Plasmodium falciparum Purine Nucleoside Phosphorylase

Abstract

Plasmodium falciparum is a purine auxotroph, and starvation of purines is known to cause purine-less death in cultured cells (1-3). Parasites cultured in human erythrocytes can be killed rapidly by blocking PNP 1 with Immucillin-H and related Immucillins; however, these agents inhibit both P. falciparum and human PNPs, albeit with higher efficiency for human PNP (2, 4). The biochemical link between PNP inhibition and purine-less death is the formation of hypoxanthine, previously identified as a primary source of purines for P. falciparum (1, 3). The amino acid sequence of P. falciparum PNP as well as its substrate specificity differs from mammalian and bacterial enzymes (4, 5). We investigated this difference by structural analysis using Immucillin-H as a ligand to stabilize the catalytic site and as a starting point for synthesis of inhibitors more specific to PfPNP.Immucillin-H is effective in causing P. falciparum cell death by purine starvation even in the purine-rich environment of human erythrocytes. Hypoxanthine but not inosine can prevent the effect (2). The transition state of PfPNP has oxacarbenium ion character and ImmH binds to PfPNP with a dissociation constant of 860 pM, making it a powerful inhibitor for this target (Refs. 4 and 6; Fig. 1). However, ImmH was designed to be a transition state analogue of mammalian PNPs and binds with a constant of 56 pM to human PNP (7). Therefore, addition of this compound to human erythrocytes infected with P. falciparum has two effects, inhibition of human PNP at lower concentrations and inhibition of both human and PfPNPs at higher concentrations.Comparison of the PfPNP amino acid sequence against the data base of all PNPs indicated that this enzyme differs from mammalian and bacterial PNPs (see below). The structure of PfPNP in complex with ImmH and SO 4 revealed that the catalytic site has capacity for binding 5Ј-substituted nucleosides. 5Ј-Methylthioinosine and inosine are good substrates for PfPNP. We hypothesized that 5Ј-methylthio-substituted Immucillin would bind preferentially to PfPNP. This hypothesis was used to design, produce, and characterize 5Ј-methylthio-Immucillin-H (MT-ImmH), a novel transition state analogue inhibitor with high specificity for the P. falciparum PNP. EXPERIMENTAL PROCEDURESPfPNP-The coding sequence for PfPNP (4) was amplified by PCR from P. falciparum 3D7 genomic DNA, placed into the pTrcHis2-TOPO vector, expressed in Escherichia coli TOP 10, and purified to Ͼ95% homogeneity by nickel affinity chromatography. ImmH was synthesized by the convergent route (8). MT-ImmH was synthesized by the desilylation of the previously reported N-tert-butoxycarbonyl-7-O-tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-D-allo-heptononitrile followed by methanesulfonylation of the 7-hydroxyl and displacement of the resulting mesylate with thiomethoxide. The resultant compound was converted into MT-Immucillin-H in an analogous fashion to that previously described (9, 10). Structure and stereochemistry of MT-ImmH was confirmed ...

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

“…The PfPNP sequence has greater similarity to E. coli than to human PNP (9), and its structure is hexameric as is the E. coli enzyme (7,20,21). PfPNP, however, has unique substrate specificity when compared with either E. coli or mammalian PNPs (9,21).…”

“…MT-ImmH is the first immucillin analogue to show high specificity for malarial PNP. Although more specific for malarial PNP, MT-ImmH binds less tightly to PfPNP than ImmH (7). Refinements in inhibitor design based upon analysis of the transition state and crystal structures of PfPNP (7,8) may permit improved PfPNP specificity and potency.…”

“…were overexpressed in Escherichia coli using the methods described (7)(8)(9). ImmH and MT-ImmH were synthesized as described (7,10,11 (12). S-Adenosylmethionine was converted to MTA by incubating S-adenosylmethionine at pH 3-5 and at 70°C for 3.5 h. All labeled compounds were purified by reverse phase-HPLC (RP-HPLC) with a 15-m, 7.8 ϫ 300-mm C 18 Deltapak column (Waters Associates), eluting isocratically with 50 mM ammonium acetate, pH 5.0, and 25% methanol at 1 ml/min.…”

“…were overexpressed in Escherichia coli using the methods described (7)(8)(9). ImmH and MT-ImmH were synthesized as described (7,10,11 (12).…”

Descriptor-and fragment-based QSAR models for a series of Schistosoma mansoni purine nucleoside inhibitors

“…[2][3][4] In particular, the parasite Schistosoma mansoni, one of the etiologic agents of human schistosomiasis, lacks the de novo pathway for purine biosynthesis and depends entirely on the salvage pathway for its purine requirements for synthesis of RNA and DNA. [5][6][7][8][9] In this context, the use of selective PNP inhibitors from S. mansoni (SmPNP) can cause purine starvation, leading to death of the parasite. Schistosomiasis is a major infectious disease that affects 200 million people in 74 endemic areas worldwide.…”