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Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins

Nature Chemical Biology volume 20, pages 1566–1576 (2024)Cite this article

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Abstract

A paradigm shift in drug development is the discovery of small molecules that harness the ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here we provide a modality for targeted protein degradation in lysosomes. We exploit an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation. We developed a heterobifunctional small molecule, methylarginine targeting chimera (MrTAC), that recruits PRMT1 to a target protein for induced degradation in lysosomes. MrTAC compounds degraded substrates across cell lines, timescales and doses. MrTAC degradation required target protein methylation for subsequent lysosomal delivery via microautophagy. A library of MrTAC molecules exemplified the generality of MrTAC to degrade known targets and neo-substrates—glycogen synthase kinase 3β, MYC, bromodomain-containing protein 4 and histone deacetylase 6. MrTAC selectively degraded target proteins and drove biological loss-of-function phenotypes in survival, transcription and proliferation. Collectively, MrTAC demonstrates the utility of endogenous lysosomal proteolysis in the generation of a new class of small molecule degraders.

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Fig. 1: MrTAC mediates proximity with PRMT1 for target degradation.
Fig. 2: MrTAC targets GSK3β to lysosomes for degradation.
Fig. 3: Arginine methylation of the protein target underscores MrTAC degradation.
Fig. 4: MrTAC degradation recapitulates loss-of-function phenotypes.
Fig. 5: Induced degradation of endogenous substrates.
Fig. 6: MrTAC regulates cell function via endogenous degradation.

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Data availability

All data reporting the findings of this study are included in the Article and within the source data found in the Supplementary Information. Proteomics data have been submitted to the ProteomeXchange Consortium in the PRIDE partner repository with the dataset identifier PXD054653. Source data are provided with this paper.

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Acknowledgements

We thank members of the Albrecht Lab for their support in this research and thank J. Leonard for assistance in modeling and conceptualization. The research was supported in part by Ono Pharmaceuticals (award to L.V.A.) and we thank the Cystinosis Research Foundation (CRF) for funding. We would like to thank the UCI Mass Spectrometry Facility and B. Katz for assistance with the collection and analysis of protein MS data. Data were collected on a Waters Acquity UPLC Xevo G2-XS QTOF system (NIH supplemental funding support received by J.S. Nowick (National Institute of General Medical Sciences (NIGMS) GM097562), V.Y. Duong (NIH GM105938) and O. Cinquin (NIGMS GM102635)). We would like to thank C. Yu and L. Huang at the UCI High-end Mass Spectrometry Facility for their help and service in MS data acquisition and analysis. We would also like to thank the Comprehensive Liver Research Center at University of California, Los Angeles (UCLA).

Author information

Authors and Affiliations

  1. Department of Developmental & Cell Biology, School of Biological Sciences, University of California, Irvine, Irvine, CA, USA

    Laurence J. Seabrook, Melissa Campos & Lauren V. Albrecht

  2. Department of Pharmaceutical Sciences, School of Pharmacy & Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA

    Carolina N. Franco, Cody A. Loy, Callie Fredlender, Steven T. Nguyen, Marian F. Khalil, Darci J. Trader & Lauren V. Albrecht

  3. Department of Chemistry, School of Physical Sciences, University of California, Irvine, Irvine, CA, USA

    Jaida Osman & Darci J. Trader

  4. Center for Neurotherapeutics, University of California, Irvine, Irvine, CA, USA

    Jan Zimak & Samantha R. Levine

  5. Department of Medicine, Division of Pulmonary & Critical Care, University of California, Los Angeles, Los Angeles, CA, USA

    Richard L. Watson

  6. Department of Genetics, Yale School of Medicine, New Haven, CT, USA

    Kaelyn Sumigray

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  1. Laurence J. Seabrook
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Contributions

L.V.A., L.J.S. and C.N.F conceived the study. L.V.A. and L.J.S. designed the experiments. L.J.S., C.N.F., M.C., S.T.N., R.L.W., M.F.K. and K.S. contributed to cell biology experiments. C.A.L., J.O., S.R.L. and D.J.T. contributed to compound synthesis. C.F. and J.Z. contributed to CRISPR cell line construction. L.V.A. and L.J.S. analyzed the data. L.V.A. and L.J.S. wrote the manuscript.

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Correspondence to Lauren V. Albrecht.

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Nature Chemical Biology thanks Cheryl Arrowsmith, Yong Tae Kwon 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 Expression and degradation of GSK3β by MrTAC.

a, Schematic of types of MrTAC compounds to mediate proximity between a protein target and a protein arginine methyltransferase (PRMT). Indirect MrTAC dimerizes HaloTag and SNAP-tag fusion proteins that are encoded alongside the PRMT and target. b, Representative immunofluorescence of HEK293 cells stably expressing GSK3β-SNAP-mCherry-FLAG (red) and Halo-PRMT1-Myc (green). c, Immunoblot analysis of GSK3β levels (left) and PRMT1 levels (endogenous, Halo-tagged monomer and Halo-tagged in MrTAC complex; right) during either a 24- or 3-h MrTACHaXS8 dose curve in stably expressing HEK293s. d, Confocal microscopy of HEK293T cells either non-transfected or transfected with GSK3β-SNAP-FLAG (red) and Halo-PRMT1-Myc (green; left). Quantification (right) shows proportion of cells expressing both constructs or just one construct relative to the total number of transfected cells over three fields of view. ****P < 0.0001, *P < 0.0217 by one-sided t-test relative to 0% (n = 3 fields of view). e, Immunoblot analysis of endogenous GSK3β and PRMT1, overexpressed GSK3β-SNAP and Halo-PRMT1 and GSK3β-MrTAC-PRMT1 complexes (0.5 µM, 1 h). f, Immunoblot analysis of HEK293T cells transiently expressing Halo-PRMT1 and GSK3β-SNAP in a 24 (left), 3 (middle) or 9-h (right) MrTAC dose curve (top). Bottom graphs show total levels of GSK3β relative to 0 µM. g, Immunoblot analysis of HeLa cells expressing Halo-PRMT1 and GSK3β-SNAP in a 3-h MrTAC dose curve (top). Bottom graph shows total levels of GSK3β relative to 0 µM. Scale bars are 10 µm. Immunoblots are representative of three independent experiments. Data are represented as means ± SEM.

Source data

Extended Data Fig. 2 VPS4-mediated GSK3β sequestration during MrTAC treatment.

a, Live-cell confocal microscopy of GSK3β-SNAP-GFP in HeLa cells incubated with Lysotracker over 60 min with 0.5 µM MrTACHaXS8. Image was captured every 10 min. Arrows note colocalization. b, Colocalization of GSK3β-SNAP-FLAG with lysosomal-associated membrane protein (LAMP-1) after MrTACHaXS8 treatment in stable HEK293 cells (0.5 µM, 3 h, mean colocalizing structures over n = 39DMSO and 54MrTAC cells; left) or transiently expressing HEK293T cells (10 µM, 3 h, mean colocalizing structures over n = 24DMSO and 45MrTAC cells; right). Arrows mark colocalization. For either experiment, graphs depict the number of colocalized GSK3β/LAMP-1 puncta within each cell. ****P < 0.0001 and ***P < 0.0010 by unpaired two-sided t-tests. c, Colocalization of HEK293T cells expressing GSK3β-SNAP-FLAG (red) with either wild-type (WT) or dominant negative (DN) VPS4-GFP after MrTACHaXS8 treatment (10 µM, 1 h). d, Colocalization of HeLa cells expressing GSK3β-SNAP-FLAG (red) with either wild-type (WT) or dominant negative (DN) VPS4-GFP after MrTACHaXS8 treatment (0.5 µM, 2 h). e, Immunoblot analysis of target protein levels of siRNA-mediated knockdown of Lamp2a, Atg7 or Vps4a (100 nM) collected on day of experiment completion (n = 3 experiments). Scale bars are 10 µm. Data are represented as means ± SEM.

Source data

Extended Data Fig. 3 Methylation of GSK3β by PRMT1.

a, Mass spectrometry of in vitro methylated GSK3β samples in presence (bottom) or absence (top) of PRMT1 and the associated protein coverage by LC–MS QTOF (left). Associated coverage map of GSK3β by LC–MS QTOF (right). b, Mass spectrometry of methylated GSK3β following FLAG immunoprecipitation of MrTAC-treated HEK293 cells that transiently express PRMT1-Halo and GSK3β-SNAP-FLAG (10 µM, 2 h with 400 nM bafilomycin and 10 µM MG132; left). Associated coverage maps of GSK3β and recruited PRMT1 by LC–MS QTOF (right).

Extended Data Fig. 4 MrTAC degradation of SNAP-tagged proteins reshapes cell signaling.

a, Proliferation following MrTACHaXS8 treatment (0.5 µM) in HeLa cells expressing either Halo-PRMT1/GSK3β-SNAP or Halo-empty/SNAP-empty over seven days (n = 4 experiments; left). **P < 0.0051 drug effect and **PD7 < 0.0010 by two-way ANOVA with Bonferroni’s multiple comparisons. Proliferation of non-transfected HeLa cells treated with MrTACHaXS8 at indicated times (n = 8 experiments), ns by unpaired two-sided t-tests (right). b, Immunoblot analysis of PRMT1 protein levels (Halo-tagged and endogenous) in MrTACHaXS8-treated HEK293 cells expressing CRISPR-integrated PRMT1-Halo/GSK3β-SNAP over 3 days (n = 3 experiments). c, Confocal microscopy of c-MYC-SNAP-FLAG colocalization with asymmetric dimethylarginine (ADMA) antibody after MrTAC treatment (1.5 h, 10 µM; left). Thick outlines mark low colocalization, and arrows mark high colocalization. Scale bar is 5 µm. Bars (right) show average Pearson correlation coefficient between FLAG/ADMA channels across all cells marked with DAPI (n = 11 fields of view). ***P < 0.0009 by two-sided unpaired t-test. d, Confocal microscopy of c-MYC-SNAP-FLAG colocalization with lysosomal-associated membrane protein (LAMP-1) in MrTAC-treated cells (1 h, 10 µM; left). Arrows indicate colocalization, scale bar is 10 µm. Bars (right) show average number of colocalized structures within a cell over 9 fields of view as normalized to DMSO condition. **P < 0.0046 by two-sided unpaired t-test. e, Immunoblot analysis of c-MYC-SNAP levels in cells treated with MrTAC (10 µM) at indicated times with cycloheximide (10 µM). Cells were treated in the presence of bafilomycin (100 nM) or MG132 (5 µM; n = 2 experiments). Data are represented as means ± SEM.

