CNAD: a potent and specific inhibitor of alcohol dehydrogenase

Abstract

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric and isomeric analogue of NAD, in which the nicotinamide ring is linked to the sugar via a C-glycosyl (C5-C1') bond. CNAD acts as a general dehydrogenase inhibitor but shows unusual specificity and affinity for liver alcohol dehydrogenase (ADH, EC 1.1.1.1). The pattern of inhibition is congruent to 4 nM, with NAD as the variable substrate. These values are 3-5 orders of magnitude smaller than those obtained for CNAD in other dehydrogenases and are comparable to values observed for the tightest binding ADH inhibitors known. The specificity and affinity of CNAD for ADH are likely due to coordination of the zinc cation at the ADH catalytic site by the CNAD pyridine nitrogen. This is supported by kinetic and computational studies of ADH-CNAD complexes. These results are compared with those for a related analogue, CPAD. In this analogue, displacement of the pyridine nitrogen to the opposite side of the ring removes the specificity for ADH.

Crystallographic Studies of Isosteric NAD Analogs Bound to Alcohol Dehydrogenase: Specificity and Substrate Binding in Two Ternary Complexes

“…In contrast, CNAD shows unusual affinity for LADH. The inhibition constant of CNAD for LADH with respect to NAD is ~4 X 10~3 /uM (pH 8; Goldstein et al, 1994). This is significantly smaller than the K\ for NADH binding to LADH (0.4 jitM) and the K& for NAD (~50 ^M) (Dalziel & Dickinson, 1975;Goldstein et al, 1994).…”

“…The inhibition constant of CNAD for LADH with respect to NAD is ~4 X 10~3 /uM (pH 8; Goldstein et al, 1994). This is significantly smaller than the K\ for NADH binding to LADH (0.4 jitM) and the K& for NAD (~50 ^M) (Dalziel & Dickinson, 1975;Goldstein et al, 1994). Further, CNAD competes with both cofactor and substrate binding in LADH (Ki ~2 X 10-3 aiM) (Goldstein et al, 1994).…”

“…CNAD acts a general dehydrogenase inhibitor, competing with cofactor but resisting hydride reduction (Goldstein et al, 1994). Inhibition constants of CNAD with respect to NAD in malate, lactate, glutamate, and inosine monophosphate dehydrogenases range between 15 and 410 jiM (Goldstein et al, 1994;H. N. Jayaram, unpublished results).…”

“…5-/3-D-Ribofuranosylnicotinamide adenine dinucleotide (CNAD, Figure lb) is a neutral isosteric analogue of NAD (Pankiewicz et al, 1993). CNAD acts a general dehydrogenase inhibitor, competing with cofactor but resisting hydride reduction (Goldstein et al, 1994). Inhibition constants of CNAD with respect to NAD in malate, lactate, glutamate, and inosine monophosphate dehydrogenases range between 15 and 410 jiM (Goldstein et al, 1994;H.…”

Reversible inhibition of sheep liver sorbitol dehydrogenase by the antidiabetogenic drug 2‐hydroxymethyl‐4‐(4‐N,N‐dimethylaminosulfonyl‐1‐piperazino) pyrimidine

“…Kinetic data and the enzyme structure model [4] enable a rationalization of the interactions of SDI 158 with enzymic groups. As in the case of CNAD, a potent inhibitor of horse liver ADH [16], the primary interaction of inhibitory nitrogen, oxygen and sulphur nucleophiles with the related SDH can be expected to involve the catalytic zinc atom. The finding that piperazine (10 mM), dimethylsulfoxide [13] and various sulfonamides [12] have no significant inhibitory effect on SDH activity implies that the pyrimidine moiety of SDI 158 is coordinated to zinc in the binary and ternary complexes.…”