Nature uses a variety of tools to mediate the flow of information in cells, many of which control distances between key biomacromolecules. Researchers have thus generated compounds whose activities stem from interactions with two (or more) proteins simultaneously. In this Perspective, we describe how these ‘bifunctional’ small molecules facilitate the study of an increasingly wide range of complex biological phenomena and enable the drugging of otherwise challenging therapeutic targets and processes. Despite their structural and functional differences, all bifunctional molecules employ Nature’s strategy of altering interactomes and inducing proximity to modulate biology. They therefore exhibit a shared set of chemical and biophysical principles that have not yet been appreciated fully. By highlighting these commonalities—and their wide-ranging consequences—we hope to chip away at the artificial barriers that threaten to constrain this interdisciplinary field. Doing so promises to yield remarkable benefits for biological research and therapeutics discovery.
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Rakhit, R., Navarro, R. & Wandless, T. J. Chemical biology strategies for posttranslational control of protein function. Chem. Biol. 21, 1238–1252 (2014).
Kraft, A. S. & Anderson, W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301, 621–623 (1983).
Rando, R. R. & Kishi, Y. Structural basis of protein kinase C activation by diacylglycerols and tumor promoters. Biochemistry 31, 2211–2218 (1992).
Wender, P. A. et al. Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C. Proc. Natl. Acad. Sci. USA 85, 7197–7201 (1988).
Che, Y., Gilbert, A. M., Shanmugasundaram, V. & Noe, M. C. Inducing protein-protein interactions with molecular glues. Bioorg. Med. Chem. Lett. 28, 2585–2592 (2018).
Winter, G. E. et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).
Bondeson, D. P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015).
Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43 (1994).
Schreiber, S. L. & Crabtree, G. R. The mechanism of action of cyclosporin A and FK506. Immunol. Today 13, 136–142 (1992).
Sabers, C. J. et al. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem. 270, 815–822 (1995).
Austin, D. J., Crabtree, G. R. & Schreiber, S. L. Proximity versus allostery: the role of regulated protein dimerization in biology. Chem. Biol. 1, 131–136 (1994). This paper contrasts the concept of induced proximity to that of the general phenomenon of allosteric regulation. The authors propose that induced co-localization of proteins via synthetic ‘dimerizers’ may be sufficient to study a variety of biological systems.
Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018). This Review discusses the breadth of biological systems that have been studied with molecular glues known as chemical inducers of proximity (CIPs), primarily focusing on cases involving protein engineering.
Ellis, R. J. & Minton, A. P. Cell biology: join the crowd. Nature 425, 27–28 (2003).
Page, M. I. & Jencks, W. P. Entropic contributions to rate accelerations in enzymic and intramolecular reactions and the chelate effect. Proc. Natl. Acad. Sci. USA 68, 1678–1683 (1971).
Kirby, A.J. in Advances in Physical Organic Chemistry (eds. Gold, V. & Bethell, D.) 17, 183–278 (Academic Press, 1980).
Tenud, L., Farooq, S., Seibl, J., Eschenmoser, A. & Endocyclische, S. N. Reaktionen am gesättigten Kohlenstoff? Vorläufige Mitteilung. Helv. Chim. Acta 53, 2059–2069 (1970). This paper describes a nucleophilic substitution reaction in which the intramolecular pathway that proceeds via an endocyclic transition state is disfavored. In doing so, it reveals that increasing the proximity of reactive species is not always sufficient to achieve rate acceleration.
Baldwin, J. E. Rules for ring closure. J. Chem. Soc. Chem. Commun. 1976, 734–736 (1976).
Good, M. C., Zalatan, J. G. & Lim, W. A. Scaffold proteins: hubs for controlling the flow of cellular information. Science 332, 680–686 (2011).
Shaw, A. S. & Filbert, E. L. Scaffold proteins and immune-cell signalling. Nat. Rev. Immunol. 9, 47–56 (2009).
Choi, K. Y., Satterberg, B., Lyons, D. M. & Elion, E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell 78, 499–512 (1994).
