as used herein and in the claims , “ comprising ” means including the following elements but not excluding others . nowadays , many researchers considered cancer as metabolic diseases . 5 ′- adenosine menophosphate - activated protein kinase ( ampk ) is a vital enzyme in regulating energy homeostasis , which is considered as one of the master control regulators of cellular and organism metabolism . many studies confirmed that ampk activators can inhibit the proliferation and migration of tumor cells . as a metabolism rate - limiting enzyme , ampk is the potential target for treatment of nsclc . ampk could be activated by activation of upstream lkb1 / camkkβ or low energy status to switch on atp producing catabolic pathways and switch off atp - consuming anabolic pathways to restore cellular energy homeostasis . it could also be allosterically activated by ampk direct activator , such as a769662 . activated ampk adjusts its downstream channels through the cascade ( e . g . acc , mtor , tsc1 / 2 ) to regulate energy metabolism . therefore , ampk is a potential new lung cancer target for drug research and development . in fact , several ampk activators have already been approved in clinical trials ( http :// clinicaltrials . gov , ids nct01210911 , nct01266486 ). in this invention , the inventors have applied high throughput screening of new ampk activator from a small - molecule library using molecular docking technique . the inventors then used kinase - based and cell - based assays to detect the ampk activation of several compounds on a gefitinib - resistant nsclc cell line with egfr double mutation ( h1975 l858r + t790m ). the result showed that d561 - 0775 is a potent ampk activator . it can directly activate ampk activity through phosphorylation of ampk at thr172 site , and it can also induce apoptosis in h1975 cells via regulation of lipogenesis . this compound has the potential to be developed as new anti - cancer drug for lung cancer treatment . h1975 was purchased from atcc ( american type culture collection ) and was cultivated with rpmi 1640 medium supplement with 10 % fetal bovine serum ( gibco , big cabin , oklahoma , me ., usa ), 100 u / ml , penicillin and 100 μg / ml and streptomycin ( gibco , big cabin , oklahoma , me ., usa ). the cells were cultured in incubator with 5 % co 2 at 37 ° c . d561 - 0775 was purchased from top science co . ltd ( shanghai , china ). the primary anti - bodies of gapdh , total / phosphor - ampk , total / phosphor - acc were purchased from cell signaling technology ( danvers , mass ., usa ). fluorescein - conjugated anti - rabbit as secondary anti - body was purchased from odyssey ( belfast , me ., usa ). cells were seeded in a 96 - well microplate with 5000 cells / well confluence , and put into the incubator overnight for cell adhesion . different concentrations of the drug d561 - 0775 were added with dimethyl sulfoxide ( dmso ) as vehicle control . the microplates were incubated for another 72 hours . the whole experiment was repeated in triplicate . 10 μl of mtt ( 5 mg / ml ) solution was added to each well . the plate was placed back into the incubator for 4 hours . after that , 100 μl of resolved solution ( 10 % sds and 0 . 1 mm hcl ) was added to each well . before dissolving the formazan crystals , the microplate was put back into the incubator for another 4 hours . the absorbance of the plate was measured at 570 nm with reference at 650 nm by a microplate reader ( tecan , morrisville , n . c ., usa ). cell viability was calculated by percentages of the absorbance of the treatment group divided by the absorbance of untreated group . at least three independent experiments were performed for data analysis and presentation . h1975 cells ( 1 × 10 5 cells / well ) were seeded in a 6 - well plate for 24 hours , and treated with the indicated concentrations of d561 - 0775 for an additional 48 hours at 37 ° c . after 48 hours , the cells were washed by ice - cold 1 × pbs once and harvested by trypsination . then cells were centrifuged , collected and re - suspended in ice - cold 1 × pbs . after removing the supernatants , cell pellets were re - suspended in 100 μl 1 × annexin - binding buffer . the cells were then double - stained with annexin - v fitc and pi ( 100 μg / ml ) of 2 μl respectively for 15 min at room temperature in dark . after that , 300 μl 1 × annexin - binding buffer was added . apoptotic cells were quantitatively counted by a bd aria iii flow cytometer ( bd biosciences , san jose , calif ., usa ) after incubating h1975 cells with d561 - 0775 for 24 hours , h1975 cells were harvested and washed with cold 1 × pbs . then , cells were lysed with ice - cold ripa lysis buffer with protease and phosphatase inhibitors added to extract the cell protein extraction . the supernatants were collected by centrifugation at 12 , 000 g , for 5 minutes . the quantitation of total protein extraction was measured by bio - rad dctm protein assay kit ( bio - rad , philadelphia , pa ., usa ). then 30 μg of protein was loaded and electrophoretically separated on 8 % sds - page gel and then transferred to nitrocellulose ( nc ) membrane . membranes were blocked with 5 % non - fat milk and pbs containing 0 . 1 % tween - 20 ( tbst ) for 1 hour at room temperature . after 1 hour , membranes were incubated with primary anti - bodies ( 1 : 1000 dilution ) against gapdh , total / phosphor - acc , total - ampk , and ( 1 : 500 dilution ) against phosphor - ampk , at 4 ° c . with gently shaking overnight . membranes were washed with tbst for 3 times ( 5 minutes / time ), and incubated with secondary fluorescent antibody ( 1 : 10000 dilutions ) for 1 hour at room temperature . after rewashing the membranes with tbst for 3 times ( 15 minutes / time ), the stripes were visualized by li - cor odessy scanner ( belfast , me ., usa ). the cyclex ampk kinase assay kit ( cyclex co ., ltd ., nagano , japan ) was used to detect the activation of ampk by d561 - 0775 . amp was used as positive control , compound c was used as ampk inhibitor , and ampk kinase was used for representing the basal level . 