Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O⁶-alkylguanine-DNA alkyltransferase insensitive to O⁶-benzylguanine

The DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O⁶-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence Likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lys¹⁶⁵, Leu¹⁶⁸ and Leu¹⁶⁹. The two positions at which changes led to the largest increase in resistance to BG were Gly¹⁵⁶ and Lys¹⁶⁵. Eleven mutants at Gly¹⁵⁶ Were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys¹⁶⁵ found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser¹⁵⁹ position (S159I and S159V) were > 80-fold more resistant than wild-type AGT. Eleven active mutants at Leu¹⁶⁹ were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys¹⁵⁰, with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asn¹⁵⁷ were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents.