Structure, function and effect of bisphosphonates on Trypanosoma cruzi hypoxanthine phosphoribosyltransferase

VALSECCHI WM; ROSSI RC; SANTOS J; DELFINO, J. M.

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

This study investigates the structure-function relationship and the effect of a family of bisphosphonates (BPs) on T. cruzi hypoxanthine phosphoribosyltransferase (TcHPRT). This enzyme catalyzes the transfer of ribose‑1-phosphate from phosphoribosyl pyrophosphate (PRPP) to hypoxanthine or guanine bases, yielding IMP or GMP, respectively, and has been proposed as a prime target for drugs aimed at treating parasitic diseases since its activity is essential for the survival of trypanosomatids. Contrary to the long-standing claim that TcHPRT is a monomer in solution, we have previously shown that the protein adopts a tetrameric arrangement. Interestingly, the proteolytic removal of the C-terminal region yields a dimer, showing enhanced activity1. Here we present a novel kinetic analysis of the TcHPRT activity, which is fully consistent with its inhibition induced by the set of BPs. The proposed kinetic scheme takes into account the occurrence of cooperativity ‑substrate binding exhibits a sigmoidal mode- and a partially ordered sequence for the binding of substrates and inhibitors. Remarkably, BPs display a biphasic behavior: activating at low concentrations and inhibiting at high concentrations, showing a competitive fashion. Cooperativity of PRPP binding, as well as the activating effect demonstrated by BPs, is more pronounced for the tetramer than for the dimer. On the contrary, the inhibitory action is less marked for the tetramer. Strikingly, the inhibitory effect of BPs is essentially null when assayed against human HPRT, a fact that might be greatly advantageous for the design of selective drugs. We also show here a structural comparison between both variants in order to underline possible reasons for such a selective effect of BPs. This new molecular knowledge becomes most relevant for the design of innovative parasite-targeted therapeutics.