George H. Hitchings

A comparison of the specificities of xanthine oxidase and aldehyde oxidase

Abstract This study directly compares the specificities of the structurally similar hydroxylating enzymes, aldehyde oxidase and xanthine oxidase. Michaelis-Menten constants for a variety of substrates of xanthine oxidase were in general lower than those of aldehyde oxidase. With respect to the rates of oxidation, the basic similarity was a preference for compounds having a substituted pyrimidine ring structure. Outstanding among the differences were the effects of the number and position of the ring substituents. Both enzymes readily oxidized a variety of unsubstituted and C-monosubstituted heterocycles, but only xanthine oxidase readily oxidized C-disubstituted derivatives. Certain N-substitutions, however, enhanced substrate activity with aldehyde oxidase, but markedly decreased it with xanthine oxidase. Although both enzymes preferred oxo over amino substituents, there were some specificity differences with respect to the chemical nature of substituents. Aldehyde oxidase, but not xanthine oxidase, tolerated 6-substitution of purine by alkyl, halogeno, cyano, or methylthio groups, while 6-hydroxyl or 6-methylamino substituents were tolerated only by xanthine oxidase. The position at which oxidation occurred was influenced by both the chemical nature and the positions of substituents. With some purines a different site was initially hydroxylated by each enzyme.

Selective inhibitors of dihydrofolate reductase

My interest in nucleic acids, their constitutents and metabolism can be traced to the discovery of adenosine triphosphate in muscle by Fiske and Subbarow (1). I had entered Harvard University as a candidate for a Ph.D. degree in 1928 and transferred to Harvard Medical School in 1929. Otto Folin, then head of the Department of Biological Chemistry, designated my first predoctoral year to be spent with Cyrus J. Fiske, then involved in momentous discoveries of phosphocreatine and the labile phosphorus compounds of muscle and other tissues. I was soon immersed in the discovery of analytical tools with which to follow the metabolism of adenosine triphosphate (2). Other lines of thought coalesced with this interest when I joined Burroughs Wellcome Co. in 1942 as the sole member of the Biochemistry Department. Meantime, the antimetabolite principle had been expressed by Woods (3) and Fildes (4). I saw the opportunity to explore nucleic acid biosynthesis in a new and revealing way by employing synthetic analogues of the purine and pyrimidine bases in a system utilizing these heterocyclic compounds for biosynthesis. I was able to interest Elvira Falco who was then an assistant in the company’s Bacteriology Department. Together we worked out a system using Lactobacillus casei, which would grow either with a then-unknown “Lcasei” growth factor or with a mixture of thymine and a purine (Fig. 1) (5). This system quickly gave us encouraging results. In a simple screening test for antibacterial activity, analogues were found to inhibit strongly not only the L. casei system but pathogenic bacteria as well. We had added toxicity testing in growing rats and other biological screening procedures and were becoming more and more excited by the results. By 1947, six or seven of us were pursuing this work, and the feeling in the group was, “Now we have the chemotherapeutic agents; we need only to find the diseases in which they will be active.” At that point I made two arrangements for collaborative studies, one with Sloan Kettering Institute for antitumor testing using sarcoma 180 in mice, and another with outside

Dihydrofolate reductases as targets for inhibitors

Abstract Species differences among dihydrofolate reductases were first detected and most strikingly illustrated by the selective binding of small molecule inhibitors. This led on the one hand to practical applications in the chemotherapy of microbial infections, and on the other hand to stimuli for sequence and conformation studies of representative enzymes. The recent availability of the requisite tools has amply confirmed the existence of large differences in sequence among bacterial enzymes and between bacterial and mammalian enzymes. The latter, although more homogeneous than bacterial enzymes, still exhibit many sequence differences. Current work has provided x-ray conformations of two bacterial enzyme complexes, but similar studies of a mammalian enzyme are not yet available. Consequently, the site and mode of binding of the selective inhibitors are still speculative sequence and conformation studies are potentially valuable for the design of selective inhibitors, but their employment in this fashion awaits further developments.

