Alexander Hampton

Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase.

The effects of a variety of 5-, 5-, and 3-substituted deoxyuridine derivatives on the cytoplasmic thymidine kinase (EC 2.7.1.21) purified from a human colon carcinoma cell line, HCT 116, were determined. Of particular interest was elucidation of the structural features important for antagonism of the feedback inhibition of thymidine kinase exerted by thymidine triphosphate. Substitutions at the 5-position altered the potency of the 5-modified compounds. The replacement of the 5-hydrogen with a methyl group or an iodine greatly increased the affinity of compounds for the thymidine kinase. This was evident for enzyme substrates with 5-hydroxyl groups [2-deoxyuridine (dUrd), 2-deoxythymidine (dThd) and 5-iodo-2-deoxyuridine (IdUrd)], feedback inhibitors with 5-triphosphate substitutions (dUTP, dTTP and IdUTP), and for 5-amino derivatives [5-amino-2,5-dideoxyuridine (5-AdUrd), 5-amino-2-5-dideoxythymidine (5-AdThd) and 5-iodo-5-amino-2,5-dideoxyuridine (5-AIdUrd)]. Qualitatively, however, the 5-substitutions did not affect the nature of the interactions with dThd kinase. For example, in the presence of dTTP, 5-AdUrd stimulated dThd kinase activity as much as 5-AdThd, but approximately a 100-fold greater concentration of 5-AdUrd was required. Similar results were obtained using intact cells in which substitutions at the 5-position affected the potency, but not the efficacy, of the 5-amino derivatives to stimulate dThd phosphorylation. In contrast, substitutions at the 5-position did alter the nature of the interaction with dThd kinase. Thus, the 5-hydroxyl compounds, dUrd, dThd and IdUrd, did not reverse the enzyme inhibition produced by dTTP nor did they stimulate dThd uptake in intact cells. 5-Deoxy-5-(ethylthio)thymidine, 5-deoxy-5-[(2-hydroxyethyl)thio]thymidine, and dTMP, but not dTDP, also antagonized the inhibition of dThd kinase produced by dTTP. In comparison to 5-AdThd, the 3-amino derivatives, 3-AdThd and 3-5-diAdThd, were much less potent, but still efficacious, antagonists of feedback inhibition. These results indicate that a wide range of dUrd derivatives can disrupt the regulation of dThd kinase and provide leads for the development of new nucleotide analogues.

Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP

Abstract A vinyl phosphonate analog of adenosine 5′-phosphate (AMP) was synthesized in which the CH 2 ue5f8Oue5f8P system of AMP is replaced by CHue5fbCHue5f8P. The V max values of this analog relative to AMP were 0.7% with rabbit muscle AMP aminohydrolase, 13.4% with rabbit muscle AMP kinase, and 6.6% with pig muscle AMP kinase. The vinyl analog of ADP produced by the kinases was a substrate of rabbit muscle pyruvate kinase. These results, together with substrate specificity properties at the AMP sites of the enzymes indicate that the C(4′)-C(5′)-O(5′)-P system of AMP is of trans character during conversion of AMP to ADP by pig or rabbit AMP kinase.

Use of a vinyl phosphonate analog of ATP as a rotationally constrained probe of the C5′O5′ torsion angle in ATP complexed to methionine adenosyl transferase

Abstract An analog of adenosine 5′-triphosphate (ATP) was synthesized in which the C4′ue5f8C5′ue5f8Oue5f8Pα system is replaced by a trans C4′ue5f8CHue5fbCHue5f8Pα system. In the form of 1:1 complexes with Mg, this analog and its counterpart with a C4′ue5f8CH2ue5f8CH2ue5f8Pα system were linear competitive inhibitors, with respect to MgATP, of the MAT-II (normal tissue) and MAT-T (hepatoma tissue) forms of rat ATP: l -methionine-S-adenosyltransferase (MAT); K m ( ATP ) K i values ranged from 0.4 to 2.4. 2′-Deoxy-ATP was a weak substrate, K m ( ATP ) K m = 0.035 , of MAT-II and a weak competitive inhibitor, K m ( ATP ) K i = 0.07 , of MAT-T. These findings, together with interactions of the MAT forms with other substrates and inhibitors, indicate that binding of ATP to these transferases is accompanied by little rotation about the C5′ue5f8O5′ bond, and that C4′ and Pα are in a trans-type relationship in enzyme-bound ATP.

Carboxylic-phosphoric mixed anhydrides isosteric with AMP and ATP as reagents for enzymic AMP and ATP sites.

Summary A carboxylic-phosphoric mixed anhydride was synthesized which differs from adenosine 5′-triphosphate (ATP) by replacement of the 5′ methylene group of ATP by a carbonyl group. This compound did not inhibit rabbit adenylate kinase, but it rapidly inactivated rabbit pyruvate kinase; the effect was prevented by ATP, ADP and phosphoenolpyruvate and therefore appears to be ATP-site-directed. The analogous anhydride isosteric with adenosine 5′-phosphate (AMP) rapidly inactivated rabbit AMP aminohydrolase; inactivation was prevented by an equimolar level of AMP. These findings, together with previous studies with this AMP analog, suggest that these anhydrides are powerful and potentially useful reagents for adenine nucleotide binding sites of enzymes.