β-Sitosterol-3- O-β- d-glucopyranoside: A eukaryotic DNA polymerase λ inhibitor

Abstract

β-Sitosterol-3- O-β- d-glucopyranoside (compound 1), a steroidal glycoside isolated from onion ( Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase λ (pol λ) in vitro. The compound did not influence the activities of replicative DNA polymerases such as α, δ and ɛ, but also showed no effect even on the activity of pol β which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of compound 1 such as β-sitosterol (compound 2) and d-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol λ inhibition. The inhibitory effect of compound 1 on both intact pol λ (i.e. residues 1–575) and a truncated pol λ lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133–575, del-1 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 μM, respectively. The compound 1-induced inhibition of del-1 pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.