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Protein Polymerization as a Novel Targeted Protein Degradation Mechanism

Cite this: Biochemistry 2021, 60, 15, 1145–1147
Publication Date (Web):March 24, 2021
https://doi.org/10.1021/acs.biochem.1c00163
Copyright © 2021 American Chemical Society
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Traditionally, biomedical scientists have sought to pharmacologically address human disease using a narrow focus–identify a key enzyme in a cellular process and block its active site with a complementary small molecule. More recently, monoclonal antibodies have proven successful at ameliorating disease states when the therapeutic target is in the extracellular space. Although both approaches have undoubtedly saved and improved lives, their utility has found limits–monoclonal antibodies are restricted to the extracellular space, and many intracellular proteins, such as protein–protein interaction targets (PPIs)–are refractory to traditional small molecule approaches due to the large and flat surfaces that mediate these interactions. Fortunately, novel approaches are emerging including targeted protein degradation (TPD), a strategy where selected proteins can be depleted via small molecules hijacking the endogenous cellular protein degradation machinery. This approach increases the druggable proteome since the small molecules can pharmacologically address their targets through a variety of binding pockets, including those outside of an active site. Indeed, this concept is clinically validated by thalidomide and its analogs as treatments for multiple myeloma and myelodysplastic syndromes. (1) These molecules, termed molecular glues, bind to the E3 ligase receptor cereblon thereby redirecting its substrate recognition to novel substrate proteins (including IKZF1, IKZF3, and CK1α), (1) resulting in their polyubiquitination and subsequent degradation in the proteasome. (1) As a twist on this theme, heterobifunctional degraders (2) similarly co-opt the cellular degradation machinery but with larger small molecules. These consist of three parts–a ligand that engages the target of interest, a second moiety that binds to an E3 ligase, and a chemical linker that connects the two. (3) The heterobifunctional degrader strategy has also been successful, including the advancement of a molecule to clinical trials. (3)

Despite advances in the TPD field, such approaches have not always worked for some important targets including BCL6, (4) a key driver of non-Hodgkin’s lymphomas. Recent work now shows that BCL6 degradation can be achieved through an alternative strategy. (5,6) Specifically, in 2017, Kerres et al. discovered a small molecule named BI-3802 which induces BCL6 degradation and shows increased pharmacological activity compared to closely related molecules. (5) However, the authors did not investigate the molecular mode of action (MOA). A recent publication from the Ebert lab (6) now shows that degradation is achieved through an entirely novel molecular glue MOA. In particular, BI-3802 binds to opposing sides of the homodimeric BTB (broad-complex, tramtrack, and bric-à-brac) domain of BCL6. This drives the reversible polymerization of the BCL6 protein using a novel molecular interface formed from both small molecule and protein components. The resulting cellular foci present multiple copies of the BCL6 protein thereby increasing its concentration within the local microenvironment. This appears to enhance its recognition as a substrate for SIAH1, an E3 ligase normally involved in the turnover of BCL6. As a result, highly specific and rapid BCL6 degradation ensues (Figure 1). This model is strongly supported by experimental evidence. First, addition of BI-3802 to the recombinant BCL6 protein resulted in large molecular weight polymers. These filaments were characterized by a combination of negative stain electron microscopy, computational modeling, and cryo-electron microscopy. The combined data set, along with the previously solved BCL6 crystal structure, led to a structural model that explains the polymerization affect–BI-3802 binds in a groove between BCL6 dimers and makes specific contacts across the interface while also inducing an interdimer salt bridge. BCL6 polymerization also seemed to occur in living cells as treatment of BI-3802 induced transient intracellular clusters of endogenous BCL6 or an eGFP-BCL6 fusion protein. These clusters later disappeared as they were degraded. A proteomic survey established the highly specific nature of this degradation as BCL6 was the only protein to show significant depletion. Importantly, degradation depended on the polymerization effect as mutation of key residues at the dimer/dimer interface (including those involved in the interdimer salt bridge) disrupted polymerization and degradation but without preventing BI-3802 binding. The first hint to the E3 ligase involved was that degradation of eGFP-BCL6 was blocked with small molecule inhibitors of polyubiquitination or the proteasome but not by pharmacological inhibition of neddylation. This indicated that the E3 ligase involved is not a cullin-RING family member since this class of E3 ligase critically requires Nedd8 for activity. A genome-scale CRISPR-Cas9 genetic screen and supporting experiments then identified SIAH1–a noncullin E3 ligase involved in BCL6 protein homeostasis–as the ligase involved in drug mediated BCL6 degradation. Furthermore, BCL6 truncation analysis and coimmunoprecipitation showed that a VxP motif on BCL6 mediates the interaction with SIAH1. Finally, a TR-FRET assay measured an increased PPI interaction between the E3 ligase SIAH1 and BCL6 in the presence of BI-3802 both in biochemical assays and in cells. These experiments validate the model that BI-3802 promotes the polymerization of BCL6 thus increasing its recognition and ubiquitination by SIAH1 resulting in its proteasomal degradation.

