Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases.

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Crystallization and preliminary X-ray crystallographic analysis of adenosine 5'-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5'-phosphate.

Adenosine 5'-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5'-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6(2)22, with unit-cell parameters a = b = 131.325, c = 208.254 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.34 A resolution from a crystal in complex with coformycin 5'-phosphate and to 4.05 A resolution from a crystal of a mercury derivative.

Enantiospecific synthesis of carbapentostatins.

In this paper we describe enantioselective syntheses of (+)-carbapentostatin (8) and its cyclopentyl analogue 12b. A new and efficient one-pot, two-step preparation of aldehyde 15 has been developed, based on the borane reduction of N-Pf-protected L-aspartic acid gamma-methyl ester (13) and Swern oxidation of the resulting alcohol. Homologation to diester 18 and ring formation by Dieckman cyclization, followed by reduction and dehydration steps, afford the 4-amino-1-cyclopentenemethanol derivative 22. Hydroboration and oxidation transform this compound stereospecifically into aminocyclopentanol 26, the key aminocyclitol component for an asymmetric synthesis of (+)-carbapentostatin.

Binding thermodynamics of the transition state analogue coformycin and of the ground state analogue 1-deazaadenosine to bovine adenosine deaminase.

Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermodynamically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are -4.7 +/- 0.8 kJ/mole-K and -1.2 +/- 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect. Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.

AMP deaminase inhibitors. 3. SAR of 3-(carboxyarylalkyl)coformycin aglycon analogues.

N3-Substituted coformycin aglycon analogues with improved AMP deaminase (AMPDA) inhibitory potency are described. Replacement of the 5-carboxypentyl substituent in the lead AMPDA inhibitor 3-(5-carboxypentyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1, 3]diazepin-8-ol (2) described in the previous article with various carboxyarylalkyl groups resulted in compounds with 10-100-fold improved AMPDA inhibitory potencies. The optimal N3 substituent had m-carboxyphenyl with a two-carbon alkyl tether. For example, 3-[2-(3-carboxy-5-ethylphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4, 5-d][1,3]diazepin-8-ol (43g) inhibited human AMPDA with a K(i) = 0. 06 microM. The compounds within the series also exhibited >1000-fold specificity for AMPDA relative to adenosine deaminase.

AMP deaminase inhibitors. 2. Initial discovery of a non-nucleotide transition-state inhibitor series.

A series of N3-substituted coformycin aglycon analogues are described that inhibit adenosine 5'-monophosphate deaminase (AMPDA) or adenosine deaminase (ADA). The key steps involved in the preparation of these compounds are (1) treating the sodium salt of 6, 7-dihydroimidazo[4,5-d][1,3]diazepin-8(3H)-one (4) with an alkyl bromide or an alkyl mesylate to generate the N3-alkylated compound 5 and (2) reducing 5 with NaBH(4). Selective inhibition of AMPDA was realized when the N3-substituent contained a carboxylic acid moiety. For example, compound 7b which has a hexanoic acid side chain inhibited AMPDA with a K(i) = 4.2 microM and ADA with a K(i) = 280 microM. Substitution of large lipophilic groups alpha to the carboxylate provided a moderate potency increase with maintained selectivity as exemplified by the alpha-benzyl analogue 7j (AMPDA K(i) = 0.41 microM and ADA K(i) > 1000 microM). These compounds, as well as others described in this series of papers, are the first compounds suitable for testing whether selective inhibition of AMPDA can protect tissue from ischemic damage by increasing local adenosine concentrations at the site of injury and/or by minimizing adenylate loss.

AMP deaminase inhibitors. 4. Further N3-substituted coformycin aglycon analogues: N3-alkylmalonates as ribose 5'-monophosphate mimetics.

AMP deaminase (AMPDA) inhibitors increase the levels of extracellular adenosine and preserve intracellular adenylate pools in cellular models of ATP depletion and therefore represent a potential new class of antiischemic drugs. Recently we reported that replacement of the ribose 5'-monophosphate component of the very potent transition-state analogue AMPDA inhibitor coformycin monophosphate (1) with a simple alkylcarboxy group resulted in potent, selective, and cell-penetrating AMPDA inhibitors. Here we report that replacement of this alkylcarboxy group with an alpha-substituted alkylmalonic acid resulted in enhanced inhibitor potency. The lead compound, 3-(5, 5-dicarboxy-6-(3-(trifluoromethyl)phenyl)-n-hexyl)coformycin aglycon (21), exhibited an AMPDA K(i) of 0.029 microM which is (3 x 10(5))-fold lower than the K(M) for the natural substrate AMP. A comparison of inhibitory potencies shows that the diacid analogues with alpha-benzyl substituents are 2-10-fold more inhibitory than similar monoacid-monoester, monoester-monoamide, or diester derivatives. Finally, these diacid analogues are 2-40-fold more potent inhibitors than the corresponding monocarboxylates.

