Amidrazone Analogues of d-Ribofuranose as Transition-State Inhibitors of Nucleoside Hydrolase

The transition state of inosine during hydrolysis by nucleoside hydrolase has been characterized by kinetic isotope effects, bond-energy/bond-order vibrational analysis, and molecular electrostatic potential surface calculations [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788–10795; Horenstein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089–7097], The heterocyclic base is protonated and the anomeric carbon of the ribofuranosyl ring is flattened to form a transition-state with extensive oxocarbenium ion character. With their delocalized charge and flattened structures, amidrazone analogues of d-ribofuranose provide both geometric and electronic mimics of the ribosyl group at the transition-state of nucleoside hydrolase. A family of riboamidrazones was synthesized with H, phenyl, and p-nitrophenyl N-substituents. The analogues were competitive inhibitors with respect to inosine and gave K_i values of 10⁻⁵,2×10⁻⁷, and 1 × 10⁻⁸ M, respectively. (p-Nitrophenyl) riboamidrazone exhibited slow-onset, tight-binding inhibition, with an overall dissociation constant of 2 × 10⁻⁹ M. The binding is reversible with an off-rate of 3 × 10⁻³ s⁻¹. Tight binding can be attributed to the close spatial match between the molecular geometry of (p-nitrophenyl)riboamidrazone and the transition-state stabilized by nucleoside hydrolase. The favorable binding interactions of the (p-nitrophenyl)riboamidrazone include oxocarbenium ion mimicry, isosteric ribosyl hydroxyls, and hydrophobic and H-bonding interactions at the nitrophenyl group. Analysis of the conformational space available to the (p-nitrophenyl)riboamidrazone indicates that the geometry that approximates the enzyme-stabilized transition state is 7–14 kcal/mol unfavorable relative to the global conformational minimum for free inhibitor. The apparent overall K_d of 2 nM represents only a fraction of the intrinsic energy available for transition-state interactions with nucleoside hydrolase. When corrected for the energy of distortion required to achieve the transition-state conformation, (p-nitrophenyl)riboamidrazone binds with an affinity near that expected for an ideal transition-state analogue.