Design, synthesis and biological evaluation of the novel MEK inhibitor and radioprotectors

Author: Ying Cheng

Cheng, Ying, 2021 Design, synthesis and biological evaluation of the novel MEK inhibitor and radioprotectors, Flinders University, College of Medicine and Public Health

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Signalling pathway dysregulation is associated with tumorgenesis. Aggressive tumour behaviour is often related with resistance to commonly used anti-cancer drugs. Cancer treatments commonly apply radio-therapy despite frequent negative side effects, including intestinal injury. The RAS/RAF/MEK/ERK signalling pathway plays a key role in regulating cell proliferation and apoptosis. This signalling pathway is deregulated in approximately one third of malignant tumours. Mutations in RAS/RAF occur in approximately 30% of malignancies and are associated with aggressive cancer behaviours and poor prognosis. To address these problems, this research consisted of two parts: 1) development and efficacy evaluation of a novel MEK inhibitor and 2) development of a series of novel compounds and evaluation of their intestinal radio-protective action and neuroprotective activity.

Based on the previously reported X-ray structure, AZD6244, a drug in phase III clinical trials, was chosen as a positive control and as a lead compound for further activity enhancement research to overcome the shortcomings of existing MEK inhibitors. As a result, a novel benzoxazole compound (KZ-001) was developed and the research determined that it is a highly potent and selective MEK 1/2 inhibitor, suitable for the potential treatment of cancer.

In vitro, KZ-001 showed approximately 27-fold and 12-fold greater inhibition of MEK1 and MEK2 kinase compared to AZD6244, respectively. Kinase selectivity studies showed that KZ-001 is a highly selective MEK1/2 inhibitor. Using the MTT- or MTS assay, KZ-001 had a 26- to 32-fold higher anti-proliferation activity in BRAF mutant cancer cell lines and 17-fold higher anti-proliferation activity in KRAS mutant cancer cell lines compared to AZD6244. In vivo xenograft models confirmed higher tumour growth inhibition activity of KZ-001 and the body weights of treated animals did not change significantly.

Like other known MEK inhibitors, KZ-001 inhibited the MAPK signalling pathway. KZ-001 completely blocked ERK phosphorylation in BRAF- and KRAS- mutant cell lines. The FITC Annexin V apoptosis assay demonstrated early apoptosis in Calu-6 and COLO 205 cell populations but not in A375 and HT-29 cells. Measurement of DNA content in A375 cells showed a significant increase of G1 phase cells and a decrease of S and G2 phase, and caused cell cycle delay of A375 cells at the G0/G1 phase.

In pharmacokinetic studies, KZ-001 showed good oral absorption and clearance from the blood stream. To determine its potential for clinical application, the synergistic effect of KZ-001 with other drug agents was investigated both in vitro and in vivo (xenograft models). KZ-001 exhibited synergistic anti-cancer effects in combination with the BRAF inhibitor vemurafenib and a microtubule-stabilizing chemotherapeutic agent docetaxel. Results obtained strongly suggest that KZ-001 is a very promising new anti-cancer drug for treatment of RAS/RAF mutant tumours.

Radiation-induced intestinal injury is one of the major side effects in patients receiving radiation therapy. There is no specific clinical treatment for radiation enteritis. This study aimed to find ROS-scavengers by rationale compound design for the treatment of radiation injury and neurodegenerative diseases.

Four compounds were designed which were expected to cleave into polyphenol and aminothiol in vivo to mitigate radiation injury. The 30-day survival experiments in C57BL/6J mice investigated radio-protective effects. Further research focused on the compound XH-105 because of its superior radio-protective effect compared to the other three compounds. XH-105 was administered by gavage to C57BL/6J mice one hour prior to total body irradiation (TBI) and survival rate was monitored. Survival rates of Lgr5+ ISCs, Ki67+ cells and villi+ enterocytes were determined histologically and lysozyme were identified and activity quantified by immunohistochemistry. DNA damage and cellular apoptosis in intestinal tissues were also evaluated. After TBI, XH-105 significantly enhanced survival rates, attenuated structural damage of the small intestine, decreased the rate of apoptosis, reduced DNA damage, maintained cell regeneration and promoted crypt proliferation and differentiation compared to vehicle-treated mice. XH-105 also reduced the expression of Bax and p53 in the small intestine. These data suggest that XH-105 protects against radiation-induced intestinal injury by inhibiting the p53-dependent apoptosis pathway.

Oxidative stress plays a central role in the common pathophysiology of neurodegenerative diseases. In Alzheimer’s disease, the aggregation of Aβ could induce ROS. HL-008, as an aminothiol compound, might be able to eliminate ROS in cell microenvironment. In this study, HL-008 showed no toxicity to PC-12 cells and protected PC-12 cells against H2O2-induced cytotoxicity. Moreover, this study showed that pretreatment with HL-008 could increase cell viability after exposure to Aβ1–42. Furthermore, all investigated concentrations of HL-008 enhanced neurite outgrowth in a dose-dependent manner.

In summary, this project developed a novel MEK inhibitor for the treatment of RAS/RAF mutant cancers and a set of radioprotectors for the mitigation of radioation therapy-induced intestinal injury. In addition, one radioprotector could potentially be used for the treatment of neurodegenerative diseases.

Keywords: MEK inhibitor, targeted therapy, combination treatment, anti-oxidants, radioprotectors

Subject: Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Professor Wei Zhang