Abstract
Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.
Highlights
Trypanosoma brucei is the protozoan parasite that is the causative agent of Human African Trypanosomiasis (HAT), known as African sleeping sickness
The kinetic constants of the naturally occurring base substrates for Trypanosoma brucei (Tbr) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are presented in Table 1, showing that guanine is the preferred substrate with the lowest Km (2.3 μM) and the most rapid turnover value, and a kcat/Km value that is 3.3-fold higher than that of hypoxanthine
Comparing the activities of the substrates of this enzyme with that of Trypanosoma cruzi (Tcr) HGPRT shows that they have similar Km values, all in the 2–10 μMrange, and similar kcat values in the range of 17–41 s−1 (Table 1)[27]
Summary
Trypanosoma brucei is the protozoan parasite that is the causative agent of Human African Trypanosomiasis (HAT), known as African sleeping sickness. Five therapeutic agents to treat this disease, i.e. pentamidine, eflornithine, nifurtimox, melarsoprol and suramin, are currently available These are far from ideal since they have numerous severe side effects including hypoglycemia, hypotension, encephalopathic syndrome, peripheral neuropathy and hepatic toxicity[3,4,5]. There is clear redundancy in the salvage pathway enzymes, but since the parasite takes up the prevailing purine precursors (i.e. hypoxanthine, xanthine and inosine) from blood serum and cerebrospinal fluid, at least some of the enzymes responsible for the synthesis of GMP, AMP and IMP should be essential in vivo[16] This is exemplified by a recent study by Li and coworkers that showed GMP synthase is essential for T. brucei virulence and viability in vivo[13]. The crystal structures of this enzyme in complex with IMP and GMP and with three ANPs have been determined with the aim to use insights from this data to improve the design of the ANP inhibitors resulting in more potent activity
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