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Extended Data Fig. 5 Degradation of endogenous targets.

a, Modeling of MrTACJQ1. MrTACJQ1 bound to BRD4 bromodomain 1 (BD1; PDB: 3MXF) and HaloTag (PDB: 6U32). Pink arrow shows direction of PRMT1 fusion (left). BRD4 BD1 binding to MrTACJQ1 compared against JQ1 ligand (gold; middle). HaloTag binding to MrTACJQ1 compared against tetramethylrhodamine-HaloTag (TMR-HT) ligand (gold; right). b, Quantification of immunoblot in Fig 5b by one-way ANOVA with Bonferroni’s multiple comparisons, *P0.1 < 0.0169, **P1 < 0.0076, **P10 < 0.0069. c, Immunoblot analysis of PRMT1-Halo levels through a 4-h MrTACJQ1 treatment (n = 3). d, Quantification of immunoblot in Fig 5f by one-way ANOVA with Bonferroni’s multiple comparisons, *P < 0.0221 (nMrTAC = 4, nControl = 3). e, Immunoblot analysis of PRMT1 levels through a 4-h MrTACSAHA treatment in HEK293s stably expressing PRMT1-Halo (n = 3; left). Right quantifies levels of total PRMT1 (Halo-tagged and monomeric, uncropped blot in source data) relative to 0 µM; ns by one-way ANOVA with Bonferroni’s multiple comparisons. f, Immunofluorescence of PRMT1-Halo in stably expressing HEK293 cells following a 4-h MrTACSAHA treatment with a lysosome-associated membrane protein 1 (LAMP-1) costain; scale bar is 5 µm. g, Analysis of HDAC6 levels through a 4-h MrTACSAHA treatment in HeLa cells transiently expressing PRMT1-Halo by immunoblot (n = 3; left) and immunofluorescence (n = 200 cells; middle); graph shows HDAC6 intensity over total cells marked by DAPI. ****P < 0.0001 by unpaired two-sided t-test (right). h, Immunoblot analysis of HDAC6 levels with MrTACSAHA (1 µM, 4 h) in HEK293s stably expressing PRMT1-Halo co-treated with lysosome inhibitor bafilomycin (400 nM, 2 h pre-treatment) or proteasome inhibitor MG132 (10 µM; n = 3; left). Right, total levels of HDAC6 in +MrTAC cells relative to either group’s -MrTAC condition. *P < 0.0445 by unpaired two-sided t-test. Immunoblots are representative of at least three independent experiments. Data are represented as means ± SEM.

Source data

Extended Data Fig. 6 Effect of endogenous MrTAC degradation on cell function.

a, Representative cell density of HEK293 cells stably expressing PRMT1-HaloTag following 6 days of 1 µM MrTACJQ1 or DMSO vehicle (n = 3 experiments); scale bar is 500 µm. b, Immunoblot analysis of Halo-tagged and endogenous PRMT1 levels through a 3-day MrTACSAHA treatment at 1 µM in HEK293s stably expressing PRMT1-Halo (left; n = 3 experiments). Middle and right quantify total levels of PRMT1-Halo and endogenous PRMT1, respectively, as normalized to the DMSO condition, ns by unpaired two-sided t-test. All quantified values are means ± SEM.

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Supplementary information

Supplementary Information

Supplementary Note.

Reporting Summary

Supplementary Video 1

Live-cell confocal microscopy of GSK3β-SNAP-GFP degradation in HeLa cells over 60 min with 0.1 µM MrTAC. Image was captured every 5 min. Scale bar is 5 µm.

Supplementary Video 2

Live-cell confocal microscopy of GSK3β-SNAP-mCherry degradation in stably expressing HEK293 cells incubated with LysoTracker over 40 min with 0.1 µM MrTAC. Image was captured every 6 min. Scale bar is 10 µm.

Supplementary Video 3

Live-cell confocal microscopy of GSK3β-SNAP-GFP degradation in HeLa cells incubated with LysoTracker over 60 min with 0.5 µM MrTAC. Image was captured every 10 min. Scale bar is 10 µm, arrows note colocalization.

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Seabrook, L.J., Franco, C.N., Loy, C.A. et al. Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins. Nat Chem Biol 20, 1566–1576 (2024). https://doi.org/10.1038/s41589-024-01741-y

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