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[Preprint]. 2024 Sep 27:2024.09.25.615094.

doi: 10.1101/2024.09.25.615094.

Discovery of electrophilic degraders that exploit S_NAr chemistry

Zhe Zhuang^{1

2}, Woong Sub Byun^{1

2}, Zuzanna Kozicka^{3

4}, Brendan G Dwyer¹, Katherine A Donovan^{5

6}, Zixuan Jiang⁷, Hannah M Jones¹, Dinah M Abeja^{5

6}, Meredith N Nix⁷, Jianing Zhong⁷, Mikołaj Słabicki^{3

4}, Eric S Fischer^{5

6}, Benjamin L Ebert^{3

4

8}, Nathanael S Gray¹

Affiliations

¹ Department of Chemical and Systems Biology, ChEM-H, and Stanford Cancer Institute, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
² These authors contributed equally: Zhe Zhuang, Woong Sub Byun.
³ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁶ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
⁷ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁸ Howard Hughes Medical Institute, Boston, MA, USA.

PMID: 39386645
PMCID: PMC11463635
DOI: 10.1101/2024.09.25.615094

Discovery of electrophilic degraders that exploit S_NAr chemistry

Zhe Zhuang et al. bioRxiv. 2024.

[Preprint]. 2024 Sep 27:2024.09.25.615094.

doi: 10.1101/2024.09.25.615094.

Authors

Affiliations

¹ Department of Chemical and Systems Biology, ChEM-H, and Stanford Cancer Institute, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
² These authors contributed equally: Zhe Zhuang, Woong Sub Byun.
³ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁶ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
⁷ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁸ Howard Hughes Medical Institute, Boston, MA, USA.

PMID: 39386645
PMCID: PMC11463635
DOI: 10.1101/2024.09.25.615094

Abstract

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly 'undruggable' targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (S_NAr) reactions in the rational design of electrophilic protein degraders. Appending an S_NAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The S_NAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an S_NAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

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Conflict of interest statement

N.S.G. is a founder, science advisory board member (SAB) and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK, Shenandoah (board member), Larkspur (board member) and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi. B.L.E. has received research funding from Novartis and Calico. He has received consulting fees from Abbvie. He is a member of the scientific advisory board and shareholder for Neomorph Inc., TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. E.S.F. is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (also board of directors). He is an equity holder and SAB member for Avilar Therapeutics, Photys Therapeutics, and Ajax Therapeutics and an equity holder in Lighthorse Therapeutics. E.S.F. is a consultant to Novartis, EcoR1 capital, Odyssey and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline, Bayer and Astellas. K.A.D. receives or has received consulting fees from Kronos Bio and Neomorph Inc. M.S. has received research funding from Calico Life Sciences LLC. All other authors declare no competing interests.

Figures

**Figure 1.. ZZ7-16-073 is a potent electrophilic BRD4 degrader dependent on the S_NAr covalent warhead.**
A. Structures of electrophilic BRD4 degrader ZZ7-16-073 and negative controls with slight chemical modifications that negate S_NAr reactivity. B. HiBiT-BRD4 results for Jurkat cells treated with the indicated compounds for 6 hours. C. Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 hours treatment with 10 pM ZZ7-16-073. D. Structure-activity relationships (SAR) of electrophilic BRD4 degraders with an S_NAr covalent warhead.

**Figure 2.. ZZ7-16-073-induced BRD4 degradation is dependent on DCAF16.**
A. HiBiT-BRD4 results for Jurkat cells treated with the indicated inhibitors for 1 hour and then ZZ7-16-073 for 6 hours. B. Ubiquitin-proteasome system (UPS)-focused CRISPR degradation screen for BRD4_BD1-eGFP and BRD4_BD2-eGFP stability in K562-Cas9 cells treated with 500 nM ZZ7-16-073 for 16 hours. C. HiBiT-BRD4 results for DCAF16-KO Jurkat cells treated with the indicated compounds for 6 hours. D. Co-immunoprecipitation of FLAG-tagged BRD4 and HA-tagged DCAF16 in the presence of the indicated compounds. E. NanoBRET assay suggesting the induced interaction between HaloTag-DCAF16 and Nanoluciferase-BRD4 fusion proteins with the indicated compounds. F. Western blots of BRD4 degradation in WT or DCAF16-KO and mutants K562 cells treated with 100 pM ZZ7-16-073 for 6 hours.

**Figure 3.. ZZ7-17-033-induced BRD4 degradation is primarily dependent on DCAF11.**
A. Structures of electrophilic BRD4 degrader ZZ7-16-078, ZZ7-17-033, and negative controls of ZZ7-17-033 with slight chemical modifications that negate S_NAr reactivity. B. HiBiT results for Jurkat cells treated with the indicated compounds for 6 hours. C. HiBiT-BRD4 results for Jurkat cells treated with the indicated inhibitors for 1 hours and then ZZ7-17-033 for 6 hours. D. Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 hours treatment with 100 nM ZZ7-17-033. E. Ubiquitin-proteasome system (UPS)-focused CRISPR degradation screen for BRD4_BD1-eGFP or BRD4_BD2-eGFP stability in K562-Cas9 cells treated with 1 μM ZZ7-17-033 for 16 hours. F. HiBiT-BRD4 results for WT, DCAF11-KO, or DCAF1-KO Jurkat cells treated with ZZ7-17-033 for 6 hours.

**Figure 4.. Development of electrophilic kinase degraders dependent on the S_NAr covalent warhead.**
A. Structures of electrophilic promiscuous kinase degraders ZZ7-18-043, ZZ7-18-045, and a negative control of ZZ7-18-045 with slight chemical modification that negate S_NAr reactivity. B. Western blots of kinases degradations in MOLT-4 cells treated for 3 hours with 1 μM indicated compounds. C. Quantitative proteome-wide mass spectrometry in MOLT-4 cells after 3 hours treatment with 1 μM ZZ7-18-045. D. Western blots of LIMK2, Aurora A, and Aurora B degradations in Jurkat cells treated with the indicated inhibitors for 1 hour and then ZZ7-18-045 for 3 hours. E. Structures of electrophilic CDK4/6 degrader ZZ7-18-084 and negative controls with slight chemical modifications that negate S_NAr reactivity. F. Western blots of CDK4 and CDK6 degradations in Jurkat cells treated with 1 μM indicated compounds for 6 hours. G. Quantitative proteome-wide mass spectrometry in MOLT-4 after 3 hours treatment with 1 μM ZZ7-18-084. H. Western blots of CDK4 and CDK6 degradations in Jurkat cells treated with the indicated inhibitors for 1 hour and then ZZ7-18-084 for 6 hours.

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References

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This is a preprint.

Discovery of electrophilic degraders that exploit S_NAr chemistry

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Discovery of electrophilic degraders that exploit S_NAr chemistry

Authors

Affiliations

Abstract

Conflict of interest statement

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