The design and synthesis of inhibitors of adenosine 5'-monophosphate deaminase.

Carbocylic coformycin (4) is a potent herbicide whose primary mode of action involves inhibition of adenosine 5'-monophosphate deaminase (AMPDA) following phosphorylation of the 5'-hydroxyl group in vivo. The search for more stable and accessible structures led to the synthesis of carbocyclic nebularine (8) and deaminoformycin (10). The latter compound is a good herbicide and its corresponding 5'-monophosphate 14 is a strong inhibitor of plant AMPDA (IC50 100 nM).

Design, synthesis, and structure-activity relationships of the first highly potent, selective, and bioavailable adenosine 5'-monophosphate deaminase inhibitors.

Structure-activity studies have been performed to optimize the potency of this novel series of AMPDA inhibitors. Conformational rigidification of the N-3 sidechain resulted in substantial effect on the potency. Addition of the hydrophobic groups provided further benefit. The most potent compound identified, 4g (Ki = 3 nM), bears little structural resemblance to AMP and exhibits a remarkable improvement (10(3) and 10(5)) in binding affinity relative to the original lead and AMP, respectively. The application of prodrug strategy achieved a large improvement (benzyl ester 5d) in oral bioavailability, resulting in compounds that should be useful in evaluating the role of AMPDA in normo- and pathophysiological states.

Design and synthesis of the first potent, selective, and cell penetrating adenosine 5'-monophosphate deaminase inhibitors.

A major milestone in purine metabolism research has been achieved with the discovery of these potent and selective AMPDA inhibitors. These inhibitors of AMPDA are based on carboxypentyl substitution on N-3 of the coformycin aglycon. They are simpler than coformycin ribose 5'-monophosphate, more stable, selective against other AMP binding enzymes as well as ADA and have good cell penetration and good oral bioavailability. These compounds and their more potent analogs are the first compounds with suitable characteristics to allow a definitive analysis of the role of AMPDA in cellular metabolism and AMPDA as a therapeutic target.

How important is the N-3 sugar moiety in the tight-binding interaction of coformycin with adenosine deaminase?

Preliminary findings on the possible important role of the N-3 sugar moiety of coformycin in its tight-binding interaction with adenosine deaminase (ADA) are reported. The compound 3-beta-D-Ribofuranosyl-5,6,7,8-tetrahydro-4H-imidazo[4,5-d][1,3]diaze pin-5-one-8-ol (1), its 3-benzyl analogue (6), and the aglycon (7) served as probes. The first two were both found to be competitive inhibitors of ADA with Ki's in the range of 10(-5) M, while the last one was inactive.

Electrostatic potential surfaces of the transition state for AMP deaminase and for (R)-coformycin, a transition state inhibitor.

The transition state for the hydrolysis of AMP by AMP deaminase has been characterized by heavy atom kinetic isotope effects (Merkler, D.J., Kline, P.C., Weiss, P., and Schramm, V.L. (1993) Biochemistry 32, 12993-13001). The experimentally established transition state includes a bond order of 0.8 to the attacking water nucleophile, a full bond order to the exocyclic 6-amino group, rehybridization of C-6 of the purine ring to sp3 and protonation of N-1 by Glu633. The transition state is one the path to formation of an unstable tetrahedral intermediate in which the exocyclic amine undergoes rapid protonation followed by its departure. In this mechanism, the highest energetic barrier on the reaction coordinate is the attack of the zinc-activated water. In a further test of this transition state structure, the electrostatic potential surface for the purine ring of the transition state has been determined by molecular orbital calculations and compared to that of the base of (R)-coformycin 5'-monophosphate, a slow onset, tight binding inhibitor of AMP deaminase that binds with an overall dissociation constant of 10(-11) M. The electrostatic potential surfaces of the aglycones of the transition state and (R)-coformycin are compared to the adenine ring of the substrate and to an alternative transition state structure in which the transition state is late, with fully bonded hydroxyl and fully protonated exocyclic amine. The results indicate a near-match of the electrostatic potential surfaces for the early transition state and (R)-coformycin. The electrostatic nature of the late transition state with a protonated amine leaving group differs both from the transition state determine by kinetic isotope effects and from that of (R)-coformycin analogues. The results provide evidence that the nature of the enzyme-stabilized transition state for adenine deamination involves an early transition state with a partially bonded hydroxyl group. The observed tight binding inhibition by (R)-coformycin analogues as transition state inhibitors results from the similarity of the partial charges on the inhibitors to that of the enzymatic transition state stabilized by AMP deaminase.

Herbicidal nucleosides from microbial sources.

The structures of five naturally-occurring herbicidal nucleosides have been determined by spectral analysis. Three (5'-deoxyguanosine, coaristeromycin and 5'-deoxytoyocamycin) are novel natural products while the remaining two (coformycin and adenine 9-beta-D-arabinofuranoside) are known natural products which have not previously been reported to be herbicidal.