Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent

Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase

SummaryPurine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization

Pre-Steady-State Kinetic Analysis of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase from <i>Mycobacterium tuberculosis</i> Reveals Partially Rate-Limiting Product Release by Parallel Pathways

As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-<i>C</i>-methyl-d-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with <i>Mycobacterium tuberculosis</i> DXR (<i>Mt</i>DXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the <i>Escherichia coli</i> orthologue, <i>Mt</i>DXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of <i>Mt</i>DXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP⁺ followed by MEP. The partially rate-limiting release of MEP occurs via two pathwaysdirectly from the binary complex and indirectly via the <i>Mt</i>DXR·NADPH·MEP complexthe partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism

DXP Reductoisomerase: Reaction of the Substrate in Pieces Reveals a Catalytic Role for the Nonreacting Phosphodianion Group

The role of the nonreacting phosphodianion group of 1-deoxy-d-xylulose-5-phosphate (DXP) in catalysis by DXP reductoisomerase (DXR) was investigated for the reaction of the “substrate in pieces”. The truncated substrate 1-deoxy-l-erythrulose is converted by DXR to 2-<i>C</i>-methylglycerol with a <i>k</i>_cat/<i>K</i>_m that is 10⁶-fold lower than that for DXP. Phosphite dianion was found to be a nonessential activator, providing 3.2 kcal/mol of transition state stabilization for the truncated substrate. These results implicate a phosphate-driven conformational change involving loop closure over the DXR active site to generate an environment poised for catalysis

Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3‑Nitropropionate

Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including <i>Mycobacterium tuberculosis</i>, where it has been shown to be essential for cell survival during chronic infection. The pH–rate and pD–rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for ^D₂O<i>V</i> and 0.56 ± 0.07 for ^D₂O(<i>V</i>/<i>K</i>_succinate). Whereas the ^D₂O<i>V</i> is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse ^D₂O(<i>V</i>/<i>K</i>_succinate) indicates that substantially different kinetic parameters exist when the enzyme is saturated with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analogue, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a <i>K</i>_i of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a <i>K</i>_i* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for association and unity for dissociation. In contrast, itaconate, a succinate analogue lacking an acidic α-proton, did not display slow-binding behavior and yielded a ^D₂O<i>K</i>_i of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191–His193 pair to the thiolate–imidazolium form, a process that is favored in D₂O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the “true inhibitor” of ICL, does not bind directly and must be generated enzymatically

Arsenate and phosphate as nucleophiles at the transition states of human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has a low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1′-(14)C, 1′-(3)H, 2′-(2)H, 9-(15)N, and 5′-(3)H(2) KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5′-(3)H(2), and some slight difference between the 2′-(2)H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5′-(3)H(2) KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNP-catalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions