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.
Plasmodium falciparum hypoxanthine−guanine−xanthine phosphoribosyltransferase (Pf HGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of Pf HGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic K i of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to Pf HGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to Pf HGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-D-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18 O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to Pf HGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.
Plasmodium falciparum
purine nucleoside phosphorylase (
Pf
PNP) catalyzes an essential step in purine salvage for parasite growth. 4′-Deaza-1′-Aza-2′-Deoxy-1′-(9-Methylene)-Immucillin-G (DADMe-ImmG) is a transition state analog inhibitor of this enzyme, and
P. falciparum
infections in an
Aotus
primate malaria model can be cleared by oral administration of DADMe-ImmG.
P. falciparum
cultured under increasing DADMe-ImmG drug pressure exhibited
Pf
PNP gene amplification, increased protein expression, and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (∼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of
Pf
PNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant
Pf
PNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed
Pf
PNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid
Pf
PNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid
Pf
PNP gave near-native
K
m
values for substrate, but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately ninefold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits is responsible for altered properties of the hybrid protein. Thus, gene amplification of
Pf
PNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies.
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