IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell (1535-6108) 2011 imex curation Sakamoto H., Tsukaguchi T., Hiroshima S., Kodama T., Kobayashi T., Fukami TA., Oikawa N., Tsukuda T., Ishii N., Aoki Y. Cancer - Interactions investigated in the context of cancer sakamotohrs@chugai-pharm.co.jp 26-Sep-2011: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Sakamoto H., Tsukaguchi T., Hiroshima S., Kodama T., Kobayashi T., Fukami TA., Oikawa N., Tsukuda T., Ishii N., Aoki Y. Cancer - Interactions investigated in the context of cancer EBI-3956565: (refers to Kd determination - no figure) cannot have as Interaction Type = Phosphorylation - as ATP cannot be phosphorylated! There presumably must be a protein substrate ??- not stated- but ATP does bind at the active site of ALK so is an interactor - Kinase Inhibitory Assay: Either time-resolved fluorescence resonance energy transfer (TR-FRET) or fluorescence polarization (FP) were used to measure the inhibitory ability against each kinase. To determine the Kd of ALK, a competition-binding assay (Ambit) was used (Kinase Screening assay) . The authors confirm that ALK is human in origin. TABLE 1: only Participants with an IC50 < 100 nM have been entered. sakamotohrs@chugai-pharm.co.jp Accepted 2011-JUL-01 by JYOTI 26-Sep-2011: Contacted by JYOTI. Cancer Cell (1535-6108) 2011 jyoti Only protein-protein interactions imex curation CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell (1535-6108) 2011 imex curation Sakamoto H., Tsukaguchi T., Hiroshima S., Kodama T., Kobayashi T., Fukami TA., Oikawa N., Tsukuda T., Ishii N., Aoki Y. Cancer - Interactions investigated in the context of cancer sakamotohrs@chugai-pharm.co.jp 26-Sep-2011: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Sakamoto H., Tsukaguchi T., Hiroshima S., Kodama T., Kobayashi T., Fukami TA., Oikawa N., Tsukuda T., Ishii N., Aoki Y. Cancer - Interactions investigated in the context of cancer sakamotohrs@chugai-pharm.co.jp Accepted 2011-JUL-01 by JYOTI 26-Sep-2011: Contacted by JYOTI. Cancer Cell (1535-6108) 2011 jyoti Only protein-protein interactions imex curation gastrintide Gastrintide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) EGPWLEEEEEAYGWMDF derived from Human Gastrin ch5424802 CH5424802 small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) a TKI a TKI alk_human ALK tyrosine kinase receptor ALK Anaplastic lymphoma kinase protein protein human Homo sapiens Human MGAIGLLWLLPLLLSTAAVGSGMGTGQRAGSPAAGPPLQPREPLSYSRLQRKSLAVDFVVPSLFRVYARDLLLPPSSSELKAGRPEARGSLALDCAPLLRLLGPAPGVSWTAGSPAPAEARTLSRVLKGGSVRKLRRAKQLVLELGEEAILEGCVGPPGEAAVGLLQFNLSELFSWWIRQGEGRLRIRLMPEKKASEVGREGRLSAAIRASQPRLLFQIFGTGHSSLESPTNMPSPSPDYFTWNLTWIMKDSFPFLSHRSRYGLECSFDFPCELEYSPPLHDLRNQSWSWRRIPSEEASQMDLLDGPGAERSKEMPRGSFLLLNTSADSKHTILSPWMRSSSEHCTLAVSVHRHLQPSGRYIAQLLPHNEAAREILLMPTPGKHGWTVLQGRIGRPDNPFRVALEYISSGNRSLSAVDFFALKNCSEGTSPGSKMALQSSFTCWNGTVLQLGQACDFHQDCAQGEDESQMCRKLPVGFYCNFEDGFCGWTQGTLSPHTPQWQVRTLKDARFQDHQDHALLLSTTDVPASESATVTSATFPAPIKSSPCELRMSWLIRGVLRGNVSLVLVENKTGKEQGRMVWHVAAYEGLSLWQWMVLPLLDVSDRFWLQMVAWWGQGSRAIVAFDNISISLDCYLTISGEDKILQNTAPKSRNLFERNPNKELKPGENSPRQTPIFDPTVHWLFTTCGASGPHGPTQAQCNNAYQNSNLSVEVGSEGPLKGIQIWKVPATDTYSISGYGAAGGKGGKNTMMRSHGVSVLGIFNLEKDDMLYILVGQQGEDACPSTNQLIQKVCIGENNVIEEEIRVNRSVHEWAGGGGGGGGATYVFKMKDGVPVPLIIAAGGGGRAYGAKTDTFHPERLENNSSVLGLNGNSGAAGGGGGWNDNTSLLWAGKSLQEGATGGHSCPQAMKKWGWETRGGFGGGGGGCSSGGGGGGYIGGNAASNNDPEMDGEDGVSFISPLGILYTPALKVMEGHGEVNIKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLAEDGVSCIVSPTPEPHLPLSLILSVVTSALVAALVLAFSGIMIVYRRKHQELQAMQMELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIGDFGMARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILERIEYCTQDPDVINTALPIEYGPLVEEEEKVPVRPKDPEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEISVRVPRGPAVEGGHVNMAFSQSNPPSELHKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHDRGNLGLEGSCTVPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEAATAPGAGHYEDTILKSKNSMNQPGP 0733D6C4FD212F41 alk-ch5424802 1 3 enzyme target enzyme target substrate substrate neutral component neutral component n-terminal biotin tag biotin tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 enzyme enzyme neutral component neutral component 4 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Table 1/Table S1 IC 50 values for ALK F1174L =1.0nM, and ALK R1275Q = 3.5 nM. alk-ch5424802-2 1 5 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection 4 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 3 enzyme target enzyme target substrate substrate neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Fig 4B CH5424802 had substantial inhibitory potency against both native ALK (Ki = 0.83 nM) and L1196M (Ki = 1.56 nM). alk-ch5424802-3 2 4 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 5 enzyme enzyme neutral component neutral component kinase_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection direct interaction direct interaction Fig 6B 1.75A R working = 16.92% R free = 20.59% CH5424802 binds to the ATP site of ALK in the DFG-in mode