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Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains

Nature Biotechnology volume 41, pages 541–551 (2023)Cite this article

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Abstract

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein–protein and protein–DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.

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Fig. 1: Modular design and synthesis of MAX-derived synthetic transcriptional repressors.
Fig. 2: STRs bind E-box DNA with high affinity and specificity.
Fig. 3: Synthetic modifications improved the stability of STRs in biological environments.
Fig. 4: STRs are cell permeable and oppose MYC-dependent gene expression and phenotypes in cells.
Fig. 5: X-ray crystal structure of a STR:E-box DNA complex.
Fig. 6: Reprogramming STRs for alternative, specific DNA binding.

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Data availability

X-ray structure coordinates are deposited in the Protein Data Bank under accession number 7RCU. Raw proteomics data are deposited in the Proteome Xchange Consortium through MassIVE under accession number MSV000089749. A representative EMSA gel for each compound and experiment type used in figure preparation is included in the Extended Data figure files. Source data are provided with this paper.

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Acknowledgements

We thank S. Ahmadiantehrani for assistance with figure and text editing and C. He, M. Rosner, E. Ozkan, P. Rice, S. Oakes and J. Montgomery for helpful discussions. P493-6 cells were kindly provided by S. Abdulkadir at Northwestern University. Jurkat cells were kindly provided by S. Tay at the University of Chicago. X-ray structure results shown in this report are derived from work performed at the Argonne National Laboratory, Structural Biology Center, at the Advanced Photon Source, under US Department of Energy, Office of Biological and Environmental Research contract DE-AC02-06CH11357. We are grateful for financial support of this work from the following: the Virginia and D. K. Ludwig Fund for Cancer Research (to S.W.F. and G.L.G.); National Institutes of Health (NIH) grant T32 CA009594-34 (to R.E.M. and C.L.) and Medical Scientist Training Program grant T32GM007281 to J.S.C.; the V Foundation for Cancer Research (to R.E.M.); NIH grant DP2GM128199-01 (to R.E.M.); and American Cancer Society-North Central Research Scholar grant RSG-17-150-01-CDD (to R.E.M.).

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Author notes

  1. These authors contributed equally: Thomas E. Speltz, Zeyu Qiao, Colin S. Swenson.

Authors and Affiliations

  1. Department of Chemistry, University of Chicago, Chicago, IL, USA

    Thomas E. Speltz, Zeyu Qiao, Colin S. Swenson, Xianghang Shangguan, John S. Coukos, Christopher W. Lee, Deborah M. Thomas, Jesse Santana & Raymond E. Moellering

  2. Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL, USA

    Thomas E. Speltz, Zeyu Qiao, Colin S. Swenson, Xianghang Shangguan, John S. Coukos, Christopher W. Lee, Deborah M. Thomas, Jesse Santana & Raymond E. Moellering

  3. Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA

    Sean W. Fanning & Geoffrey L. Greene

  4. Department of Cancer Biology, Loyola University Chicago, Chicago, IL, USA

    Sean W. Fanning

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Contributions

T.E.S., Z.Q., C.S.S., X.S. and R.E.M. contributed to the design and synthesis of all compounds, performed biochemical and cellular experiments, analyzed data and wrote the manuscript, with input from all authors. Z.Q. performed biochemical experiments, collected X-ray structure data and analyzed data. S.W.F. performed biochemical experiments, collected X-ray structure data and analyzed data. J.S.C. performed proteomics sample preparation and analysis. C.W.L., J.S. and D.M.T performed biochemical and cell-based experiments and analyzed data. G.L.G. supervised research related to X-ray structure data collection and analysis. R.E.M. conceived of the study and supervised all research.

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Correspondence to Raymond E. Moellering.