FOLATE ANTAGONISTS AS ANTIBACTERIAL and ANTIPROTOZOAL AGENTS

Elsewhere in this volume, Dr. Burchall has presented data which suggest that the enzyme dihydrofolate reductase has undergone manifold subtle alterations in the course of evolution. Its function has been relatively constant, however, and such changes as have occurred are nearly unmeasurable by the usual parameters of enzyme kinetics. That other changes have occurred is revealed strikingly by the diverse binding of small molecule inhibitors to dihydrofolate reductases prepared from tissues of different species. It is a reasonable interpretation that these small molecule inhibitors are bound to the enzyme partly within the active site, and partly within neighboring, supportive, hydrophobic regions. If so, it follows that regions of the enzyme outside the active site may vary considerably (presumably in amino acid sequence) without significantly altering the function of the active site. Such a pattern is now emerging as a general phenomenon from studies of hemoglobins, insulins, and other oligopeptides. The small molecule inhibitors of dihydrofolate reductase were detected first as diaminopyrimidine derivatives that had the general property of inhibiting the utilization of folic acid by Lactobacillus casei.’ Two additional characteristics soon became apparent: (1) they were in some way interfering with the reduction of folates, i.e., “the conversion of folic to folinic acid,” and ( 2 ) they were inherently selective in their action; “substituents that increased the activity against plasmodia diminished it against bacteria and vice versa.” Out of this emerged the concept that we were dealing with a group of active substances that all had the same mechanism of action in a biochemical sense but were cxhibiting selectivity because the cell receptor varied from species to species “in geometry and charge.” Such selectivity of action appeared, rather obviously, to form a basis for the development of chemotherapeutic agents. The elaboration of the concept of the mechanism of action and its documentation required developments that came gradually over an extended period and are still incomplete, since pure enzymes, sequence determinations and construction of three-dimensional models remain for the future. However, exploitation of the selective effects could proceed, and antimalarial activity seemed a particularly promising goal, since the first two compounds tested had activities equal to, and 5-times that of quinine, respectively. Through synthesis and testing, this group of substances progressed through members with higher and higher antimalarial activities, and culminated with pyrimethamine, S-p-chlorophenyl-2,4-diamino6-ethylpyrimidine, with an antimalarial activity approximately 1,000 times

Indications for control mechanisms in purine and pyrimidine biosynthesis as revealed by studies with inhibitors.

Two contrasting responses to competitive inhibitors are presented. The first deals with inhibition of orotidylate decarboxylase in man. Although one or more strong inhibitors are involved, compensation through accumulation of precursors and some (probably) less important adjustments soon result in compensation to the presence of the inhibitor. In contrast, inhibition of dihydrofolate reductase in Escherichia coli results in cessation of growth and under some circumstances in cell death. Accumulation of the precursor of the blocked reaction could not be demonstrated.

The role of metabolites and antimetabolites in the control of folate coenzyme synthesis

Abstract A review of the recent literature reveals that at least four enzymes responsible for the interconversion of folate coenzymes are under some form of regulatory control. Preliminary evidence obtained in this laboratory suggests that the formation of tetrahydrofolate by dihydrofolate reductase also is subject to regulation. Since the formation of purines and pyrimidines is itself controlled, the existence of an additional set of regulatory devices in the folate pathway may function to divert a limited supply of coenzymes to the most immediate, critical requirements of the cell.

The purine metablism of protozoa

Protozoa possess a wealth of purine-salvage enzymes, many with unique, or unusual, substrate specificities. As a result, many opportunities for the chemotherapist exist. An exemplification is found in the conversion in schistosomes of allopurinol ribonucleoside to the corresponding ribonucleotide followed by further anabolism to the very toxic 4-aminopyrazolo(3,4-d)pyrimidine 1-ribonucleotide. The same organisms convert another inosine analog, formycin B, to the ribonucleotide, but its inhibitory effects appear to be exercised primarily by inhibition of the organisms adenylosuccinate synthase. A substantial segment of the Phylum Protozoa shows no vestigial traces of ability to synthesize purines de novo although thymidylate synthase appears to be present in many. The absence of other tetrahydrofolate catalyzed reactions suggests that these functions were never acquired.

