Carba-NAD

Two new analogs have been reported, the nicotinamide arabino analog ara-NAD (40) and the carbocyclio ribose analog carba-NAD+ (5/) (6 and 7). The arabino moiety has little impact on coenzyme function. With HL-ADH, the Vmax for )3-nra-NADH oxidation is six times faster than for /3-NADH, and for a-ara-NADH oxidation the Vmax is 1.7 times faster, both anomers are also active with... 

This value of -340 mV for carba-NAD+ is considerably higher than the previously reported redox potential of — 388 mV for the chemically related acyclic pentamethylene-iV-nicotinamide analogs (41), thus a general reassessment of these values is in order. 

SIRT2) in complex with carba-NAD and a histone H4 acetyllysine peptide (PDB code ISZD) (b) outline catalytic mechanism of lysine deacetylation by SIRT deacetylases, which proceeds by activation of the acetyl group by 0-glycosylation (c) representative SIRT inhibitor chemotypes. 

The carbocyclic dinucleotide carba-NAD [1] is designed to function as an inhibitor of ADP-ribosyl transferases. In this compound, a cyclopentane ring replaces the fiiranose of the nicotinamide mononucleotide moiety of NAD. This modification will cause the C-N bond to be resistant to enzymatic cleavage. The analog will otherwise closely resemble NAD. We expect therefore that carba-NAD and its relatives will constitute an important set of mechanism-based inhibitors for NAD glycohydrolases and for ADP-ribosyl transferases. 

After coupling, the acetyl groups are removed by brief treatment with methanolic ammonia. The product dinucleotides are purified by anion-exchange chromatography using DEAE-cellulose, and characterized spectroscopically. H-NMR spectra of the product carba-NAD is shown in Figs. 1 and 2. The spectrum of the dinucleotide exhibits the expected contributions from the pyridine nucleotide portion and from the adenosine nucleotide. 

Carba-NAD is effectively recognized by pyridine nucleotide specific binding sites. Thus, carba-NAD is reduced by yeast alcohol dehydrogenase with a Km of 1.7 X 10" M (Km for NAD is 4.5 x 1(H M) and with a maximal velocity 70% of that of NAD itself. Using equine liver alcohol dehydrogenase, a Km of 7.6 x 10" M is obtained for carba-NAD (Km for NAD is 24 X 10 M xmder identical conditions). Reduction occurs at a maximal velocity 25% of that measured for NAD itself. Carba-NAD is therefore demonstrated to be an effective substrate for this class of enzymes. 

Low concentrations of carba-NAD have been demonstrated to inhibit the NAD glycohydrolase isolated from Bungarus fasciatus venom (11). The mechanism of inhibition was determined by Lineweaver-Burk analysis of the effect of fixed inhibitor concentration on the initial rate of the reaction (Fig. 4). The inhibition of the NAD glycohydrolase by mixtures of [1] and [7] was determined to be of the competitive type. Replots of the KmW versus inhibitor concentration (not shown) were linear, and a Ki of 74 pM determined. It is our expectation that the Ki for pme carba-NAD will be lower than this value. As it stands, the Ki for carba-NAD is close to the Km of NAD, which we determine to be 33 pM. 

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