Identifications small molecules inhibitor of p53-mortalin complex for cancer drug using virtual screening

Mortalin was over expressed in tumor cells and bind to p53 protein. This interaction was suggested to promote sequestration of p53 in the cytoplasm, thereby inhibiting its nuclear activity. The p53 is a tumor suppressor that is essential for the prevention of cancer development and loss of p53 function is one of the early events in immortalization of human cells. Therefore, abrogation p53-mortalin interaction using small molecule is guaranteed stop cancer cell grow. However study interaction of p53-mortalin, and its inhibition using small molecule is still challenging because specific site of mortalin that bind to p53, vice versa, is still debatable. This study has aims to analyze the p53-binding site of mortalin using molecular docking and to screen drug-like compounds that have potential as inhibitors of p53-mortalin interaction using virtual screening. The result showed that the lowest energy binding of p53-mortalin complex is -31.89 kcal/mol, and p53 protein bind to substrate binding domain of mortalin (THR433; VAL435; LEU436; LEU437; PRO442; ILE558; LYS555). Furthermore, the p53-binding domain of mortalin was used as receptor to screen 9000 drug-like compounds from ZINC database using molecular docking program Auto Dock Vina in PyRx 0.8 (Virtual Screening Tools). Here, we have identified three drug-like compounds that are ZINC01019934, ZINC00624418 and ZINC00664532 adequate to interrupt stability of p53-mortalin complex that warrant for anticancer agent.     

Identification and Functional Characterization of Nuclear Mortalin in Human Carcinogenesis

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.     

Ashwagandha derived withanone targets TPX2-Aurora A complex: computational and experimental evidence to its anticancer activity

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug.     

Targeting p53 for Novel Anticancer Therapy

Carcinogenesis is a multistage process, involving oncogene activation and tumor suppressor gene inactivation as well as complex interactions between tumor and host tissues, leading ultimately to an aggressive metastatic phenotype. Among many genetic lesions, mutational inactivation of p53 tumor suppressor, the “guardian of the genome,” is the most frequent event found in 50% of human cancers. p53 plays a critical role in tumor suppression mainly by inducing growth arrest, apoptosis, and senescence, as well as by blocking angiogenesis. In addition, p53 generally confers the cancer cell sensitivity to chemoradiation. Thus, p53 becomes the most appealing target for mechanism-driven anticancer drug discovery. This review will focus on the approaches currently undertaken to target p53 and its regulators with an overall goal either to activate p53 in cancer cells for killing or to inactivate p53 temporarily in normal cells for chemoradiation protection. The compounds that activate wild type (wt) p53 would have an application for the treatment of wt p53-containing human cancer. Likewise, the compounds that change p53 conformation from mutant to wt p53 (p53 reactivation) or that kill the cancer cells with mutant p53 using a synthetic lethal mechanism can be used to selectively treat human cancer harboring a mutant p53. The inhibitors of wt p53 can be used on a temporary basis to reduce the normal cell toxicity derived from p53 activation. Thus, successful development of these three classes of p53 modulators, to be used alone or in combination with chemoradiation, will revolutionize current anticancer therapies and benefit cancer patients.     

Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.     

Skp2 suppresses p53-dependent apoptosis by inhibiting p300

The F box protein Skp2 is oncogenic, and its frequent amplification and overexpression correlate with the grade of malignancy of certain tumors. Conversely, depletion of Skp2 decreases cell growth and increases apoptosis. Here, we show that Skp2 counteracts the transactivation function of p53 and suppresses apoptosis mediated by DNA damage or p53 stabilization. We demonstrate that Skp2 forms a complex with p300 through the CH1 and the CH3 domains of p300 to which p53 is thought to bind and antagonizes the interaction between p300 and p53 in cells and in vitro. As Skp2 antagonizes the interaction between p300 and p53, Skp2 suppresses p300-mediated acetylation of p53 and the transactivation ability of p53. Conversely, ectopic expression of p300 rescues the transactivation function of p53 in cells overexpressing Skp2. Taken together, our results indicate that Skp2 controls p300-p53 signaling pathways in cancer cells, making Skp2 a potential molecular target for cancer therapy.     

