February 15, 2021

Jaime Franco Pinto (2021)

Strategies to inhibit O⁶-methylguanine-DNA-methyltransferase (MGMT) for combination chemotherapy of temozolomide-resistant gliomas

Abstract

Glioma is the term coined for describing a vast group of primary cancers in the Central NervousSystem (CNS), whitin this group, we can find glioblastoma or glioblastoma multiforme (GBM) as the source of 60% of brain cancer in adults, having a peak in patients between 55 and 60 years. This type of brain cancer is a highly malignant and invasive tumor (Grade IV), that is characterized by a very poor prognosis, a very short life expectancy. In clinics, no cure has been found so far, and the first line palliative treatment consists on a multimodal approach that contains surgery, radiotherapy and chemotherapy with the drug Temozolomide (TMZ). TMZ, is a prodrug that releases a methyl cation that alkylates DNA nucleobases, specially in the position O⁶ of the guanine creating the O⁶methylguanine adduct (O⁶MeG). The formation of O⁶MeG leads to the cell death by generating a plethora of effects related with cell replication. However, more than 50% of the patients show a refractivity towards this drug because they over express O⁶-methylguanine-DNAmethyltransferase (MGMT) enzyme, which is a suicide enzyme that removes the methyl group from O⁶MeG restoring the original guanine. In the context of this Thesis, we developed three different strategies to overcome this MGMT resistance by inhibiting the action of this enzyme to increase TMZ’s action. The first strategy consisted on the development of small ligands that could interact with O⁶MeG by hydrogen bonds, therefore masking the latter from MGMT action. Here, the capacity of stabilization of DNA surrogates containing O⁶MeG of eleven novel compounds plus another previously eighteen synthesized molecules was evaluated in vitro as well their cytotoxicity alone or in combination with TMZ in the multidrug resistant cell line T98G. Unfortunately for this strategy, none of the compounds was able to selectively discriminate O⁶MeG containing DNA nor to generate cytotoxicity (alone or in combination) in T98G cell lines therefore this strategy was abandoned. The second strategy was based on the synthesis of hybrid molecules containing a DNA interacting scaffold (acridine) and a well-known MGMT inhibitor (O⁶-benzylguanine, O⁶BG). The synthesis of these molecules aimed to generate compounds that interact with DNA and also inhibit MGMT. The purpose of choosing the acridine core was sustained by the fact that the scaffold showed interesting biological properties in cancerous cells that for example increase the pro-apoptotic cascade. Here, we demonstrate one molecule was able to interact with DNA, inhibit MGMT in vitro and in cellulo, to be active in T98G cells alone (GI₅₀ = 1.1 μM) and synergic when combined with TMZ. Finally, it was demonstrated that this compound created apoptosis in cells, not by generating double strand DNA breaks (DSB) but by enhancing caspases 3 and 7. The third approach was to synthesize caged (unactive and less toxic) MGMT inhibitor (O⁶BG) which, upon irradiation with light, could release the uncaged or active MGMT inhibitor in a controlled way. Unfortunatelly, the construction of this caged inhibitor turned out to be more toxic in cellulo than the parental contro O⁶BG therefore the strategy was discarded. (Defended on February 15, 2021)