DESIGN OF SPECIES-SPECIFIC ENZYME-INHIBITORS OR ISOZYME-SPECIFIC ENZYME-INHIBITORS .1. EFFECT OF THYMIDINE SUBSTITUENTS ON AFFINITY FOR THE THYMIDINE SITE OF HAMSTER CYTOPLASMIC THYMIDINE KINASE

5-(Ethylamino)- and 5-acetamido-2ʹ-deoxyuridine 5ʹ-triphosphates were synthesized; the extent and concentration dependence of their inhibitory action on the title enzyme resembled that of the feedback inhibitor TTP. This and other findings provide a tentative indication that bulk tolerance near C-5 of the thymine ring may be more extensive at the TTP site than at the thymidine site. Enzyme-inhibitor dissociation constants (Kivalues) were determined for thymidine derivatives monosubstituted at various positions. Competitive inhibition with respect to thymidine (indicative of substituent tolerance in the enzyme-thymidine complex) was produced by 3-amylthymidine (Ki= 65 µM ), trans-5-bromo-6-ethoxy-5, 6-dihydrothymidine diastereoisomers (Ki= 180 and 310 µM ), 5ʹ-C-(acetamidomethyl)-and 5-C-(propionamidomethyl)thymidine epimers (Kirange 65-1100 µM), 3ʹ-acetamido-and 3ʹ-(ethylthio)-3ʹ-deoxythymidines (Ki= 2.5 mM and 12 µM, respectively), and certain 5ʹ-(alkylamino)- and 5ʹ-(alkylthio)-5ʹ-deoxythymidines (Kirange 180-1200 µM ). Evidence indicates that bulk tolerance at some, if not most, of the aboveatoms of thymidine is found in the enzyme-thymidine complexes of human and other mammalian thymidine kinases; attachment of suitable substituents to such atoms could, in principle, lead to thymidine site directed isozyme-specific inhibitors of human cytoplasmic thymidine kinase, which is a candidate target in the design of antineoplastic drug. © 1979, American Chemical Society. All rights reserved.