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Delineating the Role of Cooperativity in the Design of Potent Protacs for BTK

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Delineating the Role of Cooperativity in the Design of Potent Protacs for BTK Delineating the role of cooperativity in the design of potent PROTACs for BTK Adelajda Zorbaa,b, Chuong Nguyenb, Yingrong Xub, Jeremy Starrb, Kris Borzillerib, James Smithb, Hongyao Zhuc, Kathleen A. Farleyb, WeiDong Dingb, James Schiemerb, Xidong Fengb, Jeanne S. Changb, Daniel P. Uccellob, Jennifer A. Youngb, Carmen N. Garcia-Irrizaryb, Lara Czabaniukb, Brandon Schuffb, Robert Oliverb, Justin Montgomeryb, Matthew M. Haywardb, Jotham Coeb, Jinshan Chenb, Mark Niosid, Suman Luthrae, Jaymin C. Shahe, Ayman El-Kattand, Xiayang Qiub, Graham M. Westb, Mark C. Noeb, Veerabahu Shanmugasundaramb, Adam M. Gilbertb, Matthew F. Brownb, and Matthew F. Calabreseb,1 aInternal Medicine Research Unit, Pfizer Worldwide Research and Development, Cambridge, MA 02139; bDiscovery Sciences, Pfizer Worldwide Research and Development, Groton, CT 06340; cComputational Sciences, Medicinal Sciences, Pfizer Worldwide Research and Development, Groton, CT 06340; dMedicine Design, Pfizer Worldwide Research and Development, Groton, CT 06340; and ePharmaceutical Sciences Small Molecule, Pfizer Worldwide Research and Development, Cambridge, MA 02139 Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved June 20, 2018 (received for review March 1, 2018) Proteolysis targeting chimeras (PROTACs) are heterobifunctional cellular compartments and for targets that have Lys residues small molecules that simultaneously bind to a target protein and accessible for ubiquitination. A second selection criterion en route an E3 ligase, thereby leading to ubiquitination and subsequent to efficacious and selective target degradation could be de novo degradation of the target. They present an exciting opportunity to rational design of PROTACs. In a recent report, Gadd et al. (18) modulate proteins in a manner independent of enzymatic or describe an elegant model of PROTAC-facilitated target–ligase signaling activity. As such, they have recently emerged as an interactions as the foundation for target selectivity of BRD4 over “ ” attractive mechanism to explore previously undruggable targets. closely related family members BRD2 and BRD3, suggesting that Despite this interest, fundamental questions remain regarding the stabilizing target–ligase PPIs may be a powerful strategy when de- parameters most critical for achieving potency and selectivity. Here signing efficient PROTACs. we employ a series of biochemical and cellular techniques to in- ’ Here we assess the requirement of cooperativity in PROTAC- vestigate requirements for efficient knockdown of Bruton s tyro- mediated degradation of Bruton’s tyrosine kinase (BTK). BTK is sine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell a nonreceptor tyrosine kinase that belongs to the tyrosine kinase maturation. Members of an 11-compound PROTAC library were expressed in hepatocellular carcinoma (Tec) family of kinases investigated for their ability to form binary and ternary complexes and is crucial in normal B cell development (19). Mutations in with BTK and cereblon (CRBN, an E3 ligase component). Results Btk were extended to measure effects on BTK–CRBN cooperative interac- cause X-linked agammaglobulinemia (20), whereas aberrant tions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulat- Significance ing PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity. Proteolysis targeting chimera (PROTAC)-based protein degrada- tion is an emerging field that holds significant promise for tar- targeted protein degradation | PROTAC | BTK | catalytic | proteasome geting the “undruggable” proteome: the vast majority of the proteins that do not exhibit enzymatic activity and are thereby roteolysis targeting chimeras (PROTACs) (1) are bivalent not amenable to classical inhibition. Despite significant progress, Pligands that recruit an E3 ligase to a target protein thus fa- a thorough mechanistic characterization of biochemical deter- cilitating polyubiquitination and subsequent target degradation minants that underpin efficient PROTAC activity is lacking. Here (2–4). A powerful technology, first introduced in 2001 (1), we address one such question: Is positive cooperativity neces- PROTACs remained largely dormant for close to a decade due sary for potent protein degradation? Through a collection of ’ to physicochemical property limitations of chemical matter and independent techniques, we show that within a Bruton s tyrosine poor cellular penetration. Notable breakthroughs, particularly kinase/cereblon PROTAC system, potent knockdown correlates the departure from poorly cell-penetrant peptides to small- with alleviation of steric clashes in the absence of thermodynamic molecule–based ligands, have led to enhanced drug uptake and cooperativity. This result broadens the scope of PROTAC appli- efficacy both in cultured cells and, most recently, in vivo (5, 6). cations and affects fundamental design criteria across the field. BIOCHEMISTRY As such, numerous proteins ranging from gene regulatory pro- α α – Author contributions: A.Z., C.N., J.M., M.F.B., and M.F.C. designed research; A.Z., C.N., teins [AR (3, 7), ER (7, 8), ERR (6), and BRD2/3/4 (5, 9 11)], Y.X., K.B., J. Smith, H.Z., W.D., J. Schiemer, X.F., and M.N. performed research; J. Starr, retinoic acid-binding proteins [CRABPI/II (12, 13) and RAR K.B., J. Smith, H.Z., K.A.F., J.S.C., D.P.U., J.A.Y., C.N.G.