NAD glycohydrolase inhibitors

The cycloalkyl lactamimide, MDL 12330A, is an inhibitor of the guanyl nucleotide stimulatory protein [17]. This compound has several inhibitory effects besides inhibiting ADP-ribosylation of the guanyl nucleotide stimulatory protein. It also inhibits adenylate cyclase and NAD-glycohydrolase. This compound was used to further clarify whether the stimulatory effect of cAMP on [ P]-ADP-ribose incorporation was... 

NIH, an inhibitor of NAD glycohydrolase blocks incorporation of adenine equivalents from NAD, not from ADPR. 

Nicotinamide (10 mM) and 3-aminobenzamide (5 mM) were also much more efficient inhibitors of NAD-glycohydrolase than of ADP-ribosyl transferase activity in brain mitochondria although the effect of 3 aminobenzamide on NAD-glycohydrolase was less potent (Fig. 1). Nevertheless, while using nicotinamide at 10 mM concentration, NAD-glycohydrolase activity was no more detectable in synaptic mitochondria and was inhibited by 90% in non-synaptic mitochondria, while the inhibition of ADP-ribosyl transferase activity was only of about 30-40%. 

ADP-ribosylation in rat liver mitochondria. Incubation of rat liver mitochondria or mitoplasts with radiolabeled NAD+ results in the incorporation of ADP-ribose into one major protein of SO kDa and a minor one of 30 kDa. In order to confirm that the acid insoluble radioactive material transferred to these proteins is in fact due to ADP-ribosyl transferase activity and not to NAD glycohydrolase activity we investigated the effects of some inhibitors of these activities. Our results demonstrate that ADP-ribosyl transferase activity is involved in the process of mitochondrial ADP-ribosylation, because when mitochondrial NAD glycohydrolase activity was inhibited by 10 mM nicotinamide or 10 mM 3-aminobenzamide, ADP-ribose transfer stUl occurred (Fig. 1). The inhibition... 

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. 

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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