Journal: Journal of medicinal chemistry

Article Title: Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression

doi: 10.1021/acs.jmedchem.8b00506

Figure Lengend Snippet: Scaffold hopping and structure-based design of 1,4-oxazepines as a new class of BET inhibitors.

Article Snippet: The University of Michigan has filed a number of patent applications on these BET inhibitors and degraders reported in this study, which have been licensed to Oncopia Therapeutics, LLC.

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression

doi: 10.1021/acs.jmedchem.8b00506

Figure Lengend Snippet: Binding affinities of our newly designed BET inhibitors and reference compounds (+)-JQ-1 and OTX-015 to BRD4 BD1 protein, as determined in our FP-based binding assay.

Article Snippet: The University of Michigan has filed a number of patent applications on these BET inhibitors and degraders reported in this study, which have been licensed to Oncopia Therapeutics, LLC.

Techniques: Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression

doi: 10.1021/acs.jmedchem.8b00506

Figure Lengend Snippet: Evaluation of the mechanism of action of BET degradation induced by 35 (QCA570). RS4;11 cells were pre-treated for 2 h with DMSO, BET inhibitor 22 (10 μM), cereblon ligand Lenalidomide (10 μM), E1 neddylation inhibitor MLN4924 (1 μM), or proteasome inhibitors MG-132 (20 μM) and Carfilzomib (0.1 μM). Cells were then treated for 3 h with BET degrader 35 (QCA570) at 0.5 nM, a concentration that induces complete degradation of all BET-BRD proteins and downregulation of c-Myc.

Article Snippet: The University of Michigan has filed a number of patent applications on these BET inhibitors and degraders reported in this study, which have been licensed to Oncopia Therapeutics, LLC.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A ) Effects of BET and CXCR2 antagonists on cell viability. The indicated BL1/2 (blue) and M/MSL (green) TNBC cell lines and MCF7 (luminal) and MCF10A (normal-like) lines were treated with the indicated doses of the drugs for 72 hours and stained with crystal violet. Representative pictures of reproducible effects from two to three independent experiments are shown. ( B , E , and F ) Dose-response curves of single agents and drug combinations in the indicated cell lines treated with varying concentrations of JQ1 and SB225022 (B) or JQ1 and BTZ (E and F) for 72 hours. Dose-response curves are presented as means of four (B) or three (E and F) repeats. ( C and G ) CI was calculated by the CompuSyn software with the Chou-Talalay equation using multiple doses and response points. CI values for three different indicated FA are shown. ( D ) Effects of BET and proteasome inhibitors on cell viability were assessed by crystal violet staining as described in (A). ( H ) Effects of drug combinations on spheroid growth. Representative images and viability assay (CellTiter-Blue, bar graph) of day 15 4T1 spheroids ( n = 6 spheroids). Control untreated and treated spheroids with single agents at the indicated doses and with the drug combinations are shown. Scale bar, 100 μm.

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Staining, Software, Viability Assay, Control

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A and B ) Effects of BET and CXCR2 inhibition (A) or BET and proteasome inhibition (B) on PARP (A and B) and caspase-3 cleavage (A) in the indicated cell lines (blue, BL1; black, M/MSL). Staurosporine (150 nM, 16 hours) was used as a positive control. The cells were treated with low doses of JQ1 and either SB225022 (A) or BTZ (B) for 24 hours as described in Materials and Methods, lysed, and assessed by Western blot (WB) for the indicated proteins. ( C ) Ferroptosis inhibitors rescue cell death induced by JQ1 and BTZ combination. The indicated cell lines were pretreated with the indicated cell death inhibitors for 1 hour and then for an additional 72 hours in the absence or presence of JQ1 and BTZ (see Materials and Methods). Cell viability (CellTiter-Blue) is presented as percentage of untreated cells. The effects of the inhibitors on their cognate death pathways are shown in fig. S6C. Mean values of three experiments are shown (means ± SD; table S6). ( D ) Iron chelators rescued cell death induced by JQ1 and BTZ treatment. The indicated TNBC cell lines were treated with JQ1 and BTZ (see Materials and Methods) for 72 hours in the absence or presence of deferoxamine (DFO) (100 μM) or 2,2′-dipyridyl (10 μM), and cell viability was assessed by crystal violet staining. ( E ) Representative confocal images of the indicated TNBC cell lines and T47D cells stained with C11-BODIPY (10 μM) as described in Materials and Methods. The cells were treated with cumene hydroperoxide (CH) (100 μM, 3 hours) as a positive control, and either with JQ1 + SB225022 or with JQ1 and BTZ (see Materials and Methods) for 16 hours. Scale bar, 10 μm. ( F ) Assessment of lipid peroxidation in breast cancer cells in response to BET and proteasome inhibition. TNBC (gray) and non-TNBC (green) cell lines were incubated with JQ1 and BTZ (see Materials and Methods) for 16 hours or with CH (100 μM, 3 hours). Where indicated, glutathione (1 mM; see Materials and Methods) was applied. Lipid peroxidation is shown as the ratio between fluorescence emission at 510 nm (green) and 590 nm (red) (see Materials and Methods). Mean values of three experiments are shown (mean values ± SD; table S7).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Inhibition, Positive Control, Western Blot, Staining, Incubation, Fluorescence

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A and B ) Inhibition of BET and the proteasome increased iron levels (A) and decreased GSH levels (B) in TNBC cell lines. TNBC and non-TNBC (MCF7, SKBR3, T47D, and BT474) cells were incubated with JQ1 and BTZ (see Materials and Methods) for 16 hours, and total iron (A) and reduced GSH (B) levels were measured as described in Materials and Methods. Mean values of three experiments are shown (mean values ± SD; table S8). ( C ) Inhibition of BET and the proteasome increased ROS in TNBC cells. The indicated breast cancer cells were treated with JQ1 and BTZ (see Materials and Methods) for 12 hours or with tert -butyl hydrogen peroxide (TBHP; 100 μM) for 2 hours. Where indicated, GSH (1 mM) was applied 1 hour before drug treatment. Cells were then incubated with CM-H 2 DCFDA to measure ROS as described in Materials and Methods. Results are expressed as folds of control in at least three experiments (mean values ± SD; table S9).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Inhibition, Incubation, Control

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A ) Box plot showing the expression of GPX4 in patients with breast cancer grouped by PAM50. The differences between the BL patients and any other PAM50 groups are significant ( t test, P < 0.001). ( B and C ) Effects of BET and proteasome inhibition on the levels of GPX4 protein (B) and transcript (C). The indicated TNBC cell lines were treated with JQ1, BTZ, or both (see Materials and Methods) for 24 hours. Levels of GPX4 protein were assessed by WB. Intensities of GPX4 bands were quantified, normalized, and presented as fold of control in the bar graph (B). GPX4 mRNA levels were assessed by qPCR. Mean values ± SD of at least two repeats are shown (C). ( D ) Effects of BET and proteasome inhibition on level of GPX4 protein and transcript in 4T1 tumors. Representative IHC staining of 4T1 tumors treated with OTX015 (25 mg/kg per day, orally), BTZ (0.25 mg/kg, IP, every fourth day), or both to detect protein expression is shown. GPX4 mRNA levels were evaluated by qPCR. Results are mean values ± SD of at least six mice per group. ( E ) Effects of BET and proteasome inhibition on level of GPX4 transcript and additional key ferroptosis genes. The indicated TNBC cell lines were treated with JQ1 and BTZ (see Materials and Methods) for 24 hours, and mRNA levels of the indicated genes were assessed by qPCR. The results are reported as fold of control. Mean values of at least three independent experiments are shown (mean values ± SD; table S11).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Expressing, Inhibition, Control, Immunohistochemistry