-I., L.C., B.S., R.O., J. Coe, J. Chen, and (7)], to enzymes [prolyl isomerase FKBP12 (5), kinases BCR- V.S. contributed new reagents/analytic tools; A.Z., Y.X., H.Z., K.A.F., W.D., J.M., M.M.H., S.L., J.C.S., A.E.-K., X.Q., G.M.W., M.C.N., V.S., A.M.G., M.F.B., and M.F.C. analyzed data; ABL/c-ABL (14), and RIPK2 (6)] have successfully been de- and A.Z., M.F.B., and M.F.C. wrote the paper. graded by small-molecule PROTACs. This breadth has led to Conflict of interest statement: All authors are or were employees of Pfizer, Inc. tremendous excitement in the drug development field, particu- CHEMISTRY This article is a PNAS Direct Submission. larly with regard to targeting the “undruggable” proteome (15). There are thousands of predicted protein–protein interactions Published under the PNAS license. (PPIs) (16) where long, shallow interfaces are, in theory, suffi- Data deposition: Proteomic data have been deposited in the ProteomeXchange via the PRIDE Archive proteomics data repository, https://www.ebi.ac.uk/pride/archive/. Refer- cient to engage a moderate-affinity PROTAC and potentially ence to this database is included in the supplementary information for the mass spec mediate the target protein’s degradation. The task of choosing proteomics work. the optimal target–ligase pair that would lead to efficacious 1To whom correspondence should be addressed. Email: [email protected]. target degradation seems daunting given the sheer number of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. possible targets and ligases (600+) (17) available. Selection could 1073/pnas.1803662115/-/DCSupplemental. be funneled for target–ligase pairs that colocalize in similar Published online July 16, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1803662115 PNAS | vol. 115 | no. 31 | E7285–E7292 Downloaded by guest on September 28, 2021 regulation of BTK signaling has been linked to numerous B cell F Target (BTK) ligand A O O H malignancies including chronic lymphocytic leukemia (CLL), Linker N – N mantle cell lymphoma, and diffuse large B cell lymphoma (20 N F N O O 22). Ibrutinib is a selective and potent irreversible (covalent) N O inhibitor of BTK that has affected the treatment landscape for B (1) Ligase (CRBN) ligand O O NH2 O cell malignancies, particularly CLL (21, 23). What confers ibru- (2) NH2 NH (3) O tinib its selectivity is the scarcity in other kinases of a Cys residue O (4) O homologous to C481 in BTK, which forms the covalent adduct O O (5) O O O (24, 25). However, recent reports on patient relapses as a result (6) O O O O of acquired resistance mutations in BTK (C481S) following (7) O O O O Ibrutinib treatment have raised the need for new avenues for (8) O O O O O (9) O O O therapeutic intervention (26). O O (10) O O O Targeted degradation of BTK is explored as a modality and O O O (11) O O O serves as a model system to further understand the biochemical O O O underpinnings of efficient, targeted protein degradation. A li- time (hr)0 0.02 0.5 1 2 4 6 8 24 32 48 100 brary of 11 PROTACs of varying linker lengths that engage BTK B BTK 75 on one end and cereblon [CRBN, a substrate-binding member of 50 the Cul4 E3 ligase family (27, 28)] on the other are initially used % control 100 68 80 65 71 56 51 46 19 18 11 %BTK 25 0 to assess BTK degradation efficacy. Through in vitro, cellular, Vinc 0816 24 32 40 48 and in vivo experiments, rapid, efficient, prolonged, and selective time (hr) BTK degradation is demonstrated. Degradation of BTK is de- [(9)], μM 0 0.001 0.01 0.03 0.1 0.3 1 3 10 30 100 pendent on ternary complex formation but does not rely on 75 BTK K C T – B 50 energetically cooperative BTK CRBN interactions. On the % control 100 60 46 30 23 16 15 23 48 76 – % 25 contrary, negative BTK CRBN cooperativity is observed with actin 0 PROTACs of shorter linkers, and relief of steric clashes is de- 0 0.001 0.1 10 [(9)], μM termined to describe the transition between ineffective and potent 1μM (9) - + - + + BTK degradation within this system. We thus describe a case where D 1μM BTK ligand - - + - + + BTK ligand robust thermodynamic cooperativity appears unnecessary for 10μM CRBN ligand - - + + - +CRBN ligand + (9) - + BTK ligand efficient degradation. 100 + (9) BTK 75 + CRBN ligand % control 100 15 113 73 92 50 Results %BTK actin 25 + (9) PROTACs Facilitate BTK Degradation in Cultured Cells.
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