RESEARCH ARTICLE 2,000 500- 000 P<0.000 1.000 500 ging of m EH ath of prin 20 d in s d (n=7) 97 urs e from ant antitat asks are n (mo +)-IOI (red har is 2 MC ival (i,k)in p t-derived 1100(h)and Per3 m0 10 in all groups)Asg 0) 11060 fts to (+)-01 (50mgko erallsurvival by a log-rank test (00001)Black c asdemonstrated by PET hicle. inhibitor of the beT-family of bromodomains.The a into th readers for ligand discovery. flank of 6 METHODS SUMMARY gri an Women's Hospital itches signal transduction and urce beamline X10SA. igaku FR-E 1.Ptashne,M 15.209) d by 2骑 ein.B. 3.M 73 391(9 4 10721NArURE1voL46i12130DEcE%2 n Publishers Limited.All rights reserved inhibitor of the BET-family of bromodomains. The approach outlined herein further establishes the feasibility of abrogating protein–protein interactions with small molecules, and targeting additional epigenetic readers for ligand discovery. METHODS SUMMARY The inhibitor JQ1 was synthesized in both racemic and enantiomerically pure format using the synthetic route outlined in scheme 1 and scheme 2 (Supplemen￾tary Methods) and its structure was fully characterized. Human bromodomains were expressed in bacteria as His-tagged proteins and were purified by nickel￾affinity and gel-filtration chromatography. Protein integrity was assessed by SDS–PAGE and electro-spray mass spectrometry on an Agilent 1100 Series LC/MSD TOF. All crystallizations were carried out at 4 uC using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected at the Swiss Light source beamline X10SA, or using a Rigaku FR-E generator. Structures were determined by molecular replacement. Isothermal titration calorimetry experi￾ments were performed at 15 uC on a VP-ITC titration microcalorimeter (MicroCal). Thermal melting experiments were carried out on an Mx3005p RT–PCR machine (Stratagene) using SYPRO Orange as a fluorescence probe. Dose-ranging small-molecule studies of proliferation were performed in white, 384-well plates (Corning) in DMEM media containing 10% FBS. Compounds were delivered with a JANUS pin-transfer robot and proliferation measurements were made on an Envisionmultilabel plate-reader (PerkinElmer).Murine xenografts were established by injecting NMC cells in 30% Matrigel (BD Biosciences) into the flank of 6-week-old female NCr nude mice (Charles River Laboratories). Tumour measurements were assessed by caliper measurements, and volume was calculated using the formula Vol 5 0.53 L3W2 . All mice were humanely killed, and tumours were fixed in 10% formalin for histopathological examination. Quantitative immunohistochemistry was performed using the Aperio Digital Pathology Environment (Aperio Technologies) at the DF/HCC Core Laboratory at the Brigham and Women’s Hospital. Received 5 May; accepted 17 September 2010. Published online 24 September 2010. 1. Ptashne, M. Binding reactions: epigenetic switches, signal transduction and cancer. Curr. Biol. 19, R234–R241 (2009). 2. Schreiber, S. L. & Bernstein, B. E. Signaling network model of chromatin. Cell 111, 771–778 (2002). 3. Marushige, K. Activation of chromatin by acetylation of histone side chains. Proc. Natl Acad. Sci. USA 73, 3937–3941 (1976). 4. Dey, A., Nishiyama, A., Karpova, T., McNally, J. & Ozato, K. Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Mol. Biol. Cell 20, 4899–4909 (2009). a d JQ1 Vehicle Keratin Ki67 b Vehicle JQ1 Pre Post 0 500 1,000 1,500 2,000 Vehicle JQ1 Tumour volume (mm3) P = 0.039 Vehicle JQ1 5 10 15 20 % ID per g e Pre Post P = 0.0001 JQ1 Veh. Keratin Mask Vehicle JQ1 0 20 40 60 80 100 Positivity (%) c f g 0 10 20 30 0 1,000 2,000 3,000 Day of treatment 0 10 20 30 Day of treatment 0 10 20 30 Day of treatment 0 10 20 30 Day of treatment Tumour volume (mm3) 0 50 100 Per cent survival Tumour volume (mm3) Per cent survival 0 500 1,000 1,500 2,000 0 50 100 P < 0.0001 P < 0.0001 h i j k P < 0.0001 P < 0.0001 Figure 5 | JQ1 promotes differentiation, tumour regression and prolonged survival in murine models of NMC. a, PET imaging of murine NMC 797 xenografts. FDG uptake in xenograft tumours is reduced by 50 mg kg21 JQ1 treatment compared to vehicle control. Arrows indicate the anatomical location of tumour xenograft. b, Tumour volume is reduced in mice with established disease (NMC 797 xenografts) treated daily with 50 mg kg21 JQ1 compared to vehicle control. A significant response to therapy is observed by a two-tailed t-test at 14 days (P 5 0.039). Data represent the mean 6 s.d. (n 5 7). c, Histopathological analysis of NMC 797 tumours excised from animals treated with JQ1 reveals induction of keratin expression (mouse anti￾cytokeratin clone AE1/AE3, 340) and impaired proliferation (Ki67, 340), as compared to vehicle-treated animals (scale bar is 20 mm). d, Viability of patient￾derived NMC 11060 xenografts was confirmed by PET imaging. Arrow indicates the anatomical location of tumour xenograft. e, Therapeutic response of primary 11060 NMC xenografts to (1)-JQ1 (50 mg kg21 daily for 4 days) was demonstrated by PET imaging. Integrated signal encompassed within the tumour volume is presented as the per cent injected dose per gram (% ID per g). f, Histopathological analysis of primary NMC 11060 tumours excised from animals treated with (1)-JQ1 reveals induction of keratin expression (mouse anti-cytokeratin clone AE1/AE3,320; scale bar is 20 mm), compared to vehicle￾treated animals. Quantitative analysis of keratin induction was performed using image masking (f, right panel) and pixel positivity analysis (g). A significant response to therapy is observed by a two-tailed t-test (P 5 0.0001). Data represent the mean 6 s.d. of three independent wide microscopic fields. Comparative images of stained excised tumours and quantitative masks are provided in Supplementary Fig. 14. h–k, (1)-JQ1 (red circles and lines; 50 mg kg21 daily for 18 days) produces a decrease in tumour volume (h, j) and promotes prolonged survival (i, k) in patient-derived 11060 (h, i) and Per403 (j, k) NMC xenograft models (n 5 10 in all groups). A significant response to therapy is observed for tumour volume by a two-tailed t-test (P , 0.0001) and for overall survival by a log-rank test (P , 0.0001). Black circles and lines, vehicle. RESEARCH ARTICLE 1072 | NATURE | VOL 468 | 23/30 DECEMBER 2010 ©2010 Macmillan Publishers Limited. All rights reserved