O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts.

Tumour resistance to chemotherapy involving methylating agents such as DTIC (dacarbazine) and temozolomide is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT). There is considerable interest in improving the efficacy of such O(6)-alkylating chemotherapy by the prior inactivation of MGMT. We have examined the effect of the modified guanine base, O(6)-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib) on MGMT activity and cell or xenograft tumour growth inhibition by temozolomide in the human breast carcinosarcoma cell line, MCF-7. PaTrin-2 effectively inactivated MGMT in MCF-7 cells (IC(50) approximately 6 nM) and in xenografts there was complete inactivation of MGMT within 2 h of dosing (20 mg kg(-1) i.p.) and only slight recovery by 24 h. MGMT inactivation in a range of murine host tissues varied between complete and approximately 60%, with extensive recovery by 24 h. PaTrin-2 (10 microM) substantially increased the growth inhibitory effects of temozolomide in MCF-7 cells (D(60)=10 microM with PaTrin-2 vs 400 microM without). In MCF-7 xenografts, neither temozolomide (100 mg kg(-1) day(-1) for 5 days) nor PaTrin-2 (20 mg kg(-1) day(-1) for 5 days) had any significant effect on tumour growth. In contrast, the PaTrin-2-temozolomide combination produced a substantial tumour growth delay: median tumour quintupling time was increase by 22 days (P<0.005) without any significant increase in toxicity as assessed from animal weight. A PaTrin-2-temozolomide combination may therefore be beneficial in the treatment of human breast cancers.

O6-alkylguanine-DNA alkyltransferase inactivation in cancer chemotherapy.

The protein O(6)-alkylguanine-DNA alkyltransferase is the basis of an important process for repairing damage to cellular DNA, which renders cells resistant to drugs that alkylate at the O(6)-position of guanine residues. The development of various pseudosubstrates which inactivate this protein is reviewed, from a chemical standpoint. Study of the influence of pseudosubstrate molecular structure on their interaction with the active site cysteine has progressed together with direct investigation of protein structure. Combination therapy using a powerful inactivator with a suitable alkylating agent shows great clinical promise in the treatment of cancer, particularly when some degree of selectivity is possible.

O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts.

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.

Inactivation of O6-alkylguanine-DNA alkyltransferase. 1. Novel O6-(hetarylmethyl)guanines having basic rings in the side chain.

A number of novel guanine derivatives containing heterocyclic moieties at the O6-position have been synthesized using a purine quaternary salt which reacts with alkoxides under mild conditions. Initially O6-substituents were investigated in which the benzene ring of the known agent, O6-benzylguanine, was replaced by unsubstituted heterocyclic rings. The ability of these agents to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase), both as pure recombinant protein and in the human lymphoblastoid cell line Raji, has been compared with that of O6-benzylguanine. The present paper focuses on O6-substituents with basic rings, and under standard conditions several of them proved more effective than benzyl for inactivation of both recombinant and Raji ATase. Among the pyridine derivatives, the 2-picolyl compound 7 is not very active in contrast to the 3- and 4-picolyl compounds, and this influenced our choice of isomers of other basic ring systems for study. Since halogen substitution in the thiophene ring considerably increased the activity (17 versus 6), similar modifications in the pyridine series were examined. The more polar O6-substituents in this study are on the whole compatible with the stereochemical requirements of the ATase protein, and their pharmacological properties may be valuable in subsequent in vivo investigations, particularly the thenyl (6), 5-thiazolylmethyl (12), 5-bromothenyl (17), and 2-chloro-4-picolyl (21) derivatives.