Compound (SMILES): CN1C[C@H](Nc2nc3ccccn3c(=O)c2Br)C[C@H](c2ccc(OCCOCCOc3cccc4c3C(=O)N(C3CCC(=O)NC3=O)C4=O)cc2)C1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KAT2A (UniProt Q92830)
POI sequence: MAEPSQAPTPAPAAQPRPLQSPAPAPTPTPAPSPASAPIPTPTPAPAPAPAAAPAGSTGTGGPGVGSGGAGSGGDPARPGLSQQQRASQRKAQVRGLPRAKKLEKLGVFSACKANETCKCNGWKNPKPPTAPRMDLQQPAANLSELCRSCEHPLADHVSHLENVSEDEINRLLGMVVDVENLFMSVHKEEDTDTKQVYFYLFKLLRKCILQMTRPVVEGSLGSPPFEKPNIEQGVLNFVQYKFSHLAPRERQTMFELSKMFLLCLNYWKLETPAQFRQRSQAEDVATYKVNYTRWLCYCHVPQSCDSLPRYETTHVFGRSLLRSIFTVTRRQLLEKFRVEKDKLVPEKRTLILTHFPKFLSMLEEEIYGANSPIWESGFTMPPSEGTQLVPRPASVSAAVVPSTPIFSPSMGGGSNSSLSLDSAGAEPMPGEKRTLPENLTLEDAKRLRVMGDIPMELVNEVMLTITDPAAMLGPETSLLSANAARDETARLEERRGIIEFHVIGNSLTPKANRRVLLWLVGLQNVFSHQLPRMPKEYIARLVFDPKHKTLALIKDGRVIGGICFRMFPTQGFTEIVFCAVTSNEQVKGYGTHLMNHLKEYHIKHNILYFLTYADEYAIGYFKKQGFSKDIKVPKSRYLGYIKDYEGATLMECELNPRIPYTELSHIIKKQKEIIKKLIERKQAQIRKVYPGLSCFKEGVRQIPVESVPGIRETGWKPLGKEKGKELKDPDQLYTTLKNLLAQIKSHPSAWPFMEPVKKSEAPDYYEVIRFPIDLKTMTERLRSRYYVTRKLFVADLQRVIANCREYNPPDSEYCRCASALEKFFYFKLKEGGLIDK
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: OCI-AML-3 (Homo sapiens)
Cell line description: African=0%; Native American=1.44%; East Asian, North=2.86%; East Asian, South=0%; South Asian=0.99%; European, North=55.94%; European, South=38.78%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02 A*02
Stable (MSS) (Sanger).
In situ; Peripheral blood; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  0.3 nM
Assay description: KAT2A (and/or KAT2B) Degradation Assay
AML3 and MOLM13 cells were purchased from ATCC and cultured under standard conditions (37 °C, 5% CO2 and humidity). Cells were cultured in RPMI cell media containing 20% FBS, 1% Pen/Strep and L-Glutamine. Proliferation assays were conducted on cells after 2 weeks of culturing post thawing from liquid nitrogen. AML3 or MOLM13 cells were seeded at 2-5x106 cells/well in a standard tissue culture 6-well plate. Upon plating, cells were treated with 100, 10, 1, 0.1, 0.01 nM compound or DMSO for 4 hours. The final DMSO concentration was equal across all treatments (0.01%). After 4 hours, cells were spun down briefly in an ice cold centrifuge and washed twice in ice cold PBS. Upon removal of PBS after the second wash, 100 μl of ice cold RIPA Lysis Buffer + protease and phosphatase inhibitors was added and the samples incubated whilst rolling at 4 °C for ~30 minutes. After 30 minutes, samples were centrifuged on a tabletop centrifuge (>12,000 rpm) at 4 °C for 20 minutes. The supernatant was collected and protein concentration determined using a DC BIORAD assay.
40 μg of protein (per sample) was run out on a 4-20% gel and transferred onto a methanol pre-soaked PVDF membrane. After the transfer, membranes were blocked using 5% BSA in TBST for at least 1 hour at room temperature. Membranes were then cut at the level of ~the 50 kDa marker band. The top half was probed overnight at 4 °C for KAT2A or KAT2B and the bottom half for Actin. The concentrations of the primary antibodies to probe for KAT2A, KAT2B and Actin were 1/500, 1/500 and 1/5000, respectively. After overnight incubation in the primary antibody, membranes were washed 3 time in TBST and incubated at room temperature for one hour with the relevant secondary antibody (HRP conjugated anti-rabbit antibody) at 1/5000. After incubation with the secondary antibody, the membranes were washed 3 times in TBST. Membranes were developed using ECL reagent and imaged.
Bands corresponding to KAT2A (or KAT2B) and Actin, on the imaged membranes, were analyzed and quantified using ImageJ image analysis software. The ratios of KAT2A over Actin were determined for all treatments and normalized to DMSO treatment. The values were then plotted using GraphPad Prism to determine DC50 values - the concentration at which 50% of KAT2A is degraded.
Reference: PROTACS FOR TARGETED DEGRADATION OF KAT2A AND KAT2B FOR THE TREATMENT OF CANCER (patent)
Source database: TPDdb