Interaction of α-thymidine Inhibitors and Natural Substrates with Thymidylate Kinase from Plasmodium falciparum

Abstract

Malaria is one of the most devastating parasite-borne infectious diseases and Plasmodium falciparum is associated with the deadliest form of malaria. Due to theemerging drug resistance against current treatments in these parasites, there is continuous need in developing novel anti-malarial drugs. Thymidylate kinase from Plasmodium falciparum (PfTMK) is an attractive druggabletarget for the development of anti-malarial agents due to its vital role in the Plasmodium life cycle and potential in drug specificity. Several groups have developed and reportedPfTMK inhibitors, however, none have entered clinical trials. The field has been limited by the lack of structure-activity relationship (SAR) studies to aid further structuralmodifications of existing inhibitors; furthermore, there has not been a study that comprehensively characterize PfTMK-substrate interaction in order to elaborate on theunique substrate specificity of PfTMK of being capable of phosphorylating both dTMP and dGMP. In our study, we aim to address these concerns in the current field. We designed and synthesized small molecules derived from a reported PfTMK inhibitor, and thoroughly characterized the synthesized compounds in terms of enzyme inhibition and binding property; we also probed for binding site of the synthesized derivatives to provide an understanding on the SAR. We acquired methyl side-chain (ILV) assignments forPfTMK. The PfTMK-substrate interaction was comprehensively characterized in terms of steady-state kinetics, binding property, thermodynamics and dynamics. These studies provide significant insights on the unique substrate specificity of PfTMK; and more importantly, revealed a unique half-site binding mechanism with dGMP, in which we showed that the binding of the first dGMP molecule results in intermonomer communication and possible conformation change that prohibits the binding of second dGMP in the other subunit of the PfTMK dimer. These observations have significant implication on the future PfTMK inhibitor design; guanosine-based small moleculescould be more optimal than their thymidine-based counterparts in terms of PfTMK inhibition and binding specificity.