Alberti, S. Phase separation in biology. Curr. Biol. 27, R1097–R1102 (2017).
Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).
Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Yang, H. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013). This paper describes several X-ray crystal structures of mTOR complexed with various regulatory small molecules and protein binding partners. Analysis of these structures reveals that rapamycin–FKBP12 inhibits mTOR by physically blocking substrates from accessing its active site.
Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017).
Kopytek, S. J., Standaert, R. F., Dyer, J. C. & Hu, J. C. Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate. Chem. Biol. 7, 313–321 (2000).
Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase. Nature 532, 127–130 (2016). This paper examines the structural underpinnings of lenalidomide’s ability to alter the substrate specificity of the E3 ubiquitin ligase cereblon (CRBN).
Woodward, A. W. & Bartel, B. Auxin: regulation, action, and interaction. Ann. Bot. 95, 707–735 (2005).
Chini, A. et al. The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448, 666–671 (2007).
Nishimura, N. et al. Structural mechanism of abscisic acid binding and signaling by dimeric PYR1. Science 326, 1373–1379 (2009).
Brown, E. J. et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758 (1994).
Liu, J. et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807–815 (1991). This paper identifies calcineurin as the shared target of cyclosporin A (complexed with cyclophilin) and FK506 (complexed with FKBP12). It also shows that the rapamycin–FKBP12 complex, cyclophilin alone, and FKBP12 alone do not inhibit calcineurin.
Pua, K. H., Stiles, D. T., Sowa, M. E. & Verdine, G. L. IMPDH2 is an intracellular target of the cyclophilin A and sanglifehrin A complex. Cell Reports 18, 432–442 (2017).
Schreiber, S. L. A chemical biology view of bioactive small molecules and a binder-based approach to connect biology to precision medicines. Isr. J. Chem. 59, 52–59 (2019).
Crabtree, G. R. & Schreiber, S. L. Three-part inventions: intracellular signaling and induced proximity. Trends Biochem. Sci. 21, 418–422 (1996).
Diver, S. T. & Schreiber, S. L. Single-step synthesis of cell-permeable protein dimerizers that activate signal transduction and gene expression. J. Am. Chem. Soc. 119, 5106–5109 (1997).
Pruschy, M. N. et al. Mechanistic studies of a signaling pathway activated by the organic dimerizer FK1012. Chem. Biol. 1, 163–172 (1994).
Belshaw, P. J., Ho, S. N., Crabtree, G. R. & Schreiber, S. L. Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc. Natl. Acad. Sci. USA 93, 4604–4607 (1996).
Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–993 (2006).
Gray, D. C., Mahrus, S. & Wells, J. A. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease. Cell 142, 637–646 (2010).
Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).
Liberles, S. D., Diver, S. T., Austin, D. J. & Schreiber, S. L. Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proc. Natl. Acad. Sci. USA 94, 7825–7830 (1997).
Di Stasi, A. et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 365, 1673–1683 (2011). This paper demonstrates the ability of a molecular glue to induce the activity of engineered caspase 9 in stem cell transplant patients. In doing so, it establishes that bifunctional-compound-induced proximity of fusion proteins can have therapeutic benefits in the clinic.
Cyrus, K. et al. Jostling for position: optimizing linker location in the design of estrogen receptor-targeting PROTACs. ChemMedChem 5, 979–985 (2010).
Krishnamurthy, V. M., Semetey, V., Bracher, P. J., Shen, N. & Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein-ligand system. J. Am. Chem. Soc. 129, 1312–1320 (2007).
Douglass, E. F. Jr., Miller, C. J., Sparer, G., Shapiro, H. & Spiegel, D. A. A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 135, 6092–6099 (2013).
Lai, A. C. & Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2017). This Review summarizes several small-molecule-based approaches for targeted protein degradation, such as PROTACs, hydrophobic tagging (HyT), and selective hormone receptor degraders (e.g., SERDs).
Chamberlain, P. P. & Hamann, L. G. Development of targeted protein degradation therapeutics. Nat. Chem. Biol. 15, 937–944 (2019).
