Inhibitors of cellular niacinamide mononucleotide formation...

Details Inhibitors of cellular niacinamide mononucleotide formation... Inhibitors of cellular niacinamide mononucleotide formation...

2001-08-23

2003-01-14

Leary, Louise N. (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S004000, C435S029000, C424S573000, C424S172100, C424S174100

Reexamination Certificate

active

06506572

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to new biologically active compounds which inhibit the cellular formation of niacinamide mononucleotide, which is one of the essential intermediates in the NAD(P) biosynthesis in the cell. The invention further concerns pharmaceutical compositions containing these compounds and their use, especially in the treatment of cancers, leukaemias or for immunosuppression. The invention also provides screening methods as a tool for detecting the above active compounds, and for examination of cell types with respect to their NAD(P) synthesis pathway.
TECHNICAL BACKGROUND OF THE INVENTION
NAD is synthesized in mammalian cells by three different pathways starting either from tryptophan via quinoline acid, from niacin (also referred to as nicotinic acid) or from niacinamide (also referred to as nicotinamide), as shown in FIG.
1
.
The addition of a phosphoribosyl moiety results in the formation of the corresponding mononucleotides, niacin mononucleotide (dNAM) and niacinamide mononucleotide (NAM). Quinoline acid is utilized in a reaction with phosphoribosyl pyrophosphate (PRPP) to form niacin mononucleotide (dNAM). The enzyme catalyzing this reaction, quinoline acid phosphoribosyl transferase (
), is found in liver, kidney and brain.
Niacin reacts with PRPP to form niacin mononucleotide (dNAM) The enzyme catalyzing this reaction is niacin phosphoribosyl transferase (
) and is widely distributed in various tissues. Both pathways starting either from tryptophan or from niacin as NAD precursors merge at the stage of the niacin mononucleotide formalin.
Niacinamide reacts with PRPP to give niacinamide mononucleotide (NAM). The enzyme that catalyses this reaction is niacinamide phosphoribosyl transferase (z,
902
). This enzyme is specific for niacinamide and is entirely distinct from niacin phosphoribosyl transferase (
). It is also widely distributed in various tissues.
The subsequent addition of adenosine monophosphate to the mononucleotides results in the formation of the corresponding dinucleotides: Niacin mononucleotide and niacinamide mononucleotide react with ATP to yield niacin adenine dinucleotide (dNAD) and niacinamide adenine dinucleotide (NAD), respectively. Both reactions, albeit taking place on two different pathways, are catalyzed by the same enzyme, NAD pyrophosphorylase (
).
A further amidation step is needed to convert niacin adenine dinucleotide (dNAD) to niacinamide adenine dinucleotide (NAD) The enzyme which catalyses this reaction is NAD synthetase (
). NAD is the immediate precursor of niacinamide adenine dinucleotide phosphate (NADP). The reaction is catalyzed by NAD kinase (
). For details see, for example, Cory, J. G. Purine and pyrimidine nucleotide metabolism. In: Textbook of Biochemistry and Clinical Correlations, 3
rd
edition, ed. Devlin, T., Wiley Brisbane 1992, pp 529-574.
Normal cells can typically utilize both precursors niacin and niacinamide for NAD(P) synthesis, and in many cases additionally tryptophan or its metabolites, which has been demonstrated for various normal tissues: Accordingly, Murine glial cells (cortex and hippocampus=brain) use: niacin, niacinamide, and quinoline acid (Grant et al. (1998), J. Neurochem. 70: 1759-1763). Human lymphocytes use niacin and niacinamide (Carson et al. (1987), J. Immunol. 138: 1904-1907; Berger et al. (1982), Exp. Cell Res. 137: 79-88). Rat liver cells use niacin, niacinamide and tryptophan (Yamada et al. (1983), Internat. J. Vit. Nutr. Res. 53: 184-191; Shin et al. (1995), Internat. J. Vit. Nutr. Res. 65: 143-146; Dietrich (1971) , Methods Enzymol. 18B: 144-149). Human erythrocytes use niacin and niacinamide (Rocchigiani et al. (1991), Purine and pyrimidine metabolism in man VII, Part B, ed. Harkness et al., Plenum Press, New York, pp 337-340. Leukocytes of guinea pigs use niacin (Flechner et al. (1970), Life Science. 9: 153-162).
