Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3‐[4‐(Reduced 3‐Pyridine Aldehyde‐Adenine Dinucleotide)]‐Pyruvate

Abstract

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase

“…Earlier studies had shown that 3-acetylpyridine nicotinamide adenine dinucleotide-pyruvate (ANAD-Pyr), a strong inhibitor of LDH, reduced lactate production in crude brain extracts, although the inhibitor was less effective in synaptosomes [9]. The main reason for the minimal effect in synaptosomes is likely associated with the hydrophilic nature of ANAD-Pyr and the low stability of the compound at physiological pH [9]. NADH-GA is also highly polar and very stable under physiological conditions and in contrast to ANAD-Pyr can not be degraded by LDH.…”

“…The increase in proton concentration initiates a cascade of events leading to apoptosis and cell death in cultured cells including cardiac myocytes [14,15,17,35,36]. Earlier studies had shown that 3‐acetylpyridine nicotinamide adenine dinucleotide‐pyruvate (ANAD‐Pyr), a strong inhibitor of LDH, reduced lactate production in crude brain extracts, although the inhibitor was less effective in synaptosomes [9]. The main reason for the minimal effect in synaptosomes is likely associated with the hydrophilic nature of ANAD‐Pyr and the low stability of the compound at physiological pH [9].…”

“…The key role of LDH in the above process stimulated the development of specific inhibitors of the enzyme, which could prevent the production of lactic acid and thus protect tissues from lactic acidosis. It has been shown that reduced APAD-Pyr strongly decreases lactate production in crude brain extracts [9]. It was, however, much less effective at inhibiting lactate production in synaptosomes.…”

“…In NAD‐Pyr the β‐carbon atom of pyruvate is covalently linked to the C‐4 position of the nicotinamide ring [6–8]. The NAD‐ and 3‐substituted NAD (3‐acetylpyridine nicotinamide adenine dinucleotide, APAD) pyruvate adducts also spontaneously form during the incubation of pyruvate and the dinucleotides at pH 11 [9,10]. Addition of these adducts to LDH results in strong suppression of enzyme activity [11,12].…”

“…Addition of these adducts to LDH results in strong suppression of enzyme activity [11,12]. These adducts are, however, relatively unstable at neutral pH and rapidly decompose into pyruvate and NAD or APAD [9,12].…”