Figure 1

Figure 1. BI-3802 is a molecular glue that induces the degradation of BCL6 using a novel mechanism of action (artist’s rendition): BCL6 is normally present in cells as a homodimer, with contacts mediated by its BTB domain (left). BI-3802 binds to BCL6 at opposing sides of the homodimer and induces BCL6 polymerization using both small molecule and protein components (second panel from the left). Polymerization effectively increases the local concentration of the BCL6 protein within its microenvironment thereby increasing affinity of BCL6 for SIAH1 (middle panel, an E3 ligase normally involved in BCL6 turnover). This may increase the probability of productive polyubiquitin transfer–either to the BCL6 protein to which SIAH6 is docked or to a neighboring BCL6 protein. In this case, degradation through the proteasome proved to be not only rapid and efficient but also highly specific, perhaps due to the lack of direct contact with the E3–thus avoiding the induction of neosubstrate targeting.

The MOA described represents a new way for achieving molecular glue-driven degradation: one where the degrader molecule interacts only with the target protein and does not make contact with the E3 ligase involved. The absence of direct contact with the E3 ligase may explain the exquisite specificity of BI-3802 since this should avoid the induction of unwanted neosubstrate targeting. In addition, as demonstrated by BI-3802, this new type of monovalent protein-degrader may prove to be fast (t1/2s of minutes), highly potent (single digit nM EC50s), and highly efficacious (>90% degradation). (5,6) These features may be a result of the increased probability of polyubiquitination in situations where the E3 ligase is bound to complexes containing multiple copies of the substrate protein. Thus, the E3 ligase may transfer ubiquitin chains not only to the substrate to which it is directly bound but also to some of the nearest neighbors (Figure 1). This approach may also offer an avenue for targeting proteasomal degradation for the clearance of the aggregated protein associated with proteinopathies. Interestingly, E3 ligases such as the Speckle-type POZ protein (SPOP) may also leverage oligomerization for efficient degradation–but from an opposing angle. Specifically, the SPOP contains a BTB domain that polymerizes to form a supramolecular complex that positions multiple copies of the E3 ligase in close proximity to its substrates. (7) This arrangement may explain why this E3 ligase is particularly successful in the context of biodegrader approaches. (8,9) With this in mind, it would be interesting to explore the effectiveness of oligomerizing E3 ligases against polymerized versions of their natural substrates.

How might drug hunters apply these lessons to additional targets? In the case of symmetrical proteins, directly searching for supramolecular inducing molecules may be an option–either in biochemical or cell-based screens. This could be conducted either using unbiased small molecule libraries or with focused screens using analogues of known binders. Such strategies will be bolstered by the tendency for symmetrical proteins to be just below the threshold for polymerization. (10) With that in mind, structure-guided drug design approaches may also be useful. The work by Słabicki et al. also suggests that additional, unexpected degradation MOAs are likely to exist: molecular glue-mediated and otherwise. Accordingly, the discovery of monovalent protein-degraders could be facilitated with high-throughput cellular assays employing target-specific degradation readouts. Clearly, there are many avenues to explore, and although the application of lessons from the BCL6 case study to additional targets is unlikely to be a simple matter of “cut and paste”, the application of molecular glues is poised to solidify.

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The authors thank Nicolas Hastings for his input and careful reading of the manuscript.

References

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This article references 10 other publications.

