Sequence-Specific Dual DNA Binding Modes and Cytotoxicities of N-6-Functionalized Norcryptotackieine Alkaloids.

Norcryptotackieine (1a) belongs to the indoloquinoline class of alkaloids isolated from Cryptolepis sanguinolenta, a plant species that has been traditionally used as an antimalarial agent. Additional structural modifications of 1a can potentially enhance its therapeutic potency. Indoloquinolines such as cryptolepine, neocryptolepine, isocryptolepine, and neoisocryptolepine show restricted clinical applications owing to their cytotoxicity deriving from interactions with DNA. Here, we examined the effect of substitutions at the N-6 position of norcryptotackieine on the cytotoxicity, as well as structure-activity relationship studies pertaining to sequence specific DNA-binding affinities. The representative compound 6d binds DNA in a nonintercalative/pseudointercalative fashion, in addition to nonspecific stacking on DNA, in a sequence selective manner. The DNA-binding studies clearly establish the mechanism of DNA binding by N-6-substituted norcryptotackieines and neocryptolepine. The synthesized norcryptotackieines 6c,d and known indoloquinolines were screened on different cell lines (HEK293, OVCAR3, SKOV3, B16F10, and HeLa) to assess their cytotoxicity. Norcryptotackieine 6d (IC50 value of 3.1 µM) showed 2-fold less potency when compared to the natural indoloquinoline cryptolepine 1c (IC50 value of 1.64 µM) in OVCAR3 (ovarian adenocarcinoma) cell lines.

Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells.

Evading the cellular apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in cancer cells, leading to the induction of ZBP1-mediated necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of necroptosis.

Substituent effect of benzyl moiety in nitroquinoxaline small molecules upon DNA binding: Cumulative destacking of DNA nucleobases leading to histone eviction.

Cooperative disruption of Watson-Crick hydrogen bonds, as well as base-destacking, is shown to be triggered by a quinoxaline-based small molecule consisting of an N,N-dimethylaminopropyl tether, and a para-substituted benzyl moiety. This events lead to superstructure formation and DNA condensation as evident from biophysical experiments and classical molecular dynamics simulations. The DNA superstructure formation by mono-quinoxaline derivatives is highly entropically favored and predominantly driven by hydrophobic interactions. Furthermore, oversupercoiling of DNA and base-destacking cumulatively induces histone eviction from in-vitro assembled nucleosomes at lower micromolar concentrations implicating biological relevance. The DNA structural modulation and histone eviction capacity of the benzyl para-substituents are in the order: -I > -CF3> -Br > -Me > -OMe > -OH, which is largely guided by the polarity of benzyl para-substituent and the resulting molecular topology. The most hydrophobic derivative 3c with para-iodo benzyl moiety causes maximal disruption of base pairing and generation of superstructures. Both these events gradually diminish as the polarity of the benzyl para-substituent increases. On the other hand, quinoxaline derivatives having heterocyclic ring instead of benzyl ring, or in the absence of N,N-dimethylamino head-group, is incapable of inducing any DNA structural change and histone eviction. Further, the quinoxaline compounds displayed potent anticancer activities against different cancer cell lines which directly correlates with the hydrophobic effects of the benzyl para-substituents. Overall, the present study provides new insights into the mechanistic approach of DNA structural modulation driven histone eviction guided by the hydrophobicity of synthesized compounds leading to cellular cytotoxicity towards cancer cells.