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 (PfHGXPRT), 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.

Figure 1

Reactions catalyzed by (A) HGPRT, EC 2.4.2.8. and (B) HGXPRT, EC 2.4.2.8. (C) Purine metabolism in Trypanosoma sp. Reactions catalyzed by HGPRT and HGXPRT are depicted in red or blue text, respectively, over the reaction arrow. IMP: inosine monophosphate, AMPS: adenylosuccinate, AMP: adenosine monophosphate, ADP: adenosine diphosphate, ATP: adenosine triphosphate, XMP: xanthosine monophosphate, GMP: guanosine monophosphate, GDP: guanosine diphosphate, and GTP: guanosine triphosphate.

Figure 2

TcC operates via an ordered Bi Bi mechanism in which PRPP binds first followed by the 6-oxopurine (Hx or Gua). Once the E-PRPP-Purine complex forms, the flexible loop closes over the active site (k₅ step), converting the central complex to E′-PRPP-Purine. TcC catalysis converts E′-PRPP-Purine to E′-PPi-NMP for which k₇ ≫ k₈, followed by opening of the flexible loop (k₉ step) to afford the E-PPi-NMP complex, which is followed by the ordered release of PPi and NMP (IMP or GMP).

Figure 3

ITC binding assays of (A) PRPP and (B) IMP to free TcC enzyme, and (C) thermodynamic profiles of PRPP and IMP binding to free TcC enzyme.

Figure 4

(A) Viscosity effects on k_cat/K_PRPP of TcC. In the presence of glycerol (blue), k_cat/K_PRPP increased, thus exhibiting an inverse effect. Data were fit to eq 11, which provided a slope of −0.5 ± 0.3. The macroviscosogen control (PEG_10,000) is shown in red. (B) Viscosity effects on k_cat of TcC. Increasing levels of glycerol (blue) decreased the apparent k_cat of TcC, and fitting of the data for the plot of k_cat/(k_cat)_ηvs η_rel to eq 11 resulted in a slope of 2.2 ± 0.2. The macroviscosogen control (PEG_10,000) is shown in red. (C) TcC viscosity effects on k_cat fit to eq 12, providing a slope of 1.8 ± 0.3.

Figure 5

Isotope partitioning of E-PRPP* (EA*) to IMP* (Q*) increased with increasing concentrations of Hx to a limiting value (Hx ⇒ ∞) for IMP*/E-PRPP* (Q*/EA*) of 0.98 ± 0.01.

Figure 6

(A) PfHGXPRT S_N1-like reaction mechanism, showing the oxocarbenium TS structure.^,PfHGXPRT TSAI Immucillin-HP (8) is compared to the proposed S_N1-like TS. The iminoribitol ring of 8 (when protonated) mimics the positive charge in the oxocarbenium TS. (B) Experimental KIEs on TcC with radiolabeled PRPP. The remote labels (5-¹⁴C and 5-³H) are shown in pink and green, respectively; the anomeric proton label (1-³H) is shown in blue, and the anomeric carbon label (1-¹⁴C) is shown in red.

Chart 1. TSAIs of PfHGXPRT Evaluated as Inhibitors of T. cruzi HGPRTsa