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In this plenary symposium address, the author describes historically his progress from the antimetabolite 2,6-diaminopurine to 6-mercaptopurine as a useful drug against tumours and leukaemia. Subsequently a 6-mercaptopurine derivative, and eventually azathioprine, were discovered to be useful drugs to ensure kidney transplant acceptance. Several of his group's diaminopurines proved to be selective antifolates in combatting pathogenic bacteria when combined with sulfanilamide, a useful drug combination already estimated to have saved a million lives.

A program in chemotherapy is described in which antimetabolites to constituents of nucleic acids were used as probes of enzymes and metabolic pathways. These studies led to a number of insights into nucleic acid biosynthesis and related biochemical fields. They also provided exploitable information that was developed into drugs for the treatment of leukemia, the prevention of rejection of organ transplants, and a new therapy for hyperuricemia and gout. Studies on infectious diseases provided insights into the modeling of inhibitor-enzyme complexes and led to new and effective antimicrobial and antiviral agents. Basic research and relative absences of specific targeting are shown to have greatly facilitated the progress of the program.

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.

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 organism's 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.

Immunosuppressive agents have diverse (although often multiple) sites of action in the cell sequences that are involved in immune responses. New routes to selectivity are apparent at both the cellular and the biochemical level. Meanwhile, clinical work is finding new uses and more selective employment of the currently available agents.

A thioglycosidase capable of catalyzing the hydrolysis of 6-purine beta-d-glucothiopyranoside and other thioglycosides has been found in a number of mammalian tissues (including tumors) as well as in almond emulsin. The substrate specificity of mammalian thioglycosidase involves both the aglycone and the glycosyl moieties.