Inhibitors of Folate Biosynthesis and Utilization – Evolutionary Changes as a Basis for Chemotherapy

Publisher Summary This chapter elaborates the importance of the inhibitors of folate biosynthesis and utilization in chemotherapy. The exploration of enzymes and metabolic pathways by means of antimetabolites has been productive of both new medicinal agents and advances in fundamental knowledge. The rationale for its use in combination with a sulfonamide is stated. The target is the microbial pool of tetrahydrofolate cofactors that are essential to the metabolism and multiplication of the microbial cell. Such cofactors occur ubiquitously and perform the synthesis of serine and the methylation of homocysteine to form methione. Microorganisms employ similar reactions in the biosynthesis of riboflavin, pantothenate, thiamin, and dihydrofolate itself. The application of a spectrum of inhibitors to a selection of partially purified reductases documented their mechanism of action and revealed the magnitude of their differential affinities for the reductases of different species. Trimethoprim represents the culmination of an effort to tailor a molecule for maximum binding to bacterial reductases with minimum binding to mammalian reductase.

Resistance to inhibitors of dihydrofolate reductase in strains of Lactobacillus casei and Proteus vulgaris.

SUMMARY: Strains of Lactobacillus casei and Proteus vulgaris resistant to small molecule inhibitors of dihydrofolate reductase were isolated under various nutritional conditions. When thymine and/or purines were available in the media, several of the isolated strains had nutritional requirements for these metabolites, but resistant lines could still be isolated in their absence. Thus, the biochemical alteration accompanying resistance could be predetermined to a major extent by the design of the experiment. The wild-type strain of Proteus vulgaris did not incorporate exogenous thymine, and was insensitive to thymine antagonists, while the thymine-requiring strain was highly sensitive to 5-bromouracil, dithiothymine and 2-thiothymine. This suggested that resistance was accompanied by the appearance of a permeability or transport system for thymine.

Substrate-inhibitor cooperative interactions with microbial dihydrofolate reductases

Cooperativity in the binding of two substrates to an enzyme is a now well-established phenomenon. The x-ray crystallographic structure of the E. coli DHFR binary TMP complex compared with the ternary enzyme-NADPH-TMP complex suggests without too imaginative extrapolation, that the conformational changes resulting from the binding of one ligand aid in favorably positioning potential binding sites for the second ligand. Of greater importance is the fact that the extent to which inhibitor binding is enhanced by the binding of NADPH varies from species to species. To a significant extent, for example, the selectivity of TMP is enhanced by the increase in its binding to the E. coli enzyme when NADPH is present as compared with several mammalian enzymes. The reverse, negative cooperativity (a decrease in binding of a substance when moving from the binary to a ternary complex), is perhaps less common and certainly less well studied. The present paper deals with one such enzyme, the DHFR from C. albicans, and by reference to another, that from S. cerevisiae, where it is shown that the binding of substrates exhibit strong negative cooperativity. It was of interest also to determine the relationship between inhibitor/NADPH cooperativity and the relative insensitivity of N. gonorrhoeae to TMP. Equilibrium studies show that the binding of TMP in binary complex with this enzyme is exceedingly poor and that a 2,200-fold cooperative effect brings the gonococcal enzyme Ki within one order of magnitude of the E. coli enzyme Ki. Even so, it takes synergism of another sort (with sulfamethoxazole) and high doses to make co-trimoxazole therapy feasible for treating gonorrhoeae. The comparative results on the gonococcal enzyme for a family of near relatives of TMP are of interest also for the reason that the structure-activity relationships with this enzyme are quite different from those of the E. coli and other microbial enzymes. Finally, it should be pointed out that although the negative cooperativity found for the candida and saccharomyces enzymes is relatively large, it is the values of the substrate Michaelis constants that are physiologically relevant. The Km values of the yeast enzymes are within the range for other DHFR and therefore the intracellular activity of the enzymes should not be compromised.