#Bill chase transcriptions full#
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( A) Scheme for in-well GFP/TdTomato competition assay. ( H and I) Z scores of Fluc/Rluc ratio of 293FT IKZF1-Fluc-IRES-Rluc cells after screening with the Selleck BioActive Library for 8 hours (H) or 4 days (I).

( G) Scheme for negative selection screening using the dual-luciferase reporter assay. ( F) Corrected z scores obtained by subtracting z scores in (D) from z scores in (E). LEN and POM are indicted by the blue circle and red triangle, respectively.

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( D and E) Z-distribution of GFP fluorescence of DCK* cells (D) and DCK*-IKZF1 cells (E) screened with the full Selleck BioActive Library. Library wells containing POM and DiP are indicated by the red and white arrows, respectively. Columns 23 and 24 contained 10 μM POM and 12.5 μM dipyridamole (DiP), respectively. ( B and C) Representative fluorescence data of 384-well plates containing 293FT cells expressing DCK* (B) or DCK*-IKZF1 (C) treated with compounds in the Selleck BioActive Library (one compound per well), followed 24 hours later by the addition of BVdU at the EC 85 (10 and 100 μM, respectively) for 4 days. ( A) Scheme for positive selection protein degradation assay. ( H) Fluorescence data of 384-well plate containing 293FT cells expressing DCK*-IKZF1 treated with DMSO (columns 1 to 11 and 24) or 1 μM POM (columns 12 to 23), followed 24 hours later by the addition of 100 μM BVdU for 4 days (columns 1 to 24). ( G) Immunoblot analyses of cells treated as in (E) and (F). POM was added 24 hours before treatment with BVdU for 4 days. ( E and F) Number of GFP-positive 293FT cells infected to produce DCK* (E) or DCK*-IKZF1 (F) using the vectors in depicted in (A) and then treated with indicated concentrations of POM and BVdU in 384-well plate format. In (D), cells were also treated with 1 μM (POM) starting 24 hours before BVdU was added. ( C and D) Relative survival of 293FT cells infected with the lentiviral vectors depicted in (A) and then treated with the indicated concentrations of BVdU for 4 days. ( B) Immunoblot analysis of 293FT cells infected with the lentiviral vectors depicted in (A) and then treated with 1 or 10 μM POM, as indicated by the triangles, for 24 hours. DCK*, variant deoxycytidine kinase with Ser74Glu, Arg104Met, and Asp133Ala substitutions V5, V5 epitope tag GGS, Gly-Gly-Ser spacer IRES, internal ribosomal entry site.

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Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.Ĭopyright © 2021 The Authors, some rights reserved exclusive licensee American Association for the Advancement of Science. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. Here, we describe a gain of signal ('up') assay for degraders. Current loss of signal ('down') assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules.

13 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

12 Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA.

11 Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

10 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

9 Departments of Pediatrics and Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

8 Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

7 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

6 Linde Program in Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

5 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

4 Ludwig Cancer Center, Boston, MA 02115, USA.

3 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

2 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.1 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.