Cholesterol hemisuccinate (compound 5), which consists of succinic acid esterified to the beta-hydroxyl group of cholesterol, selectively and strongly inhibited the activities of mammalian DNA polymerases (pols) such as pot beta, pot lambda, and terminal deoxynucteotidyltransferase (TdT), which are family X pols, in vitro, and the IC50 values were 2.9, 6.3, and 6.5 mu M, respectively. The compound moderately suppressed the activities of other mammalian pols such as pot A (i.e., pol gamma), pol B (i.e., pols alpha, delta, and epsilon), and pol Y (i.e., pots i, eta, and kappa) with 50% inhibition observed at concentrations of 131, 89.2-98.0, and 120-125 mu M, respectively. The compound had no influence on the activities of plant pols a and P, prokaryotic pols and other DNA metabolic enzymes tested. Since other cholesterol-related compounds such as cholesterol, cholesteryl chloride, cholesteryl bromide, cholesteryl acetate, and cholesteryl-5 alpha, 6 alpha-epoxide (compounds 1-4 and 6, respectively) did not influence the activities of any enzymes tested, the hemisuccinate group of compound 5 could be important for inhibition of the pot X family. Surface plasmon resonance analysis demonstrated that compound 5 bound selectively to the C-terminal 31 kDa domain of pol beta and pol lambda containing a pot beta-like region. On the basis of these results, the inhibitory mechanism of compound 5 on the pot X family was discussed. (c) 2007 Elsevier Inc. All rights reserved.