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Bifunctional small molecules that mediate the degradation of extracellular proteins

Nature Chemical Biology volume 17, pages 947–953 (2021)Cite this article

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Abstract

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin–proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

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Fig. 1: Bifunctional MoDE-A molecule design and chemical structures.
Fig. 2: Bifunctional MoDE-A molecules mediate endocytosis of target proteins.
Fig. 3: Target protein α-DNP antibody is trafficked to lysosomes and degraded.
Fig. 4: D-MoDE-A mediates the accelerated depletion of α-DNP antibodies from serum in vivo.

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All data generated or analyzed during this study are included in this published article (and its Supplementary information files). Source data are provided with this paper.

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Acknowledgements

We thank C. Paulsen and S. Miller for use of equipment and the Eshhar laboratory for providing hybridomas for antibody production. We are thankful for financial support from the National Institute of Health Yale Chemical Biology Training grants to J.D.R. (no. 2T32GM06754 3-17), E.M.J.B. (5T32GM06754 3-12), R.A.H. (T32 GM067543) and D.F.C. (T32GM067543), the Department of Defense (BC120554) and Yale University (Michele Dufault Summer Research Fellowship to A.Z.G.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, CT, USA

    David F. Caianiello, Mengwen Zhang, Jason D. Ray, Rebecca A. Howell, Jake C. Swartzel, Emily M. J. Branham, Egor Chirkin, Venkata R. Sabbasani, Angela Z. Gong, David M. McDonald & David A. Spiegel

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Mengwen Zhang

  3. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA

    Jake C. Swartzel

  4. Yale Center for Precision Cancer Modeling, Yale University School of Medicine, New Haven, CT, USA

    Viswanathan Muthusamy

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Contributions

D.F.C., M.Z., V.M. and D.A.S. conceived the project and designed experiments. D.F.C., M.Z., E.C., A.Z.G. and J.C.S synthesized compounds. D.F.C., J.D.R., J.C.S., E.M.J.B. and R.A.H. performed in vitro and cellular experiments. V.R.S. performed in vivo experiments. D.F.C. and D.M. performed statistical analysis. D.F.C., J.D.R., D.M.M. and D.A.S. wrote the manuscript with input from all authors. D.A.S. provided supervision.

Corresponding author

Correspondence to David A. Spiegel.

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Competing interests

This work is the subject of two published PCT patent applications: PCT/US2019/026239 and PCT/US2019/026260. D.A.S. holds equity in Biohaven Pharmaceuticals, and also receives honoraria for consulting for Biohaven. Biohaven has licensed intellectual property from Yale University pertaining to the subject matter of this study.

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Peer review information Nature Chemical Biology thanks Padma Devarajan, Joshiawa Paulk and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1

a) Chemical structure of α-DNP antibody binding control molecule DNP-OH3. b) D-MoDE-A-mediated ternary complex formation is inhibited by competitive binders of either ASGPR (GalNAc, ‘GN’) or α-DNP antibody (‘DNP-OH3’). Each data point represents the mean ± SEM of a distinct biological replicate (n = 3). The line represents the mean. c) Endocytosis of α-DNP antibody in hepatocytes is dependent on both α-DNP antibody and D-MoDE-A concentration (12 hour time point). Each value represents a single biological replicate. d) Endocytosis of fluorescently labeled MIF is dependent on M-MoDE-A concentration. Each data point represents a distinct biological replicate (n = 4). The line represents the mean. e) Representative gating strategy for flow cytometry experiments.

Source data

Extended Data Fig. 2

a) Intracellular AF488 signal in colocalization studies is dependent on the presence of α-DNP antibody and D-MoDE-A. In cells treated with an isotopye control primary antibody, no EEA1 or LAMP2 signal is observed. The scale bar is 10 μm. These images are representative of three replicates. b) The majority of AF488 signal in cell culture supernatants is associated with full-length antibody. Experiments were performed in HepG2 cells. This blot is representative of three biological replicates. Uncropped blots are shown in Source data.

Extended Data Fig. 3

a) Plasma concentration of D-MoDE-A over time following a 1 mg/kg IP dose in male nude mice. b) Mouse body weight following treatment with D-MoDE-A or DNP-OH3 (data collected from the mice in the study described by Fig. 4b). Statistical differences were analyzed by ANOVA with Kruskall-Wallis test for post-hoc comparison between each treatment group and the PBS group. No significant differences were found (P > 0.9 in all cases). Each data point represents the mean ± SEM of a distinct mouse (n = 5). c) Levels of aspartate transaminase (AST) in treated mice (data from pooled serum from mice in the study described by Fig. 4b). Dashed lines represent the normal range. Each data point represents a technical replicate (n = 2). d) Levels of alanine transaminase (ALT) in treated mice (data from pooled serum from mice in the study described by Fig. 4b). Dashed lines represent the normal range. Each data point represents a technical replicate (n = 2). e) Decrease in serum levels of α-DNP antibody following a single variable dose of D-MoDE-A. Each experimental group contained at least eight mice. Statistical differences were assessed by repeated measures two-way ANOVA with Dunnett’s test for post-hoc comparison of simple effects between each of the treatment groups and PBS. Each data point represents the mean ± SEM of a distinct biological replicate (n = 10 for 0.1 mpk D-MoDE-A; n = 8 for all others). f) α-DNP antibody was co-injected with MIF. Mice treated with M-MoDE-A, D-MoDE-A, and 3w do not demonstrate rapid clearance of α-DNP antibody from circulation at early time points. Each data point represents the mean ± SEM of the concentration of α-DNP antibody in serum collected from five mice. Statistical differences were assessed by repeated measures two-way ANOVA with Tukey’s tests for post-hoc comparison of simple effects between each of the treatment groups and PBS.

Source data

Supplementary information

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

a) Uncropped α-AF488 blot corresponding to Fig. 3c. b) Uncropped α-actin blot corresponding to Fig. 3c. c) Uncropped α-AF488 blot corresponding to Fig. 3d. d) Uncropped α-actin blot corresponding to Fig. 3d.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

a) Uncropped α-AF488 blot corresponding to Extended Data Fig. 2b. b) Uncropped α-actin blot corresponding to Extended Data Fig. 2b.

Source Data Extended Data Fig. 3

Statistical source data.

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Caianiello, D.F., Zhang, M., Ray, J.D. et al. Bifunctional small molecules that mediate the degradation of extracellular proteins. Nat Chem Biol 17, 947–953 (2021). https://doi.org/10.1038/s41589-021-00851-1

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