Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

Journal name:
Nature Chemical Biology
Year published:
Published online


We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

At a glance


  1. UNC1215 is a potent antagonist of L3MBTL3.
    Figure 1: UNC1215 is a potent antagonist of L3MBTL3.

    (a) Structure of UNC1021, a nanomolar antagonist of L3MBTL3 Kme-binding activity. UNC1215 is a more potent L3MBTL3 cellular antagonist than UNC1021, and UNC1079 is a structurally similar but substantially less potent antagonist and negative control. (b) ITC analysis of site-directed mutants of L3MBTL3 (3MBT) revealed strong binding of UNC1215 to wild-type (WT) protein but not to domain 2 binding pocket mutant D381A. Domain 1 (D274A) mutant bound UNC1215 with weaker affinity than the wild type. (c) Domain architecture of the full-length L3MBTL3 protein showing a putative phenylalanine-cysteine-serine nucleic acid–binding domain, a Zinc finger (ZnF) and a sterile α motif (SAM) domain, in addition to three MBT repeat sequences (pink, blue and yellow). Mono- or dimethyllysine is represented schematically by a green oval. Mutations made in the aromatic cage of the second MBT domain (D381A) and in the aromatic cage of the first MBT domain (D274A) are represented by stars. A schematic of all protein constructs and tagged fusion proteins used for in vitro and cellular studies is in Supplementary Figure 21. (d) A surface representation of the 3MBT crystal structure (PDB code 3UT1), colored as in c, with the residues composing the conserved (domain 2) or semiconserved (domains 1 and 3) aromatic cage of each repeat in red, dark blue and orange for domain 1, 2 and 3, respectively. Thus, the three presumed binding sites of 3MBT exist in a triangular arrangement, all on the same surface of the molecule.

  2. X-ray crystal structure of the UNC1215–3MBT complex.
    Figure 2: X-ray crystal structure of the UNC1215–3MBT complex.

    UNC1215 binds in a unique 2:2 binding mode, supporting the mechanism of action of UNC1215 (PDB code 4FL6). (a) Two 3MBT molecules are rotated around a pseudo–two-fold axis perpendicular to the plane of the paper. As a consequence, the domain 3 aromatic cages of each protein, domain 3′ and domain 3″, face one another, whereas domain 1′ faces domain 2″, and domain 2′ faces domain 1″. MBT domains are colored as in Figure 1; UNC1215 is shown in purple. (b) UNC1215 binding is primarily mediated by interaction with the aromatic cage of domain 2′ (blue), consisting of Phe387, Phe405, Trp408 and Tyr412, and via a key hydrogen bond between one pyrrolidine nitrogen and Asp381. The piperidine-pyrrolidine ortho to the aniline ring bridges the two proteins by interacting with domain 1″ (pink), forming a salt bridge with Asp274. A second UNC1215 molecule binds in a reciprocal fashion to domain 1′- and domain 2″-binding pockets. (c) Surface representation showing the close association of the two 3MBT subunits and the means by which UNC1215 bridges the aromatic cages of domain 1 (pink) and domain 2 (blue).

  3. UNC1215 binds a small set of Kme reader proteins with lower affinity than L3MBTL3.
    Figure 3: UNC1215 binds a small set of Kme reader proteins with lower affinity than L3MBTL3.

    (a) Structure of a biotinylated analog of UNC1215. (b) H4K20me2 pull-down experiments in the presence of increasing concentrations of UNC1215 reveal nanomolar potency for antagonism of 3MBT pulldown, whereas much weaker affinity was observed for 53BP1. MW, molecular weight.

  4. UNC1215 potently antagonizes 3MBT localization in cells.
    Figure 4: UNC1215 potently antagonizes 3MBT localization in cells.

    (a) Recovery time of a photobleached area in GFP-3MBT–expressing cells is reduced upon treatment with UNC1215 in a dose-response manner, whereas inactive compound UNC1079 shows no effect. Solid lines represent the exponential fit of the data for 8–10 nuclei with the coefficients of variation ranging from 1–30% for the individual time points. The experiments were performed three independent times with similar data resulting. Inset shows time-lapse images of photobleached nuclei for UNC1215 (1 μM) and control (water) treatments. Scale bars, 10 μm. (b) GFP fusions of 3MBT and FLMBT localize to the nucleus in HEK293 cells. UNC1215 inhibits the foci formation of GFP-3MBT in a dose-response fashion, whereas UNC1079 has no effect on the foci. In contrast, UNC1215 is relatively ineffective at inhibiting foci formation of N-terminally tagged GFP-FLMBT. Scale bars, 10 μm. (c) The GFP-3MBT D274A domain 1 mutant shows a reduction in foci formation, whereas the GFP-3MBT D381A domain 2 mutant does not form nuclear foci. WT, wild type. Error bars represent s.e.m. from 15–25 cells.

  5. UNC1215 binds and colocalizes with full-length L3MBTL3.
    Figure 5: UNC1215 binds and colocalizes with full-length L3MBTL3.