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    Krönke, J., Fink, E. C., Hollenbach, P. W., MacBeth, K. J., Hurst, S. N., Udeshi, N. D., Chamberlain, P. P., Mani, D. R., Man, H. W., Gandhi, A. K., Svinkina, T., Schneider, R. K., McConkey, M., Järås, M., Griffiths, E., Wetzler, M., Bullinger, L., Cathers, B. E., Carr, S. A., Chopra, R., and Ebert, B. L. (2015) Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (7559), 183188,  DOI: 10.1038/nature14610
  2. 2
    Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., and Deshaies, R. J. (2001) Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U. S. A. 98 (15), 85549,  DOI: 10.1073/pnas.141230798
  3. 3
    Nalawansha, D. A. and Crews, C. M. (2020) PROTACs: An Emerging Therapeutic Modality in Precision Medicine. Cell Chem. Biol. 27 (8), 9981014,  DOI: 10.1016/j.chembiol.2020.07.020
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    McCoull, W., Cheung, T., Anderson, E., Barton, P., Burgess, J., Byth, K., Cao, Q., Castaldi, M. P., Chen, H., Chiarparin, E., Carbajo, R. J., Code, E., Cowan, S., Davey, P. R., Ferguson, A. D., Fillery, S., Fuller, N. O., Gao, N., Hargreaves, D., Howard, M. R., Hu, J., Kawatkar, A., Kemmitt, P. D., Leo, E., Molina, D. M., O’Connell, N., Petteruti, P., Rasmusson, T., Raubo, P., Rawlins, P. B., Ricchiuto, P., Robb, G. R., Schenone, M., Waring, M. J., Zinda, M., Fawell, S., and Wilson, D. M. (2018) Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6. ACS Chem. Biol. 13 (11), 31313141,  DOI: 10.1021/acschembio.8b00698
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    Słabicki, M., Yoon, H., Koeppel, J., Nitsch, L., Roy Burman, S. S., Di Genua, C., Donovan, K. A., Sperling, A. S., Hunkeler, M., Tsai, J. M., Sharma, R., Guirguis, A., Zou, C., Chudasama, P., Gasser, J. A., Miller, P. G., Scholl, C., Fröhling, S., Nowak, R. P., Fischer, E. S., and Ebert, B. L. (2020) Small-molecule-induced polymerization triggers degradation of BCL6. Nature 588 (7836), 164168,  DOI: 10.1038/s41586-020-2925-1
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    Marzahn, M. R., Marada, S., Lee, J., Nourse, A., Kenrick, S., Zhao, H., Ben-Nissan, G., Kolaitis, R. M., Peters, J. L., Pounds, S., Errington, W. J., Privé, G. G., Taylor, J. P., Sharon, M., Schuck, P., Ogden, S. K., and Mittag, T. (2016) Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles. EMBO J. 35 (12), 125475,  DOI: 10.15252/embj.201593169
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    Lim, S., Khoo, R., Peh, K. M., Teo, J., Chang, S. C., Ng, S., Beilhartz, G. L., Melnyk, R. A., Johannes, C. W., Brown, C. J., Lane, D. P., Henry, B., and Partridge, A. W. (2020) bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA). Proc. Natl. Acad. Sci. U. S. A. 117 (11), 57915800,  DOI: 10.1073/pnas.1920251117
  9. 9
    Lim, S., Khoo, R., Juang, Y. C., P.Gopal, Zhang, H., Yeo, C., Peh, K. M., Teo, J., Ng, S., Henry, B., and Partridge, A. W. (2021) Exquisitely Specific anti-KRAS Biodegraders Inform on the Cellular Prevalence of Nucleotide-Loaded States. ACS Cent. Sci. 7 (2), 274291,  DOI: 10.1021/acscentsci.0c01337
  10. 10
    Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N., and Levy, E. D. (2017) Proteins evolve on the edge of supramolecular self-assembly. Nature 548 (7666), 244247,  DOI: 10.1038/nature23320

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  • Abstract

    Figure 1

    Figure 1. BI-3802 is a molecular glue that induces the degradation of BCL6 using a novel mechanism of action (artist’s rendition): BCL6 is normally present in cells as a homodimer, with contacts mediated by its BTB domain (left). BI-3802 binds to BCL6 at opposing sides of the homodimer and induces BCL6 polymerization using both small molecule and protein components (second panel from the left). Polymerization effectively increases the local concentration of the BCL6 protein within its microenvironment thereby increasing affinity of BCL6 for SIAH1 (middle panel, an E3 ligase normally involved in BCL6 turnover). This may increase the probability of productive polyubiquitin transfer–either to the BCL6 protein to which SIAH6 is docked or to a neighboring BCL6 protein. In this case, degradation through the proteasome proved to be not only rapid and efficient but also highly specific, perhaps due to the lack of direct contact with the E3–thus avoiding the induction of neosubstrate targeting.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 10 other publications.

