bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA) Shuhui Lima, Regina Khooa, Khong Ming Peha, Jinkai Teob, Shih Chieh Changa, Simon Nga, Greg L. Beilhartzc, Roman A. Melnykc,d, Charles W. Johannese, Christopher J. Browne, David P. Lanee, Brian Henrya, and Anthony W. Partridgea,1 aQuantitative Biosciences, MSD International, Singapore 138665; bPacific Translational Biomarkers, MSD International, Singapore 138665; cMolecular Medicine, Hospital for Sick Children, Toronto, ON, Canada M5G 0A4; dDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and ep53Lab, Agency for Science, Technology and Research (A*STAR), Singapore 138648 Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved February 7, 2020 (received for review November 22, 2019) Targeted degradation approaches such as proteolysis targeting localization, cell-cycle dependent regulation or tissue- or disease- chimeras (PROTACs) offer new ways to address disease through specific expression adds an additional layer of selectivity that can tackling challenging targets and with greater potency, efficacy, be leveraged with targeted degradation strategies. and specificity over traditional approaches. However, identifica- While small molecule targeted degradation approaches offer tion of high-affinity ligands to serve as PROTAC starting points compelling advantages, the discovery of corresponding clinical remains challenging. As a complementary approach, we describe a candidates is not without its challenges. For molecular glues, class of molecules termed biological PROTACs (bioPROTACs)— limited examples exist and PROTACs have thus far been only engineered intracellular proteins consisting of a target-binding applied to targets with available small molecules inhibitors (10). domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged Classically “undruggable” proteins remain challenging although proteins as model substrates, we show that there is considerable opportunities exist to repurpose previously identified small flexibility in both the choice of substrate binders (binding positions, molecule ligands that did not block protein function for a deg- scaffold-class) and the E3 ligases. We then identified a highly ef- radation strategy. Also, of more than 600 E3 ligases encoded BIOCHEMISTRY fective bioPROTAC against an oncology target, proliferating cell by the human genome, only 4 are routinely used in PROTAC nuclear antigen (PCNA) to elicit rapid and robust PCNA degrada- design—CRBN, VHL, MDM2, and cIAP (11). The choice of the tion and associated effects on DNA synthesis and cell cycle pro- E3 determines degradation efficiencies and the current selection gression. Overall, bioPROTACs are powerful tools for interrogating lacks the diversity needed to harness the full potential of the degradation approaches, target biology, and potentially for mak- ubiquitin-proteasome system (UPS). Even if small molecule li- ing therapeutic impacts. gands to the POI were available, considerable time and effort (without assurance of success) have to be invested for testing bioPROTAC | targeted degradation | PCNA various combinations of linker lengths and E3s recruited. There is currently a poor understanding of the rules that govern stable argeted degradation approaches function by inducing the Tassembly of the ubiquitination complex in close proximity to Significance a protein of interest (POI) to catalyze its selective ubiquitin- tagging and subsequent proteasome-mediated degradation (1). Several such approaches exist including molecular glues, which Intracellular proteins interact with each other to perform — functions that are critical to normal and disease states. At- remodel the surface of an E3 ligase to induce binding to and – degradation of—neo-substrates (e.g., lenolidimide, an approved tempts at altering pathological protein protein interactions with traditional approaches have largely failed. Here, we ex- therapeutic). PROTACs (proteolysis targeting chimeras), the “ ”— other major targeted degradation class, are bispecific molecules plore an emerging approach we call bioPROTACs engi- that induce substrate degradation by simultaneously binding a neered fusion proteins that consist of a target binding domain POI and an E3 ligase (e.g., ARV-110, a degrader of the andro- and an E3 ligase, an arrangement that results in the specific gen receptor and the first-in-class PROTAC to enter clinical degradation of the therapeutic target. Our systematic study trials). Pharmacologically, small molecule degraders offer several shows bioPROTAC design requirements are highly flexible in advantages over traditional inhibitor-based therapeutics. First, terms of both the binding domain and E3 ligase components. degradation can