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T.E.S., X.S. and R.E.M. are named inventors on patent applications related to this work. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Optimization of synthetic transcriptional repressors.

a-e, Representative EMSA gels for compounds in this study. Titrations of compounds consist of 3-fold serial dilutions stating from 2 µM or 200 nM. The symbol (-) indicates a control well with no STR. EMSAs were performed as described in methods. The binding curves for the Kd values listed are shown in Figs. 2b,c. g, Ni-NTA resin pulldown gel for expressed his-tagged bHLH domains of MYC and MAX association in the presence of STR116. h, Uncropped version of gel shown in Fig. 4. i, Uncropped version of gel shown in Extended Data Fig. 4 f. Red channel shows CCNB. Green channel shows c-MYC, LDHA, and actin loading control.

Source data

Extended Data Fig. 2 STR affinity and specificity to E-Box DNA matches that of MYC and MAX protein.

a, Binding curves from EMSA experiments for dual-stabilized MAX-STRs used in this study. Data shown represent mean and s.d. from n = 3 independent replicates. Apparent Kd values represent mean and 95% C.I. from n = 3 independent replicates. b, Representative EMSA gels for recombinant MYC:MAX and MAX:MAX and associated binding curves. Kdapp values are from n = 3 independent replicates. c, Representative EMSA gels for DNA binding specificity experiments in the presence of increasing doses of unlabeled competitor oligos and associated plots of relative bound fraction determined by quantified band intensities. d, e, Representative EMSA gels for STR/protein competition experiments and associated competition curves. The indicated concentration of STR was incubated with 15 nM MYC:MAX (d) or 15 nM MAX:MAX (e) and 0.5 nM Ebox-IRD probe. The relative protein-bound E-Box DNA was quantified from n = 3 (e) or n = 2 (d) replicates and plotted to determine IC50 values. Data shown represent mean and s.d.

Source data

Extended Data Fig. 3 Hydrocarbon stapling promotes enhanced stability and cellular uptake.

a, Circular dichroism spectra for STR118 was measured at 25 °C (blue). The sample was heated to 95 °C for 5 min, cooled back down to 25 °C and the circular dichroism spectra of the same sample was obtained a second time (red). b, LCMS quantification of major fragments observed upon exposure of B-Z to trypsin. The area under the curve for extracted ion chromatograms of B-Z and indicated fragments was measured using a window of m/z ± 1. The plot shows the ratio of the area under the curve for each extracted ion chromatogram relative to the area under the curve for the extracted ion chromatogram of B-Z at time = 0 s. c, Representative EMSA gels from conditioned media stability assay. The assay was performed as described in methods. The images show 3 replicates for each STR. The experiment was repeated 3 times with similar results. d, e, Membrane integrity and viability of HeLa cells treated with FITC conjugated STR. d, Analysis of LDH release after 1-hour treatment of HeLa cells with 5 µM STR, vehicle (DMSO) or SDS lysis buffer. e, Cell-Titer-Glow viability analysis of HeLa cells treated with 5 µM STR or vehicle (DMSO) for 24 hours. Experiments were performed as described in methods. Data represent mean and s.d. of (n = 5, LDH) or (n = 2, CTG) biological replicates. Statistical analyses are by unpaired, two-sided t test. ns: not significant.

Source data

Extended Data Fig. 4 P-BioSTR118 photocrosslinks to E-Box DNA and occupies genomic DNA.

a, The chemical structure of P-BioSTR118. b, Representative EMSA gel and binding curve for P-BioSTR118 show high affinity binding for E-Box oligo. c, Denatured SDS-PAGE gel shows higher molecular weight E-Box oligo adducts are only formed upon exposure to UV at 365 nm. d, ChIP-qPCR quantification of endogenous MYC occupancy at control and E-box-containing target genes in HeLa cells. e, Photo-ChIP-qPCR quantification of P-BioSTR118 occupancy at control and E-box-containing target genes in P493-6 cells treated with 10 µM STR for 24 hr. ChIP-qPCR data represent the mean and s.e.m. of n = 2 (d) and n = 3 (e) independent biological replicates. Statistical analyses are by unpaired, two-sided t test.