    (a) UNC1215 conjugated to the cell-permeable merocyanine dye mero76 (mero76-UNC1215, (5)) localizes together with GFP-FLMBT in HEK293 cells (scale bars, 10 μm; green is GFP-FLMBT, red is mero76-UNC1215, and blue is Hoechst dye). (b) FLMBT binds biotin-UNC1215 (5 nmol). The presence of increasing concentrations (1 or 10 equivalents relative to biotin-UNC1215) of untagged UNC1215 results in a decreased amount of bound FLMBT. IP, immunoprecipitation; IB, immunoblotting.

  6. Identification of BCLAF1 as a new L3MBTL3 protein interactor.
    Figure 6: Identification of BCLAF1 as a new L3MBTL3 protein interactor.

    (a) Top: in U2OS cells, 3MBT colocalizes with BCLAF1 (green is GFP-3MBT, red is BCLAF1, and blue is DAPI). Bottom: upon treatment with UNC1215, the 3MBT and BCLAF1 nuclear foci are noticeably disrupted. Scale bars, 10 μm. (b) Immunoprecipitation (IP) experiments in cells transfected with Flag-3MBT or Flag-FLMBT show that UNC1215 disrupts the interaction between 3MBT and BCLAF1 and also reduces the interaction between FLMBT and BCLAF1. U, untransfected cells.


5 compounds View all compounds
  1. 2-Phenylamino-1,4-bis(4-(pyrrolidinyl)piperidinyl)benzamide
    Compound 1 2-Phenylamino-1,4-bis(4-(pyrrolidinyl)piperidinyl)benzamide
  2. 1,4-Bis(4-(pyrrolidinyl)piperidinyl)benzamide
    Compound 2 1,4-Bis(4-(pyrrolidinyl)piperidinyl)benzamide
  3. 1,4-Bis(4-(piperidinyl)piperidinyl)benzamide
    Compound 3 1,4-Bis(4-(piperidinyl)piperidinyl)benzamide
  4. 2-(4-Biotin-dPEG11-aminobenzoate)-1,4-bis(4-(pyrrolidinyl)piperidinyl)benzamide
    Compound 4 2-(4-Biotin-dPEG11-aminobenzoate)-1,4-bis(4-(pyrrolidinyl)piperidinyl)benzamide
  5. Merocyanine76-UNC1215
    Compound 5 Merocyanine76-UNC1215

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Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


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Author information


  1. Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    • Lindsey I James,
    • Victoria K Korboukh,
    • J Martin Herold,
    • Christopher J MacNevin,
    • Jacqueline L Norris,
    • Cen Gao,
    • Dmitri B Kireev,
    • Jian Jin,
    • William P Janzen &
    • Stephen V Frye
  2. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

    • Dalia Barsyte-Lovejoy,
    • Nan Zhong,
    • Liubov Krichevsky,
    • Wolfram Tempel,
    • Peter J Brown &
    • Cheryl H Arrowsmith
  3. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

    • Liubov Krichevsky &
    • Cheryl H Arrowsmith
  4. Ontario Cancer Institute–Campbell Family Cancer Research Institute, University of Toronto, Toronto, Ontario, Canada.

    • Liubov Krichevsky,
    • Shili Duan &
    • Cheryl H Arrowsmith
  5. Department of Pharmacology, University of North Carolina at Chapel Hill Medical School, Chapel Hill, North Carolina, USA.

    • Christopher J MacNevin &
    • Xi-Ping Huang
  6. Department of Carcinogenesis, M. D. Anderson Cancer Center, University of Texas, Smithville, Texas, USA.

    • Cari A Sagum &
    • Mark T Bedford
  7. Banting and Best Department of Medical Research, Donnelly Centre, Toronto, Canada.

    • Edyta Marcon,
    • Hongbo Guo,
    • Andrew Emili &
    • Jack F Greenblatt
  8. National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina at Chapel Hill Medical School, Chapel Hill, North Carolina, USA.

    • Xi-Ping Huang


L.I.J. synthesized all compounds and related analogs and performed ITC studies; D.B.-L. and L.K. performed immunofluorescence FRAP, affinity purification and coimmunoprecipitation studies; N.Z. and W.T. solved and analyzed the X-ray crystal structure of the UNC1215L3MBTL3 complex; V.K.K. and W.P.J. performed and analyzed AlphaScreen studies; C.J.M. synthesized the mero76-UNC1215 conjugate; J.L.N. purified proteins and performed mutagenesis; C.A.S. and M.T.B. performed protein array and protein pull-down experiments; E.M., H.G., A.E. and J.F.G. performed MS-based studies; S.D. cloned mammalian expression vectors for all cellular studies; X.-P.H. performed and analyzed GPCR selectivity studies; L.I.J., D.B.-L., N.Z., L.K., J.M.H., V.K.K., C.G., D.B.K., J.J., W.P.J., P.J.B., M.T.B, C.H.A. and S.V.F. designed studies and discussed results; L.I.J., C.H.A. and S.V.F. wrote the paper.

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