6 . molecular docking study on the interaction between d561 - 0775 and ampk . molecular docking calculation is performed to study the binding mode of d561 - 0775 to the adenosine 5 ′- monophosphate ( amp )- activated protein kinase ( ampk ) using glide programming schrodinger software ( schrodinger , inc ., new york , n . y ., 2009 ). the structure of d561 - 0775 was processed by the ligprep based on opls - 2005 force field . the 3d structure of ampk for molecular docking was retrieved from the protein data bank ( pdb id : 4cfe ) and was processed by the protein preparation wizard module . d561 - 0775 was docked into the binding site of the ampk with the standard precision ( sp ) scoring mode . the docking grid box was defined by centering on the compound 991 in the complex of ampk and ligand ( pdb id : 4cfe ). all the data was presented as mean ± sd of 3 individual experiments . differences were analyzed by one - way anova using graph prism 5 . the chemical structure of d561 - 0775 and dose response curve were shown in fig1 a and fig1 b respectively . mtt assay showed that d561 - 0775 can inhibit cell proliferation on h1975 cells . the ic50 value of d561 - 0775 is 9 . 59 ± 1 . 73 μm after 72 h treatment . 2 . d561 - 9775 significantly induced apoptosis in h1975 cells as examined by quantitative annexin v / pi flow cytometry analysis by morphology observation , after treating h1975 cells with d561 - 0775 for 48 h , the treated cells floated and rounded - up starting at 20 μm as shown in fig2 a , which is an indication of apoptosis . then by using quantitative apoptosis measurement method , flow cytometry analysis showed that d561 - 0775 induced significant level of apoptosis in a dose - dependent manner as shown in fig2 b . on comparing with the control group , d561 - 0775 showed significant higher percentage of apoptotic cells and lower percentage of viable cells in the 20 μm treatment group (** p & lt ; 0 . 01 , n = 3 ) as shown in fig2 c . 3 . d561 - 0775 dose - dependently increases ampk phosphorylation as well as fatty acid oxidation . previous studies demonstrated that phosphorylation ampk signal can activate acetyl - coa carboxylase ( acc ), while cancer cell proliferation requires lipids for cell membrane synthesis . as an ampk downstream component , acc is an important regulator of fatty acid oxidation . when ampk was activated by phosphorylation on thr172 site , acc was phosphorylated to increase fatty acid oxidation . western blot analysis as shown in fig3 illustrated that d561 - 0775 activated phosphorylation of both ampk and acc in a dose - dependent manner , indicating that the anti - cancer efficacy is mediated by suppression of lipogenesis . β - actin was used a loading control while aicar ( 5 - aminoimidazole - 4 - carboxamide ribonucleotide ) was used a positive control because aicar was previously reported to be an ampk activator . ampk kinase activity assay was performed by treating h1975 cells with indicated concentrations of d561 - 0775 . as shown in fig4 , ampk kinase activity was enhanced in a dose - dependent manner ( n = 3 , ** p & lt ; 0 . 005 , *** p & lt ; 0 . 001 ). ampk kinase was used to indicate the basal activity level , and all enzyme activities were presented as fold of the ampk kinase alone group . compound c , the ampk inhibitor , was used as negative control . amp was also used as positive control . the binding mode of d561 - 0775 docked into the active site of ampk was shown in fig5 . the interactions between ampk and d561 - 0775 mainly consist of hydrophobic , polar and hydrogen bond interactions . the hydrophobic groups of d561 - 0775 form hydrophobic interactions with the side chain of l18 , l31 , i46 and t106 . the polar groups of d561 - 0775 form polar interactions with the side chain of i46 , l31 , n48 , r83 , d88 , s108 and n11 . in addition , d561 - 0775 also form hydrogen bonds with the backbone of i46 and n48 and the side chain of l31 . further , as illustrated in fig5 , the ampk protein is represented as cartoon . ampk and key residues around the binding pocket are shown as sticks . the hydrogen bond is labeled as red dashed lines . in this invention , the inventors have identified a new ampk activator d561 - 0775 , which targets the metabolism switch - ampk kinase , and shows potent anti - cancer activity in gefitinib - resistant nsclc cell with egfr double mutation ( h1975 ). d561 - 0775 is also illustrated to directly activate ampk kinase , induce apoptosis and phosphorylates downstream acc in h1975 cells . in summary , this compound has great potential to be developed as new anti - cancer drug for gefitinib - resistant nsclc patients by targeting ampk kinase . the exemplary embodiments of the present invention are thus fully described . although the description referred to particular embodiments , it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details . hence this invention should not be construed as limited to the embodiments set forth herein .
the present invention discloses a method of treating gefitinib - resistant non - small - cell lung cancer , comprising administering an effective amount of d561 - 0775 . a pharmaceutical composition comprising d561 - 0775 admixed with a pharmaceutical carrier for treating gefitinib - resistant non - small - cell lung cancer is also disclosed therein .