| The RAS/RAF/MEK/ERK pathway plays a key role in regulating the cell proliferation,apoptosis.This pathway deregulated in approximately one third malignant tumor.Mutations in RAS/RAF occur in large portion of malignancies and are associated with aggressive clinical behaviors and poor prognosis.Therefore,we developed a novel benzoxazole compound(KZ-001)as a highly potent and selective MEK 1/2 inhibitor.Based on the previously reported X-ray structure,we choose AZD6244 as lead compound.Our efforts were focused on enhancing the activity of the known MEK inhibitor AZD6244 and overcoming the shortcomings existing in current MEK inhibitors.In vitro,KZ-001 and AZD6244 inhibited the MEK1 kinase activity,with IC50 at 7.40 ± 1.96 nM and 199.73 ± 38.25 nM with ATP concentration at 45 μM in the Z’-LYTE assay.These two compounds inhibited the MEK2 kinase activity with IC50 at 64 nM and 742 nM,respectively,indicating that KZ-001 is a MEK 1/2 inhibitor.Here we showed that compound KZ-001 exhibited approximately 30-fold greater inhibition against BRAF-and KRAS-mutant tumor cells than that of AZD6244.KZ-001 against 14 representative protein kinases were evaluated,the results showed that no inhibition was observed at 10 μM against other 12 protein kinases examined,except for MEK1/2,suggesting that KZ-001 is a highly selective MEK1/2 inhibitor.Using the MTT or MTS assay,compared to the positive drug AZD6244 with an IC50 74.5 ± 3.0 nM-184.3 ± 5.0 nM and over 3000 nM on the BRAF and KRAS mutant cancer cell line,KZ-001 showed much more potent inhibition of proliferation,with an IC50 of 2.9 ± 0.6 nM-5.7 ± 0.3 nM and 169.9 ± 97.7 nM for the BRAF-and KRAS-mutant cancer cell line,while the RAFWT and RASWT cell lines A431 are resistant to KZ-001.KZ-001 improved 26-to 32-and over 17-fold compared to AZD6244 using the BRAF-and KRAS-mutant cancer cell lines,respectively.These results were also demonstrated using in vivo xenograft model,with the body weight no significant change.Chronic dosing of mice with KZ-001(6 mg/kg)significantly inhibited the growth of A375 tumors(TGI 91.0%;P<0.001),while AZD6244 at 10 mg/kg QD resulted in 45.8%inhibition(P<0.01).Calu-6 xenografts were also inhibited by 3 mg/kg KZ-001(TGI 63.2%;P<0.01),while the effect of AZD6244 was also observed under 10 mg/kg(TGI 54.8%;P<0.05).Chronic dosing with 3 mg/kg and 6 mg/kg KZ-001 completely inhibited the growth of Colo-205 tumors(TGI 103.9%and 108.8%,respectively;P<0.01),while AZD6244 at 10 mg/kg resulted in a significant inhibition of 83.3%(P<0.01)compared to the vehicle control.KZ-001 inhibited MAPK pathway like other known MEK inhibitors.In BRAF-mutant A375,Colo-205,and HT-29 cell line,MEK phosphorylation levels did not change significantly at concentrations ranging from 1 nM to 625 nM,but the ERK phosphorylation was completely blocked by KZ-001 at concentration ranging from 5 nM to 625 nM.Interestingly,in the Calu-6(KRAS-mutant cell line),MEK phosphorylation increased at concentrations ranging from 1 nM to 625 nM,whereas ERK phosphorylation was still blocked by KZ-001.By using FITC Annexin V apoptosis detection assay,this compound induced 7.1%and 37.0%early apoptosis of Calu-6 cell population at 10 nM and 10 pM,respectively.Likewise,KZ-001 induced 24%and 33.4%early apoptosis of Colo-205 cells at population at 10 nM and 10 μM.In contrast,KZ-001 at neither 10 nM nor 10 μM induced apoptosis in A375 and HT-29 cells.By using DNA content quantitation assay in A375 cells,KZ-001 induced a significant increase at the G1 phase,a decrease at S phase and G2 phase,and caused cell cycle delay of A375 cells at G0/G1 phase at both 10 nM and 10pM.Furthermore,pharmacokinetics(PK)analysis was performed for KZ-001,and this compound showed good orally bioavailability(28%)and exposure(AUC0-∞=337±169 ng hr/mL).To determine its potential clinical application,the synergistic effect of KZ-001 with other agents was investigated both in vitro and in vivo(xenograft models).KZ-001 exhibited synergistic anti-cancer effect in combination with BRAF inhibitor vemurafenib and a microtubule-stabilizing chemotherapeutic agent docetaxel.In summary,KZ-001,a structurally novel,potent anti-cancet benzoxazole compound,was developed as a MEK inhibitor that has potential for cancer treatment.Radiation-induced intestinal injury is one of the major side effects in patients receiving radiation therapy.There is no specific treatment for radiation enteritis in the clinic.We designed and synthesized four compounds which were expected to cleave into polyphenol and aminothiol in vivo to mitigate radiation injury and investigated the radioprotictive effect by 30 days survival experiments in C57BL/6J mice.Further researches focused on the compound XH-105 because of its superiority for the radioprotective effect.In the following studies,we described the beneficial effects of XH-105 against radiation-induced intestinal injury.C57BL/6J mice were treated by gavage with XH-105 one hour before total body irradiation(TBI).The results showed that XH-105 improved the survival rate of mice.After 7.5 Gy TBI,100 m/kg XH-105-treated mice group had a 30%survival rate beyond 30 days,while vehicle-treated group had 100%mortality within 20 days.When the mice were treated with three doses of XH-105(50 mg/kg,100 mg/kg and 200 mg/kg)and exposed to 9.0 Gy TBI,all doses improved the mice survival rate compared to that of the vehicle-treated group,and in particular,the 100 mg/kg dose dramatically improved the median survival.When exposed to 11.0 Gy TBI,there was 20%survival in vehicle-treated mice at 6 days,while 60%of mice survived in the 100 m/kg XH-105-treated group.The morphological changes of the small intestine in mice were evaluated.Compared with vehicle-treated mice,XH-105-treated mice showed more survival crypts(p<0.01),an increased villous height(p<0.01).The survival of Lgr5+intestinal stem cells,Ki67+ cells,villi+ enterocytes and lysozymes were determined by immunohistochemistry.At 3.5 days after 9.0 Gy TBI,the number of villi+ enterocytes,Lgr5+ ISCs,Ki67+ positive cells,and lysozyme+ positive cells were significantly increased in the XH-105-treated mice than in the vehicle-treated group.These data clearly indicate that XH-105 enhances the regenerative response of radiation-induced intestinal injuries by promoting the differentiation and proliferation of ISCs in the small intestine.DNA damage and cellular apoptosis in intestinal tissue were also evaluated by immunofluorescence and TUNEL assay,respectively.Compared to vehicle-treated mice after TBI,XH-105 significantly enhanced the survival rate,attenuated structural damage of the small intestine,the apoptotic rate,and DNA damage,maintained cell regeneration and promoted crypt proliferation and differentiation.Bax and p53 were evaluated by Western blot and immunofluorescence,and the results showed that XH-105 also reduced the expression of Bax and p53 in the small intestine.These data suggest that XH-105 is beneficial for the protection of radiation-induced intestinal injury by inhibiting the p53-dependent apoptosis signaling pathway. |
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