Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine–Guanine Phosphoribosyltransferases

Abstract

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi , affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo , and relies on the salvage of preformed purines from the host. Hypoxanthine–guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro : TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase ( Pf HGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors

“… a All assays were performed at 37 °C (pH 8.3) in the presence of 12 mM MgCl 2 . Data were fit to eq to afford apparent kinetic parameters. b Glockzin et al, 2022 c Wenck et al, 2004 …”

“…The selectivity index (SI) was calculated as the app K i for human HGPRT ( Hs HGPRT) divided by the app K i for Tc HGXPRT. The synthesis of the Tc HG­(X)­PRT inhibitors presented in this study, the inhibition assays, and data for Tc HGPRT, Hs HGPRT, and Pf HGXPRT have been previously described. ,, …”

“…35 We have previously demonstrated the conservation of this reaction mechanism in TcHGPRT, along with the feasibility of repurposing high affinity TSAIs developed against the plasmodial enzyme as potent inhibitors of TcHGPRT. 43 These inhibitors contain a secondary amine that upon protonation structurally mimics the putative ribooxacarbenium ion predicted in the TS of the reaction catalyzed by Pf HGXPRT (Figure 8, center) as well as the P. falciparum and human purine nucleoside phosphorylases. [39][40][41][42]78 Considering the active site conservation between TcHGXPRT and Pf HGXPRT as well as the experimental evidence of the importance of inhibiting, concomitantly, both the HGPRT and HGXPRT isozymes in Trypanosoma, 32 we reevaluated this set of compounds (Figure 8) as potential inhibitors of TcHGXPRT.…”

“…cruzi genome encodes four HG­(X)­PRT isoforms. We recently characterized two isoforms as HGPRT ( Tc HGPRTsisoforms TcA and TcC), demonstrating their activity and catalytic equivalence, substrate preferences for Hx and Gua, kinetic mechanism, and identification of the rate-limiting step(s) . We also reported on a subset of Immucillins that are inhibitors of Tc HGPRT activity in vitro , the most potent of which exhibited low nanomolar affinity …”

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferase

“…Synthesis of Transition State Analogue Inhibitors. Compounds 1, 4, 8, and 19, 46 2, 3, and 9-12, 24 and 5 and 6 23 were synthesized as reported. The synthetic procedures and NMR spectra for all new compounds are in the Supporting Information.…”