Investigation of Molecular Mechanism, and Pre-clinical Evaluation of Multiple Classes of Novel Compounds for Treating Glioblastoma

Abstract

Glioblastoma multiforme (GBM) is the most complex, treatment-resistant, and non-curative brain cancer, accounting for 45% of all brain cancers. Despite the progressive discernment of the biological landscape of the disease, and numerous therapeutic agents in use or in trials, a lack of progress persists in GBM therapy. GBM possesses multiple sub-networks and extensive cross-talk between oncogenic pathways, promoting gliomagenesis, phenotypic variations, as well as adaptive mechanisms waning current treatment modes. A prospective outlook in GBM cure is to target the cross-talks between numerous sub-networks in GBM which promote tumorigenesis, therapeutic blockade, and phenotypic heterogeneity. The presence of adaptive mechanisms that help to bypass therapeutic blockade by mono-targeting agents, necessitates synergistic targeting of multiple oncoproteins or pathways. A multi-targeted, single drug molecule that horizontally inhibits various channels of tumorigenicity, such as angiogenesis or infiltration, represents a logical and alternative approach to drug cocktails to combat GBM, and can provide a guaranteed improvement in the clinical benefits. This thesis is a quest for identifying a potent multi-channel agent against GBM, which can bypass the current challenges of drug resistance and tumor recurrence.

This manuscript comprises of four publications, each detailing the extensive evaluation of novel compounds from four different chemical classes- diols, hydrazones, borons and thioesters, for their anti-GBM activity, <i>in vitro</i>. Each of the molecular classes has been evaluated for their cytotoxicity, apoptotic activity, cellular oxidative potential and caspase activity accounting for cell death. The possible targets were identified using docking studies and the detailed mechanism of action of the top compounds were analyzed using transcriptome studies.

The initial class of compounds described in this thesis is the decane-1,2-diol derivatives. The novel derivatives demonstrated fair cytotoxicity to GBM cell in the initial screening. Among a panel of twelve novel diol derivatives, the drug DBT (decane-1,2-diyl bis-(p-toluenesulfonate)) was found to be the most cytotoxic to human cancer cell lines U-87 and LN229. DBT has shown cytotoxic activity with IC₅₀ of 52 µM in U-87 cell line and 270 µM in LN229 cell line. Additionally, DBT increased sensitivity to apoptotic pathways via caspase independent pathways, induced oxidative stress, prevented migratory activity effectively up to 6hrs post treatment at IC50 concentration and arrested cell cycle transition at G1/S phase. Molecular docking study identifies a strong interaction between NMDA receptor and DBT suggesting its possible mode of action.

The next class of compounds analyzed was arylhydrazones of active methylene compounds (AHAMCs). Among 23 novel derivatives, R234 (2-(2-(2,4-dioxopentan- 3-ylidene)hydrazineyl)benzonitrile) was identified to possess the best anti-cancer features. R234 displayed an IC50 of 107 µM in U-87 cells and 87 µM in LN229 cells. R234 was found to reduce cell viability and proliferation, interrupt cell cycle at G1/S phase, drive apoptotic cell death, increase chemosensitivity and affect RTK pathways. Molecular profiling also supported the drug’s characteristic as a strong cell cycle inhibitor. Docking studies cues that R234 acts via NGFR receptor pathways.

The third class of compounds under study, detailed in this thesis, is boroxazolidones. The experimental results detected compound JRB115 (2,2- bis(2,4-difluorophenyl)-4-isopropyl-1,3,2λ4-oxazaborolidin-5-one as the best candidate among an array of 6 novel L-valine derived boroxazolidones. JRB115 exhibited dose-dependent cytotoxic effects in LN229 and SNB19 GBM cell lines with IC50 value of 53 µM and 49 µM, respectively. The compound JRB115 was induced apoptosis via caspase 3/7 activation, caused toxicity via oxidative stress and instigated cell cycle arrest at G2/M phase.

The last class of compounds reported in this project is thioester derivatives. On analysis of 12 novel compounds containing α-thioether ketones and orthothioester moieties, our results highlights 5a (1,2-bis(4-hydroxy-3-methoxyphenyl)-2-((3((2- (4-hyroxy-3-methoxyphenyl)-1,3-dithian-2-yl)thio)propyl)thio)ethan-1-one, as the most efficacious chemotherapeutic agent against GBM. 5a was proven to possess an IC50 value of 27 µM in U-87 cell line and 23 µM in LN229 cell line. The top compound inhibited GBM proliferation, promoted caspase3/7 activation, inhibited cell cycle, and restricted MAPK signaling cascade. RNA-seq analysis of 5a treated cells evidenced disfavoring of various tumorigenic pathways, notably proliferative and angiogenic signaling, and suggested inhibition of RTK signaling via EGFR. Docking studies indicated a solid interaction between 5a and EGFR endorsing the mode of action via EGFR.

In general, our findings suggest that all the chemical classes investigated possess strong bioactivity against Glioblastoma, and had derivatives more potent than the current standard drug Cisplatin. Of the four different drugs considered for comprehensive evaluation, the study indicates that, the novel thioester derivative 5a possesses multiple characteristics confronting various aspects of GBM oncogenesis. Specifically, 5a is potent cytotoxic, genotoxic and a multi-kinase inhibitor leading to anti-angiogenic, anti-invasive effects, posing it as a best anti- GBM chemotherapy agent. Designing a precision medicine based on characteristics of this deadly tumor such as tumor subtype, mutagenic variations, specific molecular signatures, cross-talk of pathways involving in proliferation, angiogenesis, and drug resistance can be promising in anti-GBM therapy.