Effects of substituents on the 5-position of deoxyuridinemonophosphate on the thiolate addition in thymidylate synthase using QM/MM technique

Abstract

Thymidylate synthase (TS) is an enzyme that catalyses the reductive methylation of dUMP to produce dTMP in the thymine nucleotide synthesis. The Michael addition and ternary covalent complex formation are considered as important steps involved in the inhibition mechanism of a known anticancer drug, 5-FU. The effect of functional group substitution on the C-5 position of dUMP substrate with halogen atoms (F-, Cl- and Br- ) and electron withdrawing (CN-, NO₂-) and donating (NH₂-, OH-) groups towards the TS stability is investigated by means of molecular dynamics simulation technique. The simulated results showed that an additionally unique hydrogen bond between the substituted group of dUMP analogues and the hydroxyl group of Y94 was observed in most systems except CldUMP and BrdUMP analogues. The MM/PBSA binding free energy provides the orders of averaged binding affinity are: CldUMP ~ FdUMP > dUMP > BrdUMP for halogen analogues and CNdUMP ~ NO₂dUMP > OHdUMP ~ NH₂dUMP for electron withdrawing and donating analogues. Based on the B3LYP/6-31+G*-CHARMM hybrid method, the Michael addition and ternary covalent complex formation between dUMP and mTHF substrates to form the methylene bridging intermediate is determined via a concerted mechanism. By following the several tests of QM/MM approaches, the SCS-MP2 method with aug-cc-pVTZ basis sets is selected for reaction energy profile evaluation. Due to the close of activation energy with FdUMP anticancer drug and high affinity of the binding in CldUMP analogue, therefore, we proposed that the CldUMP analogue has a potent to be the new candidate inhibitor against thymidylate synthase function.