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Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.

Immucillin and DADMe-Immucillin inhibitors are tight binding transition state mimics of purine nucleoside phosphorylases (PNP). 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is proposed to form a similar transition state structure as PNP. The companion paper describes modifications of the Immucillin and DADMe-Immucillin inhibitors to better match transition state features of MTAN and have led to 5'-thio aromatic substitutions that extend the inhibition constants to the femtomolar range (Singh, V., Evans, G. B., Lenz, D. H., Mason, J., Clinch, K., Mee, S., Painter, G. F., Tyler, P. C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L. (2005) J. Biol. Chem. 280, 18265-18273). 5'-Methylthio-Immucillin A (MT-ImmA) and 5'-methylthio-DADMe-Immucillin A (MT-DADMe-ImmA) exhibit slow-onset inhibition with K(i)(*) of 77 and 2 pm, respectively, and were selected for structural analysis as the parent compounds of each class of transition state analogue. The crystal structures of Escherichia coli MTAN complexed with MT-ImmA and MT-DADMe-ImmA were determined to 2.2 A resolution and compared with the existing MTAN inhibitor complexes. These MTAN-transition state complexes are among the tightest binding enzyme-ligand complexes ever described and analysis of their mode of binding provides extraordinary insight into the structural basis for their affinity. The MTAN-MT-ImmA complex reveals the presence of a new ion pair between the 4'-iminoribitol atom and the nucleophilic water (WAT3) that captures key features of the transition state. Similarly, in the MTAN-MT-DADMe-ImmA complex a favorable hydrogen bond or ion pair interaction between the cationic 1'-pyrrolidine atom and WAT3 is crucial for tight affinity. Distance analysis of the nucleophile and leaving group show that MT-ImmA is a mimic of an early transition state, while MT-DADMe-ImmA is a better mimic of the highly dissociated transition state of E. coli MTAN.

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.

Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP.Immucillin-H (ImmH).SO(4) reveals a homohexamer with ImmH and SO(4) bound at each catalytic site. A solvent-filled cavity close to the 5'-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5'-carbon. Assays established 5'-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5'-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with K(d) values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP.MT-ImmH.SO(4) shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5'-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action.

Genetic deficiency of human purine nucleoside phosphorylase (PNP) causes T-cell immunodeficiency. The enzyme is therefore a target for autoimmunity disorders, tissue transplant rejection and T-cell malignancies. Transition state analysis of bovine PNP led to the development of immucillin-H (ImmH), a powerful inhibitor of bovine PNP but less effective for human PNP. The transition state of human PNP differs from that of the bovine enzyme and transition state analogues specific for the human enzyme were synthesized. Three first generation transition state analogues, ImmG (Kd = 42 pM), ImmH (Kd = 56 pM), and 8-aza-ImmH (Kd = 180 pM), are compared with three second generation DADMe compounds (4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillins) tailored to the transition state of human PNP. The second generation compounds, DADMe-ImmG (Kd = 7pM), DADMe-ImmH (Kd = 16 pM), and 8-aza-DADMe-ImmH (Kd = 2.0 nM), are superior for inhibition of human PNP by binding up to 6-fold tighter. The DADMe-immucillins are the most powerful PNP inhibitors yet described, with Km/Kd ratios up to 5,400,000. ImmH and DADMe-ImmH are orally available in mice; DADMe-ImmH is more efficient than ImmH. DADMe-ImmH achieves the ultimate goal in transition state inhibitor design in mice. A single oral dose causes inhibition of the target enzyme for the approximate lifetime of circulating erythrocytes.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.