ulodesine and forodesine

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Topics: Animals; Catalytic Domain; Cattle; Enzyme Inhibitors; Humans; Models, Molecular; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines; Thermodynamics

Neighboring-group participation in the reaction catalyzed by purine nucleoside phosphorylase involves a compression mode between the 5'- and 4'-ribosyl oxygens, facilitated by His257. The His257Gly mutant opens a space in the catalytic site. Hydrophobic 5'-substituted Immucillins are transition-state analogue inhibitors of this mutant enzyme. Dissociation constants as low as 2pM are achieved, with K(m)/K(d) as high as 400,000,000.

Topics: Catalytic Domain; Crystallography, X-Ray; Glycine; Histidine; Humans; Imidazoles; Kinetics; Mutation; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Structure-Activity Relationship

Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.

Topics: Crystallography, X-Ray; Enzyme Inhibitors; Humans; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines; Stereoisomerism; Substrate Specificity

Transition state analogues of PNP, the Immucillins and DADMe-Immucillins, were designed to match transition state features of bovine and human PNPs, respectively. The inhibitors with or without the hydroxyl and hydroxymethyl groups of the substrate demonstrate that inhibitor geometry mimicking that of the transition state confers binding affinity discrimination. This finding is remarkable since crystallographic analysis indicates complete conservation of active site residues and contacts to ligands in human and bovine PNPs.

Topics: Animals; Arsenates; Cattle; Enzyme Inhibitors; Erythrocytes; Humans; Mice; Molecular Structure; Protein Binding; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrrolidines

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

Topics: Catalytic Domain; Crystallography, X-Ray; Inosine; Kinetics; Models, Molecular; Molecular Mimicry; Mycobacterium tuberculosis; Protein Conformation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity