Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. Schematic of the traditional occupancy-based approach to develop MDM2 inhibitors that interrupt the MDM2-p53 WT interaction (left) and p53 Y220C correctors that bind to p53 Y220C and increase its thermal stability (right). B. Schematic of the event-driven transcriptional activator of p53 (TRAP) targeting p53 Y220C mutant through the recruitment of BRD4.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. Activation of p53-regulated transcription in BxPC-3 cells with published p53 Y220C “corrector” (B-1, PK9328 free amine, KG13), p300/CBP-p53 Y220C acetylation-inducing chimera (MS78), MDM2 inhibitor (nutlin-3a), and BRD4 binder (JQ1) at 24 h. The data is shown as means ± s.d. of n=2 independent experiments. B. The structure of B-1 linker to validate the exit vector strategy. C. The ternary complex formation between p53 Y220C and BRD4 BD1 mediated by weakly active or not active B-1-based compounds. The data is shown as means ± s.d. of n=2 independent experiments. D. Activation of p53-regulated transcription in BxPC-3 cells with weakly active or no active B-1-based compounds at 24 h. The data is shown as means ± s.d. of n=2 independent experiments. E. Scatter plot showing the relationship between the maximum luciferase signal and the TR-FRET area under the curve (AUC) of the B-1-based TRAPs.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Activation Assay, Plasmid Preparation, Luciferase

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. The structures of the B-1-based bivalent TRAP library. B. TR-FRET. The ternary complex formation measured by TR-FRET between purified p53 Y220C and BRD4 BD1 . The data is shown as means ± s.d. of n=2 independent experiments. C. Luciferase reporter. Activation of p53-regulated transcription in BxPC-3 (p53 Y220C ) cells after compound treatment for 24 h. The data is shown as means ± s.d. of n=2 independent experiments.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Purification, Luciferase, Activation Assay

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. The structure of the PK9328-based molecule library. B. The ternary complex formation measured by TR-FRET between purified p53 Y220C and BRD4 BD1 . The data is shown as means ± s.d. of n=2 independent experiments. C. Activation of p53-regulated transcription in BxPC-3 cells after compound treatment for 24 h. The data is shown as means ± s.d. of n=2 independent experiments.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Purification, Activation Assay

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. Structures of negative controls containing minor chemical modifications to remove BRD4 binding ( TRAP-1-Neg1 ) or p53 Y220C binding ( TRAP-1-Neg2 ). B. TR-FRET. The ternary complex formation of TRAPs and their negative controls measured by TR-FRET between p53 Y220C and BRD4 BD1 . The data is shown as means ± s.d. of n=2 independent experiments. C. Luciferase reporter. The p53-regulated transcriptional activation of TRAPs and their negative controls at 24 h in BxPC-3 cells. The data is shown as means ± s.d. of n=2 independent experiments. D. The co-immunoprecipitation of TRAPs compared with the cotreatment of p53 Y220C binder (B-1 linker) and BRD4 binder (JQ1) in HEK293T cells after 4 h of compound treatment.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Binding Assay, Luciferase, Activation Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: The ternary complex formation of TRAP-1 treatment measured by TR-FRET between purified p53 Y220C or p53 WT DNA binding domain and BRD4 BD1 . The data is shown as means ± s.d. of n=2 independent experiments.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Purification, Binding Assay

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: A. The antiproliferative activity of TRAPs compared with weaker or inactive compounds at 72 h in BxPC-3 cells. The data is shown as means ± s.d. of n=2 independent experiments. B. Heat map showing the antiproliferative potency of TRAP-1 compared with controls in BxPC-3 (p53 Y220C ), A549 (p53 WT ), and CCD 841 CoN (p53 WT ). The data is shown as means of n=2 independent experiments.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Activating p53 Y220C with a Mutant-Specific Small Molecule

doi: 10.1101/2024.10.23.619961

Figure Lengend Snippet: Heat map showing the antiproliferative activity of TRAP-1 compared with controls in A549-p53 -/- , A549-p53 WT , and A549-p53 Y220C . The data is shown as means of n=2 independent experiments.

Article Snippet: p53 luciferase reporter lentivirus (BPS Bioscience, San Diego, CA, USA) was used to transduce BxPC-3 cells cultured as described above, and tranductants were selected by growth in 1 µg/mL puromycin (Gibco Invitrogen Corp., Grand Island, NY, USA) added directly to the culture medium.

Techniques: Activity Assay