Dikic, I. Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86, 193–224 (2017).
Sakamoto, K. M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).
Silva, M. C. et al. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife 8, e45457 (2019).
Pfaff, P., Samarasinghe, K. T. G., Crews, C. M. & Carreira, E. M. Reversible spatiotemporal control of induced protein degradation by bistable photoPROTACs. ACS Cent. Sci. 5, 1682–1690 (2019).
Reynders, M. et al. PHOTACs enable optical control of protein degradation. Preprint at ChemRxiv https://doi.org/10.26434/chemrxiv.8206688.v2 (2019).
Neklesa, T. K. et al. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).
Long, M. J. C., Gollapalli, D. R. & Hedstrom, L. Inhibitor mediated protein degradation. Chem. Biol. 19, 629–637 (2012).
Banik, S., Pedram, K., Wisnovsky, S., Riley, N. & Bertozzi, C. Lysosome targeting chimeras (LYTACs) for the degradation of secreted and membrane proteins. Preprint at ChemRxiv https://doi.org/10.26434/chemrxiv.7927061 (2019).
Schellenberg, G. D. & Montine, T. J. The genetics and neuropathology of Alzheimer’s disease. Acta Neuropathol. 124, 305–323 (2012).
Gestwicki, J. E., Crabtree, G. R. & Graef, I. A. Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation. Science 306, 865–869 (2004).
Zhang, Z. & Shokat, K. M. Bifunctional small-molecule ligands of K-Ras induce its association with immunophilin proteins. Angew. Chem. Int. Ed. Engl. 58, 16314–16319 (2019).
Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A. & Shokat, K. M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013).
Koppel, G. A. Recent advances with monoclonal antibody drug targeting for the treatment of human cancer. Bioconjug. Chem. 1, 13–23 (1990).
Shokat, K. M. & Schultz, P. G. Redirecting the immune response: ligand-mediated immunogenicity. J. Am. Chem. Soc. 113, 1861–1862 (1991).
Bertozzi, C. & Bednarski, M. C-glycosyl compounds bind to receptors on the surface of Escherichia coli and can target proteins to the organism. Carbohydr. Res. 223, 243–253 (1992).
Owen, R. M. et al. Bifunctional ligands that target cells displaying the α v β3 integrin. ChemBioChem 8, 68–82 (2007).
Danhier, F., Le Breton, A. & Préat, V. RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol. Pharm. 9, 2961–2973 (2012).
Carlson, C. B., Mowery, P., Owen, R. M., Dykhuizen, E. C. & Kiessling, L. L. Selective tumor cell targeting using low-affinity, multivalent interactions. ACS Chem. Biol. 2, 119–127 (2007).
Parker, C. G., Domaoal, R. A., Anderson, K. S. & Spiegel, D. A. An antibody-recruiting small molecule that targets HIV gp120. J. Am. Chem. Soc. 131, 16392–16394 (2009).
Rullo, A. F. et al. Re-engineering the immune response to metastatic cancer: antibody-recruiting small molecules targeting the urokinase receptor. Angew. Chem. Int. Ed. Engl. 55, 3642–3646 (2016).
Jones, C. N. et al. Bifunctional small molecules enhance neutrophil activities against Aspergillus fumigatus in vivo and in vitro. Front. Immunol. 10, 644 (2019).
McEnaney, P. J., Parker, C. G., Zhang, A. X. & Spiegel, D. A. Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease. ACS Chem. Biol. 7, 1139–1151 (2012).
Adelman, K. & Lis, J. T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).
Erwin, G. S. et al. Synthetic transcription elongation factors license transcription across repressive chromatin. Science 358, 1617–1622 (2017).
Jordan, M. A. & Wilson, L. Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr. Opin. Cell Biol. 10, 123–130 (1998).
Haggarty, S. J. et al. Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis. Chem. Biol. 7, 275–286 (2000).
Ceccarelli, D. F. et al. An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell 145, 1075–1087 (2011).
