Structure-based virtual screening of chemical libraries as potential MELK inhibitors and their therapeutic evaluation against breast cancer

Breast cancer is one of the most common malignancies having high incidences and mortality rates. Among different subtypes, triple negative breast cancer (TNBC) is the most aggressive one and shows poor patient prognosis [1,2]. Since TNBC lacks the expression of both estrogen/progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2), no hormone targeted therapy works for this disease. The treatment regime for TNBC includes conventional surgery, radiation therapy and chemotherapy, however in spite of these, the disease may lead to recurrence and metastasis [3,4]. Thus, there is an urgent need to identify suitable molecular markers/targets that can be earmarked in TNBC to develop new and effective molecular targeted therapies. In this regard, inhibiting poly-ADP ribose polymerase (PARP) in case of BRCA1/2 mutated TNBC, proteins of cell cycle regulatory pathways such retinoblastoma (RB) and phosphoinositide 3-kinase (PI3K) have been tested for their efficacies towards TNBCs [5]. Multiple small molecule inhibitors against these targets have been developed but are found to be effective in limited population only. Thus, there is a constant requirement to identify new therapeutic targets/molecules for TNBC and develop safe and effective therapeutic strategies for TNBC [6].

Serine/Threonine protein kinase, maternal embryonic leucine zipper kinase (MELK) also known as murine protein serine threonine kinase 38 (MPK38) has been considered a potential molecular target in cancer therapy [7]. Initially, MELK was identified as a regulatory protein in early embryonic development [8]. Previous studies revealed overexpression of MELK in TNBC, hence was portrayed as a promising target for TNBC and cancer stem cell population [9]. MELK plays significant role in mitotic progression in TNBC and not only function as proliferation marker, but also as predictive marker for breast cancer aggressiveness, prognosis, therapy response and disease recurrence [10]. MELK mediated phosphorylation of FOXM1, an important cell cycle progression regulator, initiates its transcriptional activity thus facilitating the activity of various mitotic regulators such as CDC25B, Survivin, Aurora B [11]. Notably, silencing MELK in TNBC cells also leads to programmed cell death emphasizing its contribution to TNBC progression and its suitability as a molecular therapeutic target. Due to lack of MELK selective inhibitors, role of MELK as therapeutic target in cancer remains underestimated. One competitive type I MELK inhibitor e.g. OTSSP167 (OTS167) is currently under clinical trial for TNBC, however the selectivity data is yet to be disclosed [12]. To enhance selectivity, MELK-T1 was developed based on fragment-based drug designing strategy, which turned out to be a selective chemical probe inhibitor for MELK having high quality protein ligand structural interaction [13]. Till date, only few inhibitors have been developed against MELK and passing through the clinical trials and the process of drug development is still a long way to go.

Drug discovery being a costly affair, researchers are focusing on alternative strategies for identifying new molecules like drug repurposing or drug repositioning, which identifies new therapeutic action of pre-existing FDA approved drugs/molecules [14,15]. The already approved molecules possess much safer pharmacological, toxicological and safety profiles and the drug development process escapes initial clinical phases making the whole event cost and time effective [16,17]. Various drugs such as Aspirin, metformin etc. are plant derivatives and have been successfully repurposed [18]. Hence, repurposing of plant derived natural compounds with multiple biological activities also serves as a beneficial strategy to develop cost effective cancer therapeutics.

In this study, we explored the potential of various phytochemicals and synthetic drugs as MELK inhibitors and aimed to repurpose them as potential therapeutic molecule against breast cancer specifically TNBC. In silico docking was employed for identifying small molecules against MELK through high throughput virtual screening using Glide (Schrodinger Inc.) and molecular mechanics with generalised Born and surface area solvation (MM/GBSA) free energy calculations and best fit molecules (hits) were selected based on their chemical space, docking scores, and binding interactions at the MELK active site. Absorption, Distribution, Metabolism and Excretion (ADME) properties of best hit molecules were also predicted using Swiss ADME software. Further, anti-tumorigenic potential of selected hits was assessed using cell viability assays, trypan blue assay, qRT-PCR and Western blotting. The study identifies isoliquiritigenin and emodin as potential plant derived MELK inhibitors that show high binding affinity at the active site of MELK and also diminishes MELK protein and transcript levels in TNBC cells. Strong growth inhibitory effect of both the phytoconstituents in TNBC cells was observed compared to non-tumorigenic mammary epithelial cells. The anti-tumorigenic effect of these phytoconstituents was an outcome of induction of cell cycle arrest, accumulation of DNA damage leading to enhanced apoptosis. Thus, our study proposes isoliquiritigenin and emodin as potential anti-carcinogenic candidates for TNBC. The study outcomes also emphasize on the significance of MELK for the development of targeted therapy for TNBC.

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