ISOLATION OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE MUTANTS HIGHLY RESISTANT TO INACTIVATION BY O-6-BENZYLGUANINE

The activity of O-6-alkylguanine-DNA alkyltransferase (AGT) protects c ells from killing by methylating or chloroethylating agents, ACT is st rongly inhibited by O-6-benzylguanine (ED50, 0.2 mu M), and this drug is presently undergoing clinical trials to enhance chemotherapy by alk ylating agents. Point mutations such as P140A (ED50, 5 mu M) render AG T resistant to O-6-benzylguanine (BG), Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG trill enable the rational design o f improved inhibitors able to inactivate these mutants. BG-resistant A GT mutants may also be valuable for expression in bone marrow stem cel ls to reduce myelosuppression brought about by alkylating agents, to i ncrease the therapeutic index of therapies including BG, and for use a s a selectable marker to allow other genes to be expressed in such ste m cells. We have therefore set up a general screen to obtain such muta nts by using the ability of AGT to protect Escherichia coli GWR109 lac king endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidi ne. When the cells were rendered permeable to BG by mutating the lipop olysaccharide membrane component forming strain TRG8, the protection b y AGT expression was abolished by treating the tells with BG, The know n P140A mutant was used to test the system and was highly selected for by treatment with 50 mu M BG and 40 mu g/ml N-methyl-N'-nitro-N-nitro soguanidine, The sequence coding for PVP at positions 138-140 in AGT w as replaced with a random nucleotide sequence, and this library was us ed to transform TRG8, All of the 59 colonies analyzed having AGT activ ity that survived the selection from the pool of 36,000 transformants were resistant to BG, Many (69%) of these mutants contained lysine at position 140, and all of these show-ed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated wi th 1.2 mM BG, This result was confirmed with three mutants (P138K/V139 L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogen eity, The nest most common residues found at position 140 were arginin e (7%) and asparagine (7%). Studies carried out with purified preparat ions of mutants P140R and P140N revealed that these mutations also pro vided resistance to BG but to a lesser extent than P140K (ED(50)s of 1 90 and 7 mu M respectively). These results indicate that: (a) this scr eening method can be used to evaluate BG resistance of single or multi ple changes throughout the AGT sequence; and (b) replacement of prolin e-140 with lysine is the most effective point mutation at this site ca using BG resistance and is more than 200 times more effective than rep lacement with alanine.