Source data

Extended Data Fig. 5 STRs alter the proteome and reduce proliferation of MYC-dependent cell lines.

a, Venn diagram depicting total number of shared and unique peptides analyzed between STR116 and tetracycline treated P493-6 cells. b, Bar graph of the average median change in expression of indicated number of proteins analyzed for individual datasets. c, DAVID-GO analyses indicating clusters of relevant upregulated (c, red, top) and downregulated (d, blue, bottom) proteins shared between STR116 and tetracycline treated P493-6 cells. P-values for SILAC ratios were calculated using a background-based t-test. e, Firefly luciferase activity in HCT116 E-box reporter cells measured after STR treatment (20 μM for 24 hr). Data shown represent mean and s.d. of n = 3 independent biological replicates. Statistical analyses are by unpaired, two-sided t test. f, Representative western blot analysis of P493-6 cells after 48 hr treatment with indicated combinations of vehicle (‘MYC-ON’), tetracycline (‘MYC-OFF’) and 20 µM STR116 or STR118. g, Viability of P493-6 cells treated with STR116 under conditions of low (left, ‘MYC-OFF’) or high MYC expression (right, ‘MYC-ON’) after 72 hr. h, i, Relative viability of Ramos (h) or Jurkat (i) cells treated with STR116 after 72 hr. Viability plots show mean and s.d. for n = 3 independent biological replicates.

Source data

Extended Data Fig. 6 Analysis of B-Z:E-Box crystal structure.

a, Electron density map (top, s = 2.0), overlay of resolved structure onto density map (middle) and cartoon representation for unit cell of crystal structure (bottom). b, DNA overhang contact between neighboring DNA duplexes (top). Contacts between thymine and adenine on oligos from adjacent complexes (bottom). c-e, Hydrophobic core formed between residues from B-Z homodimer include bPhe43, bLeu46, zLeu64 and zAla67. Additional interacting residues between B-Z homodimers include bIle39 and zArg60 near DNA binding interface and zTyr70, zLys66, bPro51, and bVal50 near the c-terminus of the basic helix. f, Cartoon representation of Max homodimer (monomer 1 blue, monomer 2 pink, PDB: 1HLO) bound to DNA (left), B-Z homodimer (monomer 1 yellow with chemical crosslink green, monomer 2 orange) bound to DNA (right) and overlay of structures (middle). Structural alignment of B-Z homodimer to Max homodimer results in an RMSD of 0.847 Å for the backbone of the entire DNA binding domain held in common and 2.3 Å for entire bHLH structure. The interface between DNA binding domain of B-Z (1781 Å2) is also comparable to that of MAX homodimer (1726 Å2). g, Structural alignment for all residues in common between MAX and B-Z. h, Overview of basic helices from structure alignment.

Extended Data Fig. 7 Quantitative multiplexed EMSA (qEMSA) assay allows one-pot direct comparison of binding to a pool of unique DNA motifs.

a, Schematic depicting workflow of qEMSA. b, qEMSA profile of STR118 depicting high selectivity for canonical E-box DNA. c-g, Bar graphs indicating specific target enrichment derived from qEMSA experiments for STR118 (c) MAX/MAX (d), (e) STR116, (f) STR640 and (g) STR690. Data shown represent mean and s.d. from n = 2 independent replicates.

Extended Data Fig. 8 Design and characterization of TFAP4- and OLIG2-derived STRs.

a, Sequences of DNA probes containing target motifs E1, E2, and E3 used in b-f. b, Representative EMSA gels showing DNA binding of indicated compound with DNA consensus oligo E1, E2, and E3 in the presence of 0.01 mg/ml salmon sperm DNA. c-f, Dose-dependent target selectivity curves from quantified EMSA gels for native MAX dimer (c) or MAX- (d), TFAP4- (e) and OLIG2-derived (f) STRs binding to indicated target sequences, E1, E2, or E3.

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Speltz, T.E., Qiao, Z., Swenson, C.S. et al. Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains. Nat Biotechnol 41, 541–551 (2023). https://doi.org/10.1038/s41587-022-01504-x

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