NAD(P) is involved in a variety of biochemical reactions which are vital to the cell and have therefore been thoroughly investigated. This key function of NAD(P) has evoked also some investigations in the past on the role of this compound for the development and growth of tumors, and as to what the NAD(P) metabolism could also be utilized to combat tumors. Indeed, compounds aiming at the treatment of tumor diseases have been described which involve—concomitantly to other effects—also the decrease of NAD(P) levels in the cell. However, these compounds primarily act by initiating the cellular synthesis of dinucleotide derivatives which structurally deviate from natural NAD. The biochemical consequences of this approach and the putative mechanisms of the resulting cell-damage are, therefore, manifold as outlined in the Table 1.
Compounds
Mode of action
Ref.
6-amino-
Primary mechanism of action:
1, 2, 3
nicotin-
Synthesis of 6-amino-AND(P), a competitive in-
amide
hibitor of AND(P)-requiring enzymes, especially
of 6-phosphogluconate dehydrogenase, an enzyme
of the pentose-phosphate-pathway which provides
the precursor of the nucleotide biosynthesis
ribose-5-phosphate.
Resulting biochemical effects in the cell:
Inhibition of purine nucleotide de novo synthesis
from [
14
C]glycine. Decrease of intracellular
purine (ATP, GTP) and pyrimidine (UTP, CTP)
nucleotide pools resulting in the inhibition of
DNA and RNA synthesis.
Inhibition of PARP (an enzyme involved in the
DNA repair).
Reduction of the ATP to ADP ratio.
Depression of intracellular AND concentration.
tiazofurin,
Primary mechanism of action:
2, 4, 5
selena-
Synthesis of the AND analogs TAD, SAD which
zofurin
are potent inhibitors of inosine monophosphate
dehydrogenase, an enzyme involved in the
synthesis of purine nucleotides.
Resulting biochemical effects in the cell:
Depletion of GMP and accumulation of IMP
resulting in an inhibition of DNA and RNA
synthesis.
Stimulation of AND synthesis after short
exposure (<24 h).
Inhibition of AND synthesis after prolonged ex-
posure (>24 h), probably due to negative feedback
inhibition of AND synthesis by TAD/SAD
which accumulate in the cell.
azaserine,
Primary mechanism of action:
6, 7
6-diazo-5-
Analogs of glutamine which block the enzymatic
oxo-L-
transfer of the amido group of glutamine.
norleucine
Resulting biochemical effects in the cell:
Inhibition of IMP synthesis resulting in an inhibi-
tion of DNA and RNA synthesis.
Inhibition of AND synthesis from the precursor
niacin at the following step: dNAD → AND
Mutagen, cancerogen.
DNA-
Primary mechanism of action:
4, 8, 9,
interacting
Induction of DNA strand breaks.
10
compounds
Resulting biochemical effects in the cell:
(e.g. N-
Multiple consequences of DNA damage.
methyl-N′-
Activation of the DNA repair enzyme PARP
nitro-N-
resulting in a decline of the intracellular AND
nitroso-
content, since the substrate of PARP is AND.
guanidine)
Mutagen, cancerogen.
Abbreviations:
PARP, poly(ADP-ribose) polymerase;
AND, niacinamide adenine dinucleotide;
NADP, niacinamide adenine dinucleotide phosphate;
dNAD, niacin adenine dinucleotide;
ATP, adenosine triphosphate;
ADP, adenosine diphosphate;
GTP, guanosine triphosphate;
GMP, guanosine monophosphate;
UTP, uridine triphosphate;
CTP, cytosine trisphosphate;
DNA, desoxyribonucleic acid;
RNA, ribonucteic acid;
TAD, tiazofurin adenine dinucleotide;
SAD, selenazofurin adenine dinucleotide;
IMP, inosine monophosphate.
It is therefore not possible to make any predictions from these data on the biological effects of a primary and specific inhibition of the NAD biosynthesis in various cell types. In particular, it remains completely speculative whether this mechanism may be advantageous over the above utilization or dinucleotide derivatives with regard to tumor selectivity of the cell damaging effect, the most important feature of a potential drug for tumor therapy.
JP-459555, published in 1970, describes the extraction of a structurally unknown constituent from potatoes, baker's yeast and bovine blood which inhibits respiration of tumor cells and NAD synthesis of erythrocytes. The inventors propose the use

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