be induced via interaction sites across the POI Resulting molecules can be used as powerful tools for uncov- surface, regardless of whether the binding site is of functional ering biology, informing on the design of small molecule target consequence (2), thus expanding the chemical space for tackling degraders (e.g. PROTACs), and, if delivery issues can be otherwise intractable targets (3). Second, molecules can be addressed, potential therapeutics. recycled for multiple rounds of degradation, a substoichiometric Author contributions: S.L., S.C.C., S.N., G.L.B., R.A.M., C.W.J., C.J.B., D.P.L., B.H., and A.W.P. property which is especially useful for high-abundance targets designed research; S.L., R.K., K.M.P., and J.T. performed research; S.L. contributed new compared to stoichiometric inhibitors may become limited by the reagents/analytic tools; S.L. and A.W.P. analyzed data; S.L. and A.W.P. wrote the paper; high systemic doses required and corresponding polypharmacology- and S.L., R.K., K.M.P., J.T., S.C.C., S.N., G.L.B., R.A.M., C.W.J., C.J.B., D.P.L., B.H., and A.W.P. based toxicities (4). Third, superior pharmacological inhibition contributed to discussion of the results and next steps. can be achieved as degradation attenuates all biological activities The authors declare no competing interest. (enzymatic, transactivation, scaffolding) and inhibition is sus- This article is a PNAS Direct Submission. tained pending protein resynthesis (5). Hence, reduced drug Published under the PNAS license. dosing frequencies can potentially be realized. Finally, enhanced 1To whom correspondence may be addressed. Email: [email protected]. specificity can be attained through differences in substrate This article contains supporting information online at https://www.pnas.org/lookup/suppl/ degradability, E3 suitability. and ternary complex stability (6–9). doi:10.1073/pnas.1920251117/-/DCSupplemental. Also, the ability to engage E3s with differences in subcellular First published March 2, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1920251117 PNAS | March 17, 2020 | vol. 117 | no. 11 | 5791–5800 Downloaded by guest on September 28, 2021 ternary complex formation between substrate, PROTAC and E3, was reduced in cells expressing vhhGFP4-SPOP, as marked by making informed decisions on PROTAC design difficult. Lastly, the mCherry-positive signal. Interestingly, as vhhGFP4-SPOP ex- as PROTACs are typically composed of two ligands connected by pression increased, the degradation of H2B-GFP was attenuated. a linker, these molecules usually violate Lipinski’s rule of five and, This is consistent with the well documented “hook effect” seen thus, often suffer from permeability and metabolic liabilities (12). with small molecule-based PROTACS. Specifically, beyond a As a complementary approach to small molecule-based de- threshold concentration of a PROTAC molecule, reduced deg- graders, we sought to develop a biologic equivalent to serve both radation occurs, due to the decreased likelihood of forming the as a biological tool and as a potential therapeutic approach. prerequisite substrate:PROTAC:E3 ternary complexes in favor of Specifically, instead of using small molecules to bridge the sub- substrate:PROTAC and PROTAC:E3 binary complexes (28). strate and the E3 ligase, we have reengineered the E3 ligase by Control constructs were engineered where either one or both directly replacing its natural substrate recognition domain with a modular components were mutated. Specifically, vhhGFP4mut peptide or a miniprotein that binds a POI. These fusion proteins, lacks the complementarity determining region 3 (CDR3) and no which we term bioPROTACs (biological PROTACs), were longer recognizes GFP, whereas SPOPmut lacks the three-box expressed in cells to drive targeted degradation of POIs. Al- motif responsible for recruiting CUL3 and, thus, cannot assem- though bioPROTACs are not novel entities, the work described ble the ubiquitination machinery. In all controls, H2B-GFP levels herein represent a systematic exploration of this approach. were maintained (Fig. 1B). The selective depletion of H2B-GFP Historically, effective degraders have been described for the in mCherry-positive cells expressing vhhGFP4-SPOP but not its classically “undruggable” proteins such as β-catenin (13–15), controls was also recapitulated with confocal imaging (Fig. 1C) KRAS (16), and c-Myc (17) using domains engrafted from their and Western blot analysis of cells sorted into mCherry-positive endogenous interacting partners on to E3 ligases beyond those and mCherry-negative populations (Fig. 1D). Finally, to dem- that can be recruited by small-molecule PROTAC (e.g., βTrCP onstrate that H2B-GFP down-regulation