Effects of Bisphosphonate Compounds on the HPRT activity and its Biological Function on Trypanosoma cruzi

JAVIER SANTOS; JOSÉ MARÍA DELFINO; WANDA M. VALSECCHI; SILVIA FERNANDEZ-VILLAMIL

CABA

Congreso; Drug Discovery for Neglected Diseases International Congress 2018 4th Scientific Meeting of the Research Network Natural Products against Neglected Diseases; 2018

Hypoxanthine phosphoribosyltransferase (HPRT) is the key enzyme of the purine salvage pathway. It is essential for the survival of trypanosomatids, organisms that -unlike humans- cannot benefit from de novo nucleotide synthesis. As such, this enzyme has been proposed as a potential target for drugs aimed at treating parasitic diseases. This study underscores the effect of different ligands (analogues of the substrate phosphoribosyl pyrophosphate), bisphosphonates (BPs), on the activity and biological function of T. cruzi HPRT (TcHPRT). To assess the action of these small molecules within a cellular environment, we exposed cultures of epimastigotes to selected BPs, demonstrating significant growth inhibition. These results showed a strong correlation with those on the isolated recombinant enzyme. Strikingly, the inhibitory effect is essentially null when assayed against an isolated human HPRT.Given that the TcHPRT is an essential enzyme for the nucleotide biosynthesis, we reasoned that enzyme inhibition may affect the synthesis of DNA arresting the cell cycle. To evaluate this issue, we decided to analyze the cellular cycle of the parasite by flow cytometry. As expected, parasites exhibited a significant delay from the beginning of phase S when cultured in the presence of these compounds. Moreover, we found a correlation between these results and the progression of the infection in Vero cells. We have previously shown1 that the protein adopts a tetrameric arrangement and, interestingly, the proteolytic removal of the C-terminal region (CTR) yields a dimer, showing somewhat increased activity. The inhibitory effect of olpadronate and ibandronate at high concentrations was higher for dimer than for the tetramer. Considering that TcHPRT is imported to glycosomes, inside the organelle, the CTR which includes the import signal- might be excised, giving rise to the dimeric form. To evaluate this hypothesis, western blot analysis was carried out using extracts of epimastigotes. Additionally, we show by first time evidence of the subcellular localization of TcHPRT. This new molecular and cellular knowledge becomes most relevant for the design of innovative parasite-targeted therapeutics.1Valsecchi, W. M. et al. (2016). The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase. Biochimica et Biophysica Acta 1864 655-666.