Development and Assessment of 1,5-Diarylpyrazole/Oxime Hybrids Targeting EGFR and JNK-2 as Antiproliferative Agents: A Comprehensive Study through Synthesis, Molecular Docking, and Evaluation.

New 1,5-diarylpyrazole oxime hybrid derivatives (scaffolds A and B) were designed, synthesized, and then their purity was verified using a variety of spectroscopic methods. A panel of five cancer cell lines known to express EGFR and JNK-2, including human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line Hela, human leukemia cell line K562, human pancreatic cell line SUIT-2, and human hepatocellular carcinoma cell line HepG2, were used to biologically evaluate for their in vitro cytotoxicity for all the synthesized compounds 7a-j, 8a-j, 9a-c, and 10a-c. The oxime containing compounds 8a-j and 10a-c were more active as antiproliferative agents than their non-oxime congeners 7a-j and 9a-c. Compounds 8d, 8g, 8i, and 10c inhibited EGFR with IC50 values ranging from 8 to 21 µM when compared with sorafenib. Compound 8i inhibited JNK-2 as effectively as sorafenib, with an IC50 of 1.0 µM. Furthermore, compound 8g showed cell cycle arrest at the G2/M phase in the cell cycle analysis of the Hela cell line, whereas compound 8i showed combined S phase and G2 phase arrest. According to docking studies, oxime hybrid compounds 8d, 8g, 8i, and 10c exhibited binding free energies ranging from -12.98 to 32.30 kcal/mol at the EGFR binding site whereas compounds 8d and 8i had binding free energies ranging from -9.16 to -12.00 kcal/mol at the JNK-2 binding site.

Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB-GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein-protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60-167) or M-heat domain (∆M, aa 967-1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240-1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148-2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.

Development of an RHEB-Targeting Peptide To Inhibit mTORC1 Kinase Activity.

In cancer, the mechanistic/mammalian target of rapamycin complex-1 (mTORC1) is hyperactivated to promote survival under adverse conditions. The kinase activity of mTORC1 is activated by small-GTPase RHEB-GTP. Therefore, a new modality to inhibit mTORC1 activity has emerged, through intercepting RHEB. However, due to the relatively large contact area involved in the interaction between RHEB and mTORC1, facilitating this inhibition through small molecules has been challenging. Here, we report the development of a peptide that can inhibit the RHEB-mTORC1 interaction. The peptide, P1_WT, was designed based on the α-helix (aa 101-115) of the N-heat domain of mTOR to interact with switch II of RHEB. P1_WT bound to RHEB (K D = 0.14 µM) and inhibited RHEB-mTORN-heat interaction (IC50 = 0.33 µM) in vitro. Consequently, P1_WT inhibited mTORC1 activity at a sub-micromolar level (IC50 ∼ 0.3 µM). P1_WT was predicted to be cell-permeable due to the rich content of arginine (23%), enhancing the intracellular translocation. These results show that P1_WT is a potential compound to further develop inhibitors for mTORC1 by intercepting RHEB from mTORC1.

Mapping of mTOR drug targets: Featured platforms for anti-cancer drug discovery.

The mammalian/mechanistic target of rapamycin (mTOR) is a regulatory protein kinase involved in cell growth and proliferation. mTOR is usually assembled in two different complexes with different regulatory mechanisms, mTOR complex 1 (mTORC1) and mTORC2, which are involved in different functions such as cell proliferation and cytoskeleton assembly, respectively. In cancer cells, mTOR is hyperactivated in response to metabolic alterations and/or oncogenic signals to overcome the stressful microenvironments. Therefore, recent research progress for mTOR inhibition involves a variety of compounds that have been developed to disturb the metabolic processes of cancer cells through mTOR inhibition. In addition to competitive or allosteric inhibition, a new inhibition strategy that emerged mTOR complexes destabilization has recently been a concern. Here, we review the history of mTOR and its inhibition, along with the timeline of the mTOR inhibitors. We also introduce prospective drug targets to inhibit mTOR by disrupting the complexation of the components with peptides and small molecules.

In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Activity of mTORC1.

Cancer-specific metabolic alterations hyperactivate the kinase activity of the mammalian/mechanistic target of rapamycin (mTOR) for overcoming stressful environments. Rapalogs, which allosterically inhibit mTOR complex 1 (mTORC1), have been approved as anticancer agents. However, the immunosuppressive side effect of these compounds results in the promotion of tumor metastasis, thereby limiting their therapeutic efficacy. We first report a nonrapalog inhibitor, WRX606, identified by a hybrid strategy of in silico and in cell selections. Our studies showed that WRX606 formed a ternary complex with FK506-binding protein-12 (FKBP12) and FKBP-rapamycin-binding (FRB) domain of mTOR, resulting in the allosteric inhibition of mTORC1. WRX606 inhibited the phosphorylation of not only the ribosomal protein S6 kinase 1 (S6K1) but also eIF4E-binding protein-1 (4E-BP1). Hence, WRX606 efficiently suppressed tumor growth in mice without promotion of metastasis. These results suggest that WRX606 is a potent lead compound for developing anticancer drugs discovered by in silico and in cell methods.

Highly ordered functionalized mesoporous silicate nanoparticles reinforced poly (lactic acid) gatekeeper surface for infection treatment.

The controlled release of a drug considers the key feature of the delivery carrier that enhances therapeutic efficacy. This study was aimed at design, synthesis of nano valve and capping systems onto caged functionalized mesoporous silica nanoparticles (SBA15) with nanoflowers polylactic acid (PLA-NF). Levofloxacin (LVX) as a specific model drug was encapsulated onto series; SBA15, SBA15@NH2, and SBA15@NH2/PLA. The examined nanocarriers released in a controlled fashion by external stimuli. The delivery vehicle based on PLA-NF coated SBA15@NH2, potent conjugated with LVX with experienced a high extent of trapping content with fast releasing by pH regulating mechanism. In vial LVX released profile and in vitro antifungal forceful of the selected microbes were detected. However, SBA15@NH2/PLA exhibited pore size, surface area and pore volume 5.4 nm, 163 and 0.011 respectively, but the significantly clear zone was obtained with Staphylococcus aureus ATCC 6538 (G+ve), Escherichia coli ATCC 25922 (G-ve), Candida albicans ATCC 10231 (yeast) and Aspergillus niger NRRL A-326 (fungus). Viability test avouch that rising functionality enhanced cytocompatibility and non-toxicity profile. Based on the aforementioned promising data, this type of nanocarriers offers when functionalized with targeting cells, the accessibility to deliver antibiotics onto nanosystem for increased potency against microbes and reduce side effects.