Huang, H. et al. E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin. Nat. Chem. Biol. 10, 156–163 (2014).
Struntz, N. B. et al. Stabilization of the Max homodimer with a small molecule attenuates Myc-driven transcription. Cell. Chem. Biol. 26, 711–723.e14 (2019).
Sijbesma, E. et al. Site-directed fragment-based screening for the discovery of protein-protein interaction stabilizers. J. Am. Chem. Soc. 141, 3524–3531 (2019).
Long, X. & Nephew, K. P. Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-α. J. Biol. Chem. 281, 9607–9615 (2006).
Blake, R. A. Abstract 4452: GNE-0011, a novel monovalent BRD4 degrader. Cancer Res. 79, 4452 (2019).
Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).
Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014).
Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017).
Uehara, T. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017).
Li, Z. et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature 575, 203–209 (2019).
de Waal, L. et al. Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics. Nat. Chem. Biol. 12, 102–108 (2016).
Guo, Z. et al. Rapamycin-inspired macrocycles with new target specificity. Nat. Chem. 11, 254–263 (2019).
Bierer, B. E., Somers, P. K., Wandless, T. J., Burakoff, S. J. & Schreiber, S. L. Probing immunosuppressant action with a nonnatural immunophilin ligand. Science 250, 556–559 (1990).
Scott, D. E., Bayly, A. R., Abell, C. & Skidmore, J. Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat. Rev. Drug Discov. 15, 533–550 (2016).
McGrath, J. P. et al. Pharmacological inhibition of the histone lysine demethylase KDM1A suppresses the growth of multiple acute myeloid leukemia subtypes. Cancer Res. 76, 1975–1988 (2016).
Ishikawa, Y. et al. A novel LSD1 inhibitor T-3775440 disrupts GFI1B-containing complex leading to transdifferentiation and impaired growth of AML cells. Mol. Cancer Ther. 16, 273–284 (2017).
Maiques-Diaz, A. et al. Enhancer activation by pharmacologic displacement of LSD1 from GFI1 induces differentiation in acute myeloid leukemia. Cell Rep. 22, 3641–3659 (2018).
Vinyard, M. E. et al. CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML. Nat. Chem. Biol. 15, 529–539 (2019).
Polier, S. et al. ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system. Nat. Chem. Biol. 9, 307–312 (2013).
Jones, L. H. Small-molecule kinase downregulators. Cell Chem. Biol. 25, 30–35 (2018). This Review covers small-molecule-based methods to decrease levels of kinases in cells, particularly in the context of cancer. Both linker-containing molecules (e.g., PROTACs) and non-linker compounds are discussed, including several FDA-approved, ATP-competitive compounds (e.g., erlotinib and vemurafenib).
Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N. & Levy, E. D. Proteins evolve on the edge of supramolecular self-assembly. Nature 548, 244–247 (2017). This paper studies the phenomenon by which proteins self-assemble into symmetric multimeric complexes. The authors often observed that a single point mutation was sufficient to catalyze polymerization, which suggests that proteins are primed for self-assembly.
Zimmermann, G. & Neri, D. DNA-encoded chemical libraries: foundations and applications in lead discovery. Drug Discov. Today 21, 1828–1834 (2016).
Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541.e29 (2017).
The authors thank B. Melillo, J. Ostrem, and B. Wagner for their critical feedback on the manuscript. Research from the Schreiber laboratory in the area covered by this Perspective was generously supposed by the National Institute of General Medical Sciences.
S.L.S. serves on the Board of Directors of the Genomics Institute of the Novartis Research Foundation (“GNF”); is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics; is a shareholder and advises Decibel Therapeutics and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Exo Therapeutics, and F-Prime Capital Partners; and is a Novartis Faculty Scholar. C.J.G. is an employee of Vertex Pharmaceuticals.
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Gerry, C.J., Schreiber, S.L. Unifying principles of bifunctional, proximity-inducing small molecules. Nat Chem Biol 16, 369–378 (2020). https://doi.org/10.1038/s41589-020-0469-1
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