Although this amino acid does not directly interact with the DNA, the mutation disrupted the binding of p53 with DNA by inducing unfavorable structural changes in its DNA binding region (Fig.?2). and doxorubicin (a DNA damage inducing reagent) treated cells with anti-53BP1 and p21WAF-1 antibodies. Increase in 53BP1 and p21WAF-1 was observed in treated cells. Quantitation of 53BP-1 and p21WAF1 from immunostaining images is demonstrated in (B) and (C), respectively. Number S4. Quantitation of immunostaining images of mortalin (A) and p53 (B) demonstrated in Fig. ?Fig.5;5; p21WAF1 and HP1g (demonstrated in Fig. ?Fig.6)6) and p53 (shown in Fig. ?Fig.7).7). (PDF 8692 kb) 13046_2019_1099_MOESM1_ESM.pdf (8.5M) GUID:?F5630774-9A69-41D2-A26F-303C076D876A Data Availability StatementAll data generated or analyzed during this study are included in this published article (and its additional documents). Abstract Background Tumor suppressor p53 protein is frequently mutated in a large majority of cancers. These mutations induce local or global changes in protein structure therefore influencing its binding to DNA. The structural variations between the crazy type and mutant p53 therefore provide an opportunity to selectively target mutated p53 harboring malignancy cells. Restoration of wild type p53 activity in mutants using small molecules that can revert the structural changes have been considered for malignancy therapeutics. Methods We used bioinformatics and molecular docking tools to investigate the structural changes between the wild type and mutant p53 proteins (p53V143A, p53R249S, p53R273H and p53Y220C) and explored the therapeutic potential of Withaferin A and Withanone for Tegoprazan restoration of wild type p53 function in malignancy cells. Malignancy cells harboring the specific mutant p53 proteins were utilized Tegoprazan for molecular assays to determine the mutant or wild type p53 functions. Results We found that p53V143A mutation does not show any significant structural changes and was also refractory to the binding of withanolides. p53R249S mutation critically disturbed the H-bond network and destabilized the DNA binding site. However, withanolides did not show any selective binding to either this mutant or other comparable variants. p53Y220C mutation produced a cavity near the site of mutation with local loss of hydrophobicity and water network, leading to functionally inactive conformation. Mutated structure Tegoprazan could accommodate withanolides suggesting their conformational selectivity to target p53Y220C mutant. Using human cell lines made up of specific p53 mutant proteins, we exhibited that Withaferin A, Withanone and the extract rich in these withanolides caused restoration of wild type p53 function in mutant p53Y220C cells. This was associated with induction of p21WAF-1-mediated growth arrest/apoptosis. Conclusion The study suggested that withanolides may serve as highly potent anticancer compounds for treatment of cancers harboring a p53Y220C mutation. Electronic supplementary material The online version of this article (10.1186/s13046-019-1099-x) contains supplementary material, which is available to authorized users. Keywords: Withaferin A, Withanone, p53 mutants, Wild type p53 restoration, Malignancy therapy Introduction p53 protein has been established as a tumor suppressor and guardian of the genome. It inhibits proliferation of genetically altered or stressed cells by induction of growth arrest, senescence or apoptosis [1]. It also blocks the metastasis and angiogenesis of malignancy cells. In the absence of stress, wild type p53 (p53WT) undergoes quick degradation, regulated by HDM2 and other unfavorable regulators like Pirh2, COP1 and mortalin [2C5] accounting for its short half-life in normal cells. Besides, p53 regulates its own stability by structural modulation [6]. Under stressed conditions like genotoxic damage, oncogene activation or hypoxia, it is stabilized and activated by post-translational modifications [7, 8]. Activated p53 Tegoprazan then either induces growth arrest or apoptosis in the dividing cells [9, 10] curtailing the proliferation of genetically stressed/damaged cells that carry high risk of carcinogenesis. Inactivation of p53 protein is the key factor in uncontrolled proliferation of cells. Mutated p53 with altered function or total inactivation has been detected in over 85% of cancers [11, 12]. Genetic changes in p53 results in (i) altered interactions with proteins like ubiquitin ligases leading to modified levels of ubiquitination [13], Rabbit polyclonal to AGBL3 (ii) exclusion of p53 from nucleus [5], (iii) abrogation of p53-DNA interactions [14] or (iv) unstable tetramer structure, essential for p53 to function as a transcriptional activator [12]. More than 7500 single missense point mutations that impact the central core of p53, involved in direct binding with DNA have been reported (http://p53.iarc.fr/). The DNA binding domain (DBD) of p53 stretches from 112 to 286 amino acids (Additional?file?1: Fig. S1). Mutations in the DBD either disrupt the DNA binding directly or bring local/global switch in the p53 protein structure. It is comprised of immunoglobulin-like -sandwich, made up of two antiparallel -linens facilitating an elastic DNA-binding surface [14]. Two large loops (L2 and L3) stabilized by a zinc ion and a loopCsheetChelix motif (loop L1) collectively make the DBD. Four residues.