Krishna C. Agrawal

Characterization of the biochemical mechanism of action of α-(N)-heterocyclic carboxaldehyde thiosemicarbazones

Abstract α-(N)-Heterocyclic carboxaldehyde thiosemicarbazones have been shown to be potent inhibitors of the biosynthesis of DNA in mammalian cells. Studies with transplanted murine neoplasms have demonstrated that the enzymatic site responsible for the blockade of DNA replication is at the level of ribonucleoside diphosphate reductase, an enzyme of critical importance for the generation of the deoxynucleoside triphosphate precursors of these macromolecules. Kinetic studies were carried out with a partially purified enzyme from a rat tumor in an effort to define the molecular mechanism by which agents of this class inhibit the activity of ribonucleoside diphosphate reductase. The concentration of the nucleoside diphosphate substrate, the allosteric activator ATP, or magnesium ion did not influence the inhibition by a representative derivative of this type of agent IQ-1. Dithiothreitol, a model dithiol used in place of the natural substrate thioredoxin, was partially competitive with the inhibitor. The results appear to be most compatible with a model in which the preformed metal chelate of IQ-1 interacts with the enzyme at or near the site occupied by the dithiol substrate. To ascertain some of the structural features required for inhibition of ribonucleoside diphosphate reductase activity, the inhibitory potency of a series of methyl and benzo derivatives of PT was measured. The results suggested that position 6 of PT and position 3 of IQ-1 are equivalent with respect to orientation of the inhibitor at the enzymatic binding site and that little or no tolerance exists for structural modification at this position. In a similar manner, substitution of the terminal amino group of the thiosemicarbazone side chain decreased enzyme inhibition, supporting the presence of a low bulk tolerance zone in this position. Further studies with these substituted derivatives were interpreted to be suggestive of the existence of a hydrophobic bonding zone adjacent to the inhibitor-binding site of the enzyme. These results were employed to design MAIQ-1, a derivative of this class with strong inhibitory potency for ribonucleoside diphosphate reductase, as well as the other necessary requisite properties for clinical trial as an antineoplastic agent.

Localization in Escherichia coli B of two enzymatic sites of action by 1-formylisoquinoline thiosemicarbazone (IQ-1) on ribonucleic acid biosynthetic pathways☆

Abstract The site(s) of action of 1-formylisoquinoline thiosemicarbazone (IQ-1) on RNA biosynthetic pathways was investigated in Escherichia coli B by measuring the effects of this drug on the conversion of 3 H-uridine and 3 H-adenosine to acid-soluble nucleotide forms. The incorporation of 3 H-uridine into acid-soluble UMP and CMP was unaffected by a 50 per cent growth-inhibitory concentration of IQ-1; however, the progression of radioactivity from pyrimidine nucleoside monophosphates to di- and triphosphates was markedly depressed. In contrast, the conversion of 3 H-adenosine to purine nucleotide forms was unaffected by IQ-1. A partially purified preparation of ATP: nucleoside monophosphate phosphotransferase from E. coli was used to show that pyrimidine nucleoside monophosphate kinase activity was sensitive to IQ-1. A second locus of action appeared to be RNA polymerase; variable inhibition, which seemed to depend upon the degree of purity of the enzyme, was produced by IQ-1. Several structurally related compounds were tested for potency against pyrimidine nucleoside monophosphate kinase and RNA polymerase; the results showed close correlation between the potential for inhibition of growth and the degree of interference with the activity of these enzymes, suggesting the involvement of these lesions in the bacteriostatic action of IQ-1.

Biochemical studies of the effects of 1-formylisoquinoline thiosemicarbazone (IQ-1) in escherichia coli B

Abstract 1-Formylisoquinoline thiosemicarbazone (IQ-1) was inhibitory to the growth of Escherichia coli B. Growth inhibition was readily reversed either by washing or by the addition of ferrous or ferric ions. The biosynthesis of RNA was the most sensitive to IQ-1 of the metabolic processes measured; DNA synthesis was also inhibited, but was less susceptible, and the formation of protein and cell respiration were unaffected by growth-inhibitory concentrations of IQ-1. The incorporation of 14C-uracil into RNA and DNA was depressed to a greater degree than was the utilization of 14C-adenine for the formation of these macromolecules, suggesting the presence of two sites of action for IQ-1 in this organism, one site resulting in general blockade of RNA synthesis, and the other interferring specifically with the metabolic utilization of uracil. Results of structure-activity relationships suggest that blockade of the biosynthesis of RNA by IQ-1 is at least in part related to the growth-inhibitory potency of this agent. The findings indicate that the mechanism of action of IQ-1 in E. coli B is different from that occurring in neoplastic cells, which primarily involves inhibition of DNA synthesis.

Inhibitors of alkaline phosphatase of Sarcoma 180/TG

Abstract The inhibitory potential of a variety of agents of different chemical classes has been examined using a particulate-bound alkaline phosphatase partially purified from the 6-thioguanine-resistant variant, Sarcoma 180/TG ascites cells, in an effort to discover inhibitory potential which might be useful in blocking this enzyme activity in situ. The most potent inhibitors tested were derivatives of α-(N)-heterocyclic carboxaldehyde thiosemicarbazones, tetramisole, permanganate, and beryllium. Kinetic studies of the mode of inhibition of alkaline phosphatase by some of these inhibitors indicated that derivatives of α-(N)-heterocyclic carboxaldehyde thiosemicarbazones and those of tetramisole and beryllium inhibited the enzyme by different mechanisms. The kinetics of inhibition by beryllium and derivatives of α-(N)-heterocyclic carboxaldehyde thiosemicarbazones were mixed, while inhibition by a derivative of tetramisole was uncompetitive.