Molecular hybridization design and synthesis of novel spirooxindole-based MDM2 inhibitors endowed with BCL2 signaling attenuation:A step towards the next generation p53 activators

Despite the achieved progress in developing efficient MDM2-p53 protein-protein interaction inhibitors (MDM2 inhibitors), the acquired resistance of tumor cells to such p53 activators posed an argument about the druggability of the pathway. Combination studies disclosed that concomitant inhibition of MDM2 and BCL2 functions can sensitize the tumor cells and synergistically induce apoptosis. Herein, we employed a rapid combinatorial approach to generate a novel series of hybrid spirooxindole-based MDM2 inhibitors (5a-s) endowed with BCL2 signaling attenuation. The adducts were designed to mimic the thematic features of the chemically stable potent spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-ones MDM2 inhibitors while installing a pyrrole ring on the core via a carbonyl spacer inspired by the natural product marinopyrrole A that efficiently inhibits BCL2 family functions by various mechanisms. NCI 60 cell-line panel screening revealed their promising broad-spectrum antiproliferative activities. The NCI-selected derivatives were screened for cytotoxic activities against normal fibroblasts, MDA-MB 231, HepG-2, and Caco-2 cells via MTT assay, subjected to mechanistic apoptosis studies for assessment of p53, BCL2, p21, and caspase 3/7 status, then evaluated for potential MDM2 inhibition utilizing MST assay. The most balanced potent and safe derivatives; 5i and 5q were more active than 5-fluorouracil, exhibited low μM range MDM2 binding (KD =1.32 and 1.72 μM, respectively), induced apoptosis-dependent anticancer activities up to 50%, activated p53 by 47-63%, downregulated the BCL2 gene to 59.8%, and reduced its protein level (13.75%) in the treated cancer cells. Further downstream p53 signaling studies revealed > 2 folds p21 upregulation and > 3 folds caspase 3/7 activation. Docking simulations displayed that the active MDM2 inhibitors resided well into the p53 binding sites of MDM2, and shared key interactions with the co-crystalized inhibitor posed by the indolinone scaffold (5i, 5p, and 5q), the halogen substituents (5r), or the installed spiro ring (5s). Finally, in silico ADMET profiling predicted acceptable drug-like properties with full accordance to Lipinski's, Veber's, and Muegge's bioavailability parameters for 5i and a single violation for 5q

Corrigendum to “Novel spirooxindole based benzimidazole scaffold: In vitro, nanoformulation and in vivo studies on anticancer and antimetastatic activity of breast adenocarcinoma”

This is a critical needed correction. In Fig. 15, the image corresponding to the lung from mouse treated with 25 mg/kg was taken from the same section of the mouse that was treated with 10 mg/kg. The corrected Fig. 15 [Formula presented] Fig. 15. Microscopic pictures of H&amp;E stained lung sections from groups received (A) 4*106 cells/200 μL showing congested blood vessels (red arrows), peribronchial and interstitial aggregation (black arrows) of tumour cells admixed with MNCs. Microscopic pictures of H&amp;E stained lungs sections from treated groups (B) 10 or (c) 25 mg/kg) showing disappeared congestion with decreased numbers of perivascular and interstitial infiltration of tumour cells. Increasing dose of treatment 25 mg/kg was more efficient than 10 mg/kg. Low magnification X: 100 with 100 μm scale bar.</p

Corrigendum to “Novel spirooxindole based benzimidazole scaffold: In vitro, nanoformulation and in vivo studies on anticancer and antimetastatic activity of breast adenocarcinoma”

This is a critical needed correction. In Fig. 15, the image corresponding to the lung from mouse treated with 25 mg/kg was taken from the same section of the mouse that was treated with 10 mg/kg. The corrected Fig. 15 [Formula presented] Fig. 15. Microscopic pictures of H&amp;E stained lung sections from groups received (A) 4*106 cells/200 μL showing congested blood vessels (red arrows), peribronchial and interstitial aggregation (black arrows) of tumour cells admixed with MNCs. Microscopic pictures of H&amp;E stained lungs sections from treated groups (B) 10 or (c) 25 mg/kg) showing disappeared congestion with decreased numbers of perivascular and interstitial infiltration of tumour cells. Increasing dose of treatment 25 mg/kg was more efficient than 10 mg/kg. Low magnification X: 100 with 100 μm scale bar.</p

Corrigendum to “Novel spirooxindole based benzimidazole scaffold: In vitro, nanoformulation and in vivo studies on anticancer and antimetastatic activity of breast adenocarcinoma”

This is a critical needed correction. In Fig. 15, the image corresponding to the lung from mouse treated with 25 mg/kg was taken from the same section of the mouse that was treated with 10 mg/kg. The corrected Fig. 15 [Formula presented] Fig. 15. Microscopic pictures of H&amp;E stained lung sections from groups received (A) 4*106 cells/200 μL showing congested blood vessels (red arrows), peribronchial and interstitial aggregation (black arrows) of tumour cells admixed with MNCs. Microscopic pictures of H&amp;E stained lungs sections from treated groups (B) 10 or (c) 25 mg/kg) showing disappeared congestion with decreased numbers of perivascular and interstitial infiltration of tumour cells. Increasing dose of treatment 25 mg/kg was more efficient than 10 mg/kg. Low magnification X: 100 with 100 μm scale bar.</p

Corrigendum to “Novel spirooxindole based benzimidazole scaffold: In vitro, nanoformulation and in vivo studies on anticancer and antimetastatic activity of breast adenocarcinoma”

