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Nedd8,a core molecule of the neddylation pathway,was discovered as an ubiquitin-like molecule responsible for different kinds of regulation of its target proteins such as cellular localization,changes in function etc.Neddylation pathway,a post-translational modifications(PTMs)system,follows the similar three steps enzymatic cascade reaction like ubiquitination.To start the reaction,firstly NEDD8is activated by NEDD8activating enzyme or APPBP1-UBA3(E1).Then,activated NEDD8is received by NEDD8conjugating enzyme UBE2M/UBE2F(E2).Finally,with the help of E3ligase(RBX1/RBX2.DCN1),NEDD8conjugated to its target protein and modify the function of that molecule.Proteins involve in cell cycle and apoptosis is vastly influenced by this pathway.Thus,Neddylation plays an essential role in cancer cell survival and proliferation.
Oppositely,inhibition of neddylation by MLN4924,a first-in-class small-molecule inhibitor of NEDD8activating enzyme APPBP1-UBA3(E1),showed significant anticancer effects.The drug showed its efficacy by inhibiting cullin neddylation and inducing cell cycle arrest,apoptosis,senescence and autophagy in a different cancer cell,which leads to impeding the cancer cell growth.However,in recent studies,drug resistance against MLN4924was observed,and alternative effective treatment targeting neddylation has not been discovered yet to inhibit cancer cell growth.Extensive research work is going on to identify new and effective inhibitors against the neddylation pathway for cancer treatment.Recent days,drug repurposing holds the potential to complement traditional drug discovery by mitigating the high monetary‐and time‐related costs and risks associated with drug discovery.With a failure rate of~45%associated with safety or toxicity issues mitigating the safety risk,in addition to the saving of up to5–7years in average drug development time,confers an attractive prospect for drug developers and patients alike.
In this study,we developed an HTRF based drug screening method to identify NAE-UBE2M inhibitor which can inhibit neddylation pathway.All required proteins were purified from E.coli and they were＞98%pure and the assay was validated by evaluate the IC50of MLN4924(5.80±0.763nM),S/B ratio(3.86±0.251)and Z＇factor(0.73±0.04).Sixteen hundred(1600)FDA approved drugs that are currently using to treat different kinds of diseases were analyzed with a view to evaluate its effects on NAE-UBE2M interaction or neddylation inhibition.From drug sscreening twelve drugswere identified,which have the potency to inhibit neddylation at50μM concentrations.Among them,we have got the50%inhibitory concentration(IC50)in protein-protein interaction assay of three drugs namely Micafungin Sodium,Otilonium Bromide and Ceritinib that showed IC5016.990±1.458μM,9.019±2.264μM,and4.899±1.522μM respectively.The pharmacological analysis of Micafungin Sodium and Otilonium Bromide using gastric cancer cell lines showed that the drugs could inhibit the neddylation at the cellular level as well and impede the growth of cancer cells.Interruption of cell growth was observed due to DNA damage and cell cycle arrest in the G2/M checkpoint.Overall,our finding revealed new effects of marketed drugs and it also suggests that the compounds can be a useful model for the design and development of a new class of highly potent inhibitors for cancer treatment.Morevover it imply the the relation of the neddylation pathway with the action of those drugs.
Oppositely,inhibition of neddylation by MLN4924,a first-in-class small-molecule inhibitor of NEDD8activating enzyme APPBP1-UBA3(E1),showed significant anticancer effects.The drug showed its efficacy by inhibiting cullin neddylation and inducing cell cycle arrest,apoptosis,senescence and autophagy in a different cancer cell,which leads to impeding the cancer cell growth.However,in recent studies,drug resistance against MLN4924was observed,and alternative effective treatment targeting neddylation has not been discovered yet to inhibit cancer cell growth.Extensive research work is going on to identify new and effective inhibitors against the neddylation pathway for cancer treatment.Recent days,drug repurposing holds the potential to complement traditional drug discovery by mitigating the high monetary‐and time‐related costs and risks associated with drug discovery.With a failure rate of~45%associated with safety or toxicity issues mitigating the safety risk,in addition to the saving of up to5–7years in average drug development time,confers an attractive prospect for drug developers and patients alike.
In this study,we developed an HTRF based drug screening method to identify NAE-UBE2M inhibitor which can inhibit neddylation pathway.All required proteins were purified from E.coli and they were＞98%pure and the assay was validated by evaluate the IC50of MLN4924(5.80±0.763nM),S/B ratio(3.86±0.251)and Z＇factor(0.73±0.04).Sixteen hundred(1600)FDA approved drugs that are currently using to treat different kinds of diseases were analyzed with a view to evaluate its effects on NAE-UBE2M interaction or neddylation inhibition.From drug sscreening twelve drugswere identified,which have the potency to inhibit neddylation at50μM concentrations.Among them,we have got the50%inhibitory concentration(IC50)in protein-protein interaction assay of three drugs namely Micafungin Sodium,Otilonium Bromide and Ceritinib that showed IC5016.990±1.458μM,9.019±2.264μM,and4.899±1.522μM respectively.The pharmacological analysis of Micafungin Sodium and Otilonium Bromide using gastric cancer cell lines showed that the drugs could inhibit the neddylation at the cellular level as well and impede the growth of cancer cells.Interruption of cell growth was observed due to DNA damage and cell cycle arrest in the G2/M checkpoint.Overall,our finding revealed new effects of marketed drugs and it also suggests that the compounds can be a useful model for the design and development of a new class of highly potent inhibitors for cancer treatment.Morevover it imply the the relation of the neddylation pathway with the action of those drugs.