Role of alanine racemase in Trypanosoma and antiparisitic effect of new class of alanine racemase inhibitors.

GIRARD, RM; PAES, LS; PEREIRA, CA; DA SILVA, MS; PAVANI, RS; ELIAS, MC; SILBER, ARIEL

Santa Fe

Jornada; XXVIII Reunión Anual Sociedad Argentina de Protozoología (SAP); 2016

Sociedad Argentina de Protozoología (SAP)

In trypanosomes, L-amino acids have, in additionto its classical role on protein synthesis, variousfunctions including, not exhaustively: energymetabolism, osmoregulation, resistance to thermaland oxidative stress. Nevertheless, D-amino acidsare not found as part of the primary structure ofproteins and their biological functions remainelusive in parasites. In T. cruzi and T. vivax, theproline racemase (PR) are involved in host cellsinfection and was describe, as a potent mitogen.The occurrence of D-Alanine and an AlanineRacemase (AR) activity was also described inLeishmania amazonensis. Two copies of putativeAR gene in T. cruzi (respectively 1,167 bp (TcARA) and 759 bp (TcAR B) ) and one in T. bruceihave been identified. In silico analysis suggeststhat AR critical residues, involved in binding thecofactor pyridoxal 5?-phosphate (PLP) andracemization between L-and D-alanine, areconserved in both T. cruzi and T. brucei. ARspecific activity was measured in differentdevelopmental stages of T. cruzi and in T. bruceiprocyclic forms (PCF). AR activity was higher inepimastigotes and metacyclic trypomastigotesforms compared to amastigotes, trypomastigotesand intracellular epimastigotes. TcAR A werecloned and expressed as recombinant product(rTcAR A). rTcAR A showed a MW of 43 kDa, andVmax and Km values of 21.5 ± 6.04 mM and 60 ±6.7 μmol/min.mg. Immunofluorescence with aspecific anti-rTcAR serum showed a cytoplasmiclocalization for TcAR A. We decided to evaluateC3 which belong to a new class of AR inhibitorsagainst T. cruzi and T. brucei. C3 reduced: rTcARA activity; T. cruzi epimastigotes, procyclic andbloodstream forms growth; well as to inducephosphatidylserine exposure, DNA damage,deregulation of ROS, cytosolic Ca2+ andmitochondrial membrane potential in T. cruziepimastigote and procyclic forms. However, C3 donot produce cell cycle arrest in both organisms.