This is a critical needed correction. In Fig. 15, the image corresponding to the lung from mouse treated with 25 mg/kg was taken from the same section of the mouse that was treated with 10 mg/kg. The corrected Fig. 15 [Formula presented] Fig. 15. Microscopic pictures of H&amp;E stained lung sections from groups received (A) 4*106 cells/200 μL showing congested blood vessels (red arrows), peribronchial and interstitial aggregation (black arrows) of tumour cells admixed with MNCs. Microscopic pictures of H&amp;E stained lungs sections from treated groups (B) 10 or (c) 25 mg/kg) showing disappeared congestion with decreased numbers of perivascular and interstitial infiltration of tumour cells. Increasing dose of treatment 25 mg/kg was more efficient than 10 mg/kg. Low magnification X: 100 with 100 μm scale bar.</p

Corrigendum to “Novel spirooxindole based benzimidazole scaffold: In vitro, nanoformulation and in vivo studies on anticancer and antimetastatic activity of breast adenocarcinoma”

This is a critical needed correction. In Fig. 15, the image corresponding to the lung from mouse treated with 25 mg/kg was taken from the same section of the mouse that was treated with 10 mg/kg. The corrected Fig. 15 [Formula presented] Fig. 15. Microscopic pictures of H&amp;E stained lung sections from groups received (A) 4*106 cells/200 μL showing congested blood vessels (red arrows), peribronchial and interstitial aggregation (black arrows) of tumour cells admixed with MNCs. Microscopic pictures of H&amp;E stained lungs sections from treated groups (B) 10 or (c) 25 mg/kg) showing disappeared congestion with decreased numbers of perivascular and interstitial infiltration of tumour cells. Increasing dose of treatment 25 mg/kg was more efficient than 10 mg/kg. Low magnification X: 100 with 100 μm scale bar.</p

Design, Synthesis, Chemical and Biochemical Insights Into Novel Hybrid Spirooxindole-Based p53-MDM2 Inhibitors With Potential Bcl2 Signaling Attenuation

The tumor resistance to p53 activators posed a clinical challenge. Combination studies disclosed that concomitant administration of Bcl2 inhibitors can sensitize the tumor cells and induce apoptosis. In this study, we utilized a rapid synthetic route to synthesize two novel hybrid spirooxindole-based p53-MDM2 inhibitors endowed with Bcl2 signaling attenuation. The adducts mimic the thematic features of the chemically stable potent spiro [3H-indole-3,2′-pyrrolidin]-2(1H)-ones p53-MDM2 inhibitors, while installing a pyrrole ring via a carbonyl spacer inspired by the natural marine or synthetic products that efficiently inhibit Bcl2 family functions. A chemical insight into the two synthesized spirooxindoles including single crystal x-ray diffraction analysis unambiguously confirmed their structures. The synthesized spirooxindoles 2a and 2b were preliminarily tested for cytotoxic activities against normal cells, MDA-MB 231, HepG-2, and Caco-2 via MTT assay. 2b was superior to 5-fluorouracil. Mechanistically, 2b induced apoptosis-dependent anticancer effect (43%) higher than that of 5-fluorouracil (34.95%) in three studied cancer cell lines, activated p53 (47%), downregulated the Bcl2 gene (1.25-fold), and upregulated p21 (2-fold) in the treated cancer cells. Docking simulations declared the possible binding modes of the synthesized compounds within MDM2

Table1_Targeted ferroptotic potency of ferrous oxide nanoparticles-diethyldithiocarbamate nanocomplex on the metastatic liver cancer.docx

Existing treatments are frequently ineffective in combating liver cancer (LC) due to its rapid growth, high metastatic potential, and chemoresistance. Thus, inducing ferroptosis, a new non-apoptotic regulated cell death-dependent massive iron overload-mediated lipid peroxidation, is an alternative effective approach for treating LC. The efficient trigger of ferroptosis requires blocking cellular antioxidant (anti-ferroptosis) response and selectivity to avoid harming other healthy tissues. In this study, green chemically synthesized ferrous oxide nanoparticles (F(II) NPs) were used for enhancing selective iron accumulation in tumor tissue, while diethyldithiocarbamate (DE) was for inhibiting the antioxidant system (glutathione and aldehyde dehydrogenase (ALDH) 2) which protects the tumor from damage-dependent lipid peroxides. Thus, F(II) NPs were used with DE as nanocomplex (DF(II) NPs) and its anti-LC activity compared to ferrous oxide DF(II). DF(II) NPs outperformed the typical complex of DF(II) in eradicating metastatic LC cells in HepG2 cells and a chemically induced metastatic LC animal model, as evidenced by flow cytometry, histological and immunohistochemical analyses, and α-fetoprotein depletion. The superior therapeutic potency-dependent ferroptotic activity of DF(II) NPs, attributed to their higher selective accumulation (∼77%) than DF(II) in tumor tissues (liver and lung), resulted in a strong elevation of cellular lipid peroxidation with extreme suppression of nuclear related factor 2 (Nrf2) transcriptional activity, glutathione (GSH), glutathione peroxidase 4, and ALDH2. Subsequently, a severe inhibition in the expression of oncogenes and metastatic cancer stem cell genes was recorded in DF(II) NPs-treated LC animal group. In contrast to DF(II), DF(II) NPs were able to normalize liver functions and did not show any variations in hematological and histological parameters in the blood and tissues of DF(II) NPs-treated normal mouse group. These findings validate the potency and safety of DF(II) nanocomplex as a promising nanodrug for combating metastatic LC.</p