanking sequences are employed to measure the rate constants of these enzymes in the presence and absence of DB293 or DB75. The restriction digests were performed with a solution of supercoiled and relaxed ΦX174 under constant DB293 or DB75 concentration or in the absence of DB293 or DB75. DB293 causes enhanced cleavage and an increase in rate for Stu I, but an inhibition of cleavage and a decrease in rate constant for Mlu I. On the other hand, DB75 experiences cleavage enhancement for both enzymes. The pseudo-first order rate constant for Stu I increased from 0.020 min-1 to 0.035 min-1 in the presence of DB293 and 0.064 min-1 in the presence of DB75. The rate constant for Mlu I decreased from 0.047 min-1 to 0.023 min-1 in the presence of DB293 and increased to 0.061 min-1 in the presence of DB75. The drug effects varied whether the DNA was relaxed or supercoiled. The results from these restriction enzyme kinetic assays suggest that the nature of the binding of DB75 and DB293 to DNA depends on the DNA sequence in the vicinity of the binding sites.

Effects of heterocyclic diamidine antiparastitic agent binding to DNA on restriction enzyme kinetics

253rd National Meeting (2017)

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