Recent studies have shown that Plasmodium falciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1'-(3)H]-, [2'-(3)H]-, [1'-(14)C]-, [9-(15)N]-, and [5'-(3)H]inosines were 1.184 +/- 0.004, 1.031 +/- 0.004, 1.002 +/- 0.006, 1.029 +/- 0.006, and 1.062 +/- 0.002 for the human enzyme and 1.116 +/- 0.007, 1.036 +/- 0.003, 0.996 +/- 0.006, 1.019 +/- 0.005, and 1.064 +/- 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D(N)A(N) (S(N)1) stepwise mechanism with very little leaving group involvement. The near-unity 1'-(14)C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1'-(14)C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9-(15)N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-(15)N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small beta-secondary 2'-(3)H KIE for both enzymes indicates a 3'-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5'-(3)H KIE reveals substantial distortion at the 5'-hydroxymethyl group which causes loosening of the C5'-H5' bonds during the reaction coordinate.
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.
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.
The aza-C-nucleosides, Immucillin-H and Immucillin-G, are transition state analogue inhibitors of purine nucleoside phosphorylase, a therapeutic target for the control of T-cell proliferation. Immucillin analogues modified at the 2'-, 3'-, or 5'-positions of the azasugar moiety or at the 6-, 7-, or 8-positions of the deazapurine, as well as methylene-bridged analogues, have been synthesized and tested for their inhibition of human purine nucleoside phosphorylase. All analogues were poorer inhibitors, which reflects the superior capture of transition state features in the parent immucillins.
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 ...
Purine nucleoside phosphorylases (PNPs) catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the fixed purine and phosphate nucleophiles. As the phosphorolysis reaction progresses along the reaction coordinate, the distance between the purine and carbocation increases and the distance between carbocation and phosphate anion decreases. Immucillin-H and Immucillin-G have been shown previously to be potent inhibitors of PNP. We now report the synthesis of a second generation of stable transition state analogues, DADMe-Immucillins 2, 3, and 4, with increased distance between ribooxacarbenium and purine mimics by incorporation of a methylene bridge between these groups. These compounds are potent inhibitors with equilibrium dissociation constants as low as 7 pM against human PNP. Stable chemical analogues of enzymatic transition states are necessarily imperfect since they lack the partial bond character of the transition state. The immucillins and DADMe-Immucillins represent approaches from the product and reaction side of the transition state.
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 DADMeImmucillins 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 k cat for huPNP being 18-fold greater than k cat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k chem /k cat 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 Asp 206 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.Inhibition of human purine nucleoside phosphorylase (huPNP) 1 by transition state analogue inhibitors shows promise for the control of T-cell cancers and autoimmune diseases (1-3). The combined inhibition of human and P. falciparum PNPs kills parasites cultured in human erythrocytes by purine-less death (4, 5). Immucillin-H (ImmH) is the first transition state analogue inhibitor developed in this class and shows a 56 pM K d for huPNP (6). It has recently entered phase II clinical trials against T-and B-cell cancers (7). ImmH was designed from the early ribooxacarbenium ion transition state structure of bovine PNP (8). More recently, the transition state structures have been solved for huPNP and PfPNP, which are both characterized by symmetric near fully dissociated ribooxacarbenium ion transition states (9).The second generation transition state analogue inhibitors were synthesized to mimic these dissociated transition states and include DADMe-Immucillin-H and DADMe-Immucillin-G. These are extraordinary inhibitors of huPNP with K d values of 16 and 7 pM, respectively. DA...
Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.
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