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

Abstract

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.

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

“…One interpretation could be that inhibitor 6 is binding to Tc HGXPRT in two conformations, one with the 5′-phosphate bound as indicated in Figure and one with its 5′-phosphate bound to loop 1. In fact, design strategies of acyclic nucleoside phosphonate as Pf HGXPRT and Tbb HG­(X)­PRTs inhibitors containing a second phosphate group intended to bind to loop 1 have been proposed. , However, it is well-described that the family of TSAIs tested in this study (Table ) binds to Pf HGXPRT in the presence of a PPi molecule bound to loops 1 and 4, ,, and our inhibition data revealed the potentiation of the inhibitory activity in the presence of PPi in the in vitro assays, with a specially pronounced effect on K i values of 6 (Figure S18). These results combined with the structure of 6 bound to Tc HGXPRT in the presence of the second Pi (a PPi analogue) suggest a similar mode of binding is expected to occur in T .…”

“…The plasmodial enzyme also presents the core and hood domain architecture characteristic of the Type I PRTases; 56 however the anchor domain is absent, suggesting that this may be unique to the trypanosomal HGXPRTs. A distinction worth noting is the structure of Pf HGXPRT loop 2�the highly disordered residues in T. cruzi assume an antiparallel β-hairpin conformation in P. falciparum (for example, see Figure S16B), and the densities of these residues are well-defined in the available high resolution crystallographic structures (≤2 Å), 34,40,75 with B-factors consonant with a structurally more rigid segment of the protein. The more defined organization of the secondary structure of these residues and Pf HGXPRTdetermined intrinsic kinetic isotope effects 35 suggest that opening and closing of its active site does not play a relevant role in its kinetic mechanism as it does in T. cruzi.…”

“…82 (S)-1, the Gua-based version of (S)-2, is the most potent inhibitor of TcHGXPRT in the set evaluated, with an approximately 2.5-fold improvement when compared to (S)-2. The structure of Pf HGXPRT bound to (S)-1 has been recently determined (7TUX.pdb 34 ). All Pf HGXPRT residues that interact with (S)-1 are conserved in TcHGXPRT and adopt the same conformation within the active site (Figure S23A).…”

N2′‐Branched Acyclic Nucleoside Phosphonates Containing 9‐Deazahypoxanthine as Inhibitors of Plasmodium falciparum 6‐Oxopurine Phosphoribosyltransferase

“…Structurally, they consist of 9‐deazahypoxanthine or 9‐deazaguanine linked to an acyclic ribocation phosphonate mimic. The selectivity ratios [ K i (Hs)/ K i ( Pf )] of reported AIPs range up to 490 in favor of Pf HGXPRT [21] . However, a structural explanation for the observed selectivity values has yet to be advanced.…”

“…The selectivity ratios [K i (Hs)/ K i (Pf)] of reported AIPs range up to 490 in favor of PfHGXPRT. [21] However, a structural explanation for the observed selectivity values has yet to be advanced. The most likely reason lies in the difference in flexibility between a several loops that are part of the active site or surround the active sites in these two enzymes.…”