Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase

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.

Crystal structures of Trypanosoma brucei hypoxanthine – guanine – xanthine phosphoribosyltransferase in complex with IMP, GMP and XMP

“…This binding mode is commonly observed for the nucleoside monophosphates when in complex with the 6‐oxopurine PRTs. However, it has also been observed in other crystal structures that the 5′‐phosphate group can be located outside the 5′‐phosphate binding pocket and protruding out from the active site (Fig. ).…”

“…The quaternary structures of 6‐oxopurine PRTs from several organisms have previously been determined and have been shown to be either tetramers or dimers . For example, human HGPRT and Pf HGXPRT are tetrameric, T. cruzi HGPRT can exist as either a dimer or tetramer , while Tbr HGPRT and Tsu HGXPRT are dimers. Tbr HGXPRT was analysed by SEC‐MALLS (size exclusion chromatography multi‐angle laser light scattering) which showed that the major component has a molecular weight of 53.9 ± 0.3 kDa (Fig.…”

“…Plasmodium falciparum (Pf) HGXPRT is an example of a 6-oxopurine PRT that can use all three bases, but for this enzyme there is a > 300-fold preference for hypoxanthine and guanine over xanthine [26]. An isoform of the other 6-oxopurine PRT in T. brucei, TbrHGPRT, weakly recognizes xanthine as the substrate but the catalytic efficiency for this substrate is > 1000-fold lower than for guanine and hypoxanthine (Table 1) [24]. As indicated earlier, there are two other known 6-oxopurine PRTs that are able to use the three naturally occurring purine bases.…”

Crystal structures of APRT from Francisella tularensis – an N–H···N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases

“…The other two loops, loop II and loop IV, are observed to make a network of interactions with terminal phosphate moieties of PRPP. The PRPP binding pocket is highly conserved among all Type-1 PRTases with a canonical DD motif, which stabilizes the co-factor Mg 2+ ion together with the ribose moiety of PRPP [32][33][34].…”

Synthesis and evaluation of symmetric acyclic nucleoside bisphosphonates as inhibitors of the Plasmodium falciparum, Plasmodium vivax and human 6-oxopurine phosphoribosyltransferases and the antimalarial activity of their prodrugs

“…19 Nucleoside phosphonates bearing guanine or hypoxanthine as the nucleobase are effective inhibitors of Pf HGXPRT and Pv HGPRT. They also inhibit the bacterial 6-oxopurine PRTases from Mycobacterium tuberculosis, 20 Escherichia coli, 21,22 and the HGPRT found in the parasite Trypanosoma brucei 23 . A variety of different structural analogues of these phosphonate inhibitors have been synthesised, including the single chain ANPs, the ANbPs, the aza-ANPs, the pyrrolidine nucleoside phosphonates, the pyrrolidine nucleoside bisphosphonates and the acylic immucillin phosphonates.…”