Targeting Colon Cancer Cells with Pyrazino-Imidazolinone Derivatives: Synthesis,Molecular Docking,and in Vitro Evaluation of Anti-Proliferative and Pro-Apoptotic Activities

We report the synthesis, spectroscopic characterization, molecular docking and biological evaluation of nine pyrazino-imidazolinone derivatives. These derivatives were evaluated for their anticancer activity against three cancer cell lines: 518A2 melanoma, HCT-116, and HCT-116 p53 knockout mutant colon carcinoma. The MTT assay was employed to assess their effectiveness. Among the nine compounds tested, four compounds (5 a, 5 d, 5 g, and 5 h) exhibited promising antiproliferative activity specifically against HCT-116 p53-negative cells (IC₅₀ 0.23, 0.20, 2.07 and 58.75 μM, respectively). Interestingly, treatment with the 3,4-dimethoxyphenyl derivative 5a resulted in a significant increase (199 %) in caspase activity in HCT-116 p53-negative cells compared to untreated cells while the bromo-pyrazine derivative 5d demonstrated (190 %) increase. These findings suggest that compounds 5a and 5 d induce p53-independent apoptotic cell death. Additionally, in silico molecular docking studies with EGFR and tyrosinase proteins indicated that compounds 5 d and 5 e have the potential to bind to important anticancer drug targets.     

Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy

Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin’s binding pocket, resulting in a direct improvement in the tumor’s sensitivity to chemotherapy. Molecular modeling of human mortalin’s binding pocket and its binding to the SEP domain of UBXN2A followed by yeast two-hybrid and His-tag pull-down assays revealed that three amino acids (PRO442, ILE558, and LYS555) within the substrate-binding domain of mortalin are crucial for UBXN2A binding to mortalin. As revealed by chase experiments in the presence of cycloheximide, overexpression of UBXN2A seems to interfere with the mortalin-CHIP E3 ubiquitin ligase and consequently suppresses the C‐terminus of the HSC70‐interacting protein (CHIP)-mediated destabilization of p53, resulting in its stabilization in the cytoplasm and upregulation in the nucleus. Overexpression of UBXN2A causes a significant inhibition of cell proliferation and the migration of colon cancer cells. We silenced UBXN2A in the human osteosarcoma U2OS cell line, an enriched mortalin cancer cell, followed by a clinical dosage of the chemotherapeutic agent 5-fluorouracil (5-FU). The UBXN2A knockout U2OS cells revealed that UBXNA is essential for the cytotoxic effect achieved by 5-FU. UBXN2A overexpression markedly increased the apoptotic response of U2OS cells to the 5-FU. In addition, silencing of UBXN2A protein suppresses apoptosis enhanced by UBXN2A overexpression in U2OS. The knowledge gained from this study provides insights into the mechanistic role of UBXN2A as a potent mortalin inhibitor and as a potential chemotherapy sensitizer for clinical application.     

miR-375 targets the p53 gene to regulate cellular response to ionizing radiation and etoposide in gastric cancer cells

MicroRNAs (miRNAs) offer a new approach for molecular classification and individual therapy of human cancer due to their regulation of oncogenic pathways. In a previous report, elevated miR-375 was found in recurring gastric cancer, and it was predicted that miR-375 may be a regulator of p53 gene. However, its biological role and mechanism of actions remain unknown. In this study, we characterized the expression level of miR-375 in gastric cancer cell lines – BGC823, MGC803, SGC7901, AGS, N87, MKN45 – using RT-PCR. We found that exogenous expression of miR-375 promoted the growth of AGS cells in both liquid and soft agar media. In agreement with the previous report, overexpression of miR-375 in AGS cells reduced the p53 protein expression level. A luciferase assay demonstrated that miR-375 down-regulated p53 expression through an interaction with the 3′ UTR region of p53. In addition, the expression of miR-375 desensitizes cells to ionizing radiation and etoposide. Flow cytometry analyses showed that miR-375 abrogated the cell cycle arrest and apoptosis after DNA damage. These results demonstrate that miR-375 targets p53 to regulate the response to ionizing radiation and etoposide treatment.