    1. 1
      Krönke, J., Fink, E. C., Hollenbach, P. W., MacBeth, K. J., Hurst, S. N., Udeshi, N. D., Chamberlain, P. P., Mani, D. R., Man, H. W., Gandhi, A. K., Svinkina, T., Schneider, R. K., McConkey, M., Järås, M., Griffiths, E., Wetzler, M., Bullinger, L., Cathers, B. E., Carr, S. A., Chopra, R., and Ebert, B. L. (2015) Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (7559), 183188,  DOI: 10.1038/nature14610
    2. 2
      Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., and Deshaies, R. J. (2001) Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. U. S. A. 98 (15), 85549,  DOI: 10.1073/pnas.141230798
    3. 3
      Nalawansha, D. A. and Crews, C. M. (2020) PROTACs: An Emerging Therapeutic Modality in Precision Medicine. Cell Chem. Biol. 27 (8), 9981014,  DOI: 10.1016/j.chembiol.2020.07.020
    4. 4
      McCoull, W., Cheung, T., Anderson, E., Barton, P., Burgess, J., Byth, K., Cao, Q., Castaldi, M. P., Chen, H., Chiarparin, E., Carbajo, R. J., Code, E., Cowan, S., Davey, P. R., Ferguson, A. D., Fillery, S., Fuller, N. O., Gao, N., Hargreaves, D., Howard, M. R., Hu, J., Kawatkar, A., Kemmitt, P. D., Leo, E., Molina, D. M., O’Connell, N., Petteruti, P., Rasmusson, T., Raubo, P., Rawlins, P. B., Ricchiuto, P., Robb, G. R., Schenone, M., Waring, M. J., Zinda, M., Fawell, S., and Wilson, D. M. (2018) Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6. ACS Chem. Biol. 13 (11), 31313141,  DOI: 10.1021/acschembio.8b00698
    5. 5
      Kerres, N., Steurer, S., Schlager, S., Bader, G., Berger, H., Caligiuri, M., Dank, C., Engen, J. R., Ettmayer, P., Fischerauer, B., Flotzinger, G., Gerlach, D., Gerstberger, T., Gmaschitz, T., Greb, P., Han, B., Heyes, E., Iacob, R. E., Kessler, D., Kölle, H., Lamarre, L., Lancia, D. R., Lucas, S., Mayer, M., Mayr, K., Mischerikow, N., Mück, K., Peinsipp, C., Petermann, O., Reiser, U., Rudolph, D., Rumpel, K., Salomon, C., Scharn, D., Schnitzer, R., Schrenk, A., Schweifer, N., Thompson, D., Traxler, E., Varecka, R., Voss, T., Weiss-Puxbaum, A., Winkler, S., Zheng, X., Zoephel, A., Kraut, N., McConnell, D., Pearson, M., and Koegl, M. (2017) Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6. Cell Rep. 20 (12), 28602875,  DOI: 10.1016/j.celrep.2017.08.081
    6. 6
      Słabicki, M., Yoon, H., Koeppel, J., Nitsch, L., Roy Burman, S. S., Di Genua, C., Donovan, K. A., Sperling, A. S., Hunkeler, M., Tsai, J. M., Sharma, R., Guirguis, A., Zou, C., Chudasama, P., Gasser, J. A., Miller, P. G., Scholl, C., Fröhling, S., Nowak, R. P., Fischer, E. S., and Ebert, B. L. (2020) Small-molecule-induced polymerization triggers degradation of BCL6. Nature 588 (7836), 164168,  DOI: 10.1038/s41586-020-2925-1
    7. 7
      Marzahn, M. R., Marada, S., Lee, J., Nourse, A., Kenrick, S., Zhao, H., Ben-Nissan, G., Kolaitis, R. M., Peters, J. L., Pounds, S., Errington, W. J., Privé, G. G., Taylor, J. P., Sharon, M., Schuck, P., Ogden, S. K., and Mittag, T. (2016) Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles. EMBO J. 35 (12), 125475,  DOI: 10.15252/embj.201593169
    8. 8
      Lim, S., Khoo, R., Peh, K. M., Teo, J., Chang, S. C., Ng, S., Beilhartz, G. L., Melnyk, R. A., Johannes, C. W., Brown, C. J., Lane, D. P., Henry, B., and Partridge, A. W. (2020) bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA). Proc. Natl. Acad. Sci. U. S. A. 117 (11), 57915800,  DOI: 10.1073/pnas.1920251117
    9. 9
      Lim, S., Khoo, R., Juang, Y. C., P.Gopal, Zhang, H., Yeo, C., Peh, K. M., Teo, J., Ng, S., Henry, B., and Partridge, A. W. (2021) Exquisitely Specific anti-KRAS Biodegraders Inform on the Cellular Prevalence of Nucleotide-Loaded States. ACS Cent. Sci. 7 (2), 274291,  DOI: 10.1021/acscentsci.0c01337
    10. 10
      Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N., and Levy, E. D. (2017) Proteins evolve on the edge of supramolecular self-assembly. Nature 548 (7666), 244247,  DOI: 10.1038/nature23320

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