One arrives now at what one could call generally coenzyme- or cofactor-antagonists. As these co-factors are in certain instances loosely, in others strongly bonded on to the apoenzyme, 'fraudulent' or 'rogue' coenzymes could either compete with the normal ones for their proteinous site to form inactive holoenzymes, or interfere with their biosyntheses. As in many cases their precursors are identical with vitamins, it appears possible to utilize anti-vitamins 223,225 such as deoxypyridoxal and a riboflavin analogue as anti-tumor agents. With nicotinamide antagonists, such as 2-ethylamino-1, 3, 4-thiadiazole89 and 6-aminonicotinamide,82,124,224 the latter was shown by Dietrich, Shapiro, et al.,62-22* to be transformed into a 'fraudulent' DPN analogue, being bound to the corresponding apo-dehydrogenase and producing ineffective holoenzymes.208 Earlier examples of this kind were given by Kaplan et al. who demonstrated that the DPNases of animal tissue (diphosphopyridine nucleotidase which hydrolyses DPN to nicotinamide+ denosine-pyrophosphoryl-ribose) can catalyse an exchange reaction between the nicotinamide moiety of DPN and nicotinamide analogues such as 3-acetyl pyridine. This compound, when injected into mice carrying tumors, increased the normal coenzyme level in the liver but caused the formation of its 'fraudulent' analogue in the neoplastic tissue which represents a 'lethal synthesis.'193 While Shapiro, Dietrich, et al applied their anti-vitamins often in combination with other agents to mice with mammary carcinoma 755 which they found low in the corresponding precursors of co-factors and thus were relying on hitting the deficient cell harder than the normal one, Morton 186 proposed to tackle the problem of diminishing the level of DPN and its dependent enzymes in a different manner. It will be remembered that in the previous chapter on coenzymes and metal co-factors it was mentioned that apparently low activity levels of enzymes could be caused by deficiencies in coenzymes and that DPN and particularly TPN and their biosynthetic systems were found to be reduced in certain tumors. Morton 186 therefore suggested to attempt a further diminution of the rate of biosynthesis of DPN as a basis for carcino-chemotherapy by interfering especially with the second stage of this process (Fig. 16b) which can start with nicotinamide or nicotinic acid. The latter gives desamido-DPN which is then transformed in presence of glutamine and ATP to DPN. It is not improbable that such interfering agents may be found among quaternary pyridinium compounds as studied at present by Morton 188 and Timmis et al.237 In this connection it is interesting to note that. Holzer and Kroger 136 have reported on a drop of DPN-levels and inhibition of aerobic and anaerobic glycolysis in ascites tumor cells treated with a number of alkylating agents which under in vitro conditions will quaternize pyridine derivatives such as nicotinamide. In Holzer's experiments in vivo the anti-tumor effect of the alkylating agents in rats with Jensen sarcomas could be weakened by dosing the animals with nicotinamide.

Another series of coenzyme-antagonists, interfering in 1-carbon transfer, concerns the clinically important folic acid analogues and related compounds which may be also considered as substrate-like inhibitors for the following reasons: folic acid (see Fig. 18) is converted into tetrahydrofolic acid, the starting material for the coenzyme of 1-carbon interchanges,57 by an enzyme folic reductase using TPNH as hydrogen donor. It is, therefore, not unlikely that so-called anti-folics, such as aminopterin or amethopterin ('Methotrexate') , inhibit the reductase by acting as 'rogue' substrates. At the same time it is not impossible that these compounds are transformed to the tetrahydrofolic acid analogue and then, particularly in the case of amethopterin, prevent the formylation of N10. Whatever the true situation, the anti-leukemic effects of the antagonists are not in doubt.71

Fig. 18. Folic acid, derivatives and analogues.

To carry on with this sub-chapter, for which no claim to completeness is made, the case of L-azaserine and DON should be briefly mentioned together with that of other amino-acid analogues. The action mechanism of the diazo-derivatives has been elucidated by Buchanan et al.68 and amounts to an antagonism to glutamine which acts as a co-factor for glutamine transamidase during the de novo synthesis of inosinic acid, from which other purines and eventually NA derive (see Fig. 7). As glutamine, noted above, plays the part of an amide donor in other metabolic events, it is probable that the diazo-derivatives do block also these processes. It is not quite certain yet whether glutamine analogues act in the same way.183 The role of a cyclic amino-acid, 1-amino-cyclopentane-1-carboxylic acid, as studied among others, by Ross and Connors,78 and that of α-alkylated amino-acids and those in which a benzene ring is substituted or altered (thienylalanine) is even less clear.

For a complete story one has to consider the inhibition of enzymes through a process of elimination of their metal activators, if essential for the activity. This removal is easy when, as pointed out before, 58 they are loosely bound to the apoenzyme, and much more difficult if more firmly attached. When the enzymes or their co-factors contain heavy metals, these can under certain conditions be 'poisoned' by forming complexes with, say, cyanide in the case of the cytochrome oxidase system (see also 83). As to the removal of metals, if possible at all, this can be achieved by treatment with chelating agents, either directly or via dialysis, but none of these methods, as far as one can ascertain, has been effective undei in vivo conditions, as there the complexing agents will interact non-specifically with the metal whether it belongs to an enzyme or not. This does not mean that after more systematic studies of distribution of trace elements by tissue analysis (see references 32, 232, 239), or by injection of radio-active elements,178 more specific agents may become known which, in selected cases, could be applied to control an excess of a metal activator. On the other hand deficiency of metals in animals or man has been counteracted by dietary means and by therapeutic treatment with metal derivatives. Reference is made to the decreased content of zinc in cancerous prostate glands. This takes one to the next proposal by which alterations in enzymic activity might be achieved.