Allosteric inhibition of antiapoptotic MCL-1


MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure–function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.

Figure 1: Selective inhibition of MCL-1ΔNΔC binding activity by covalent modification of C286.
Figure 2: C286 dependence and specificity of action of MAIM1.
Figure 3: MAIM1 impairs the capacity of MCL-1ΔNΔC to protect BID BH3 from deuterium exchange.
Figure 4: BID BH3–induced decrease in deuterium exchange at the canonical groove is reversed by MAIM1 derivatization.
Figure 5: C286W mutagenesis mimics the inhibitory effects of MAIM1 on MCL-1 activity.
Figure 6: C286W mutagenesis blunts the antiapoptotic activity of MCL-1 in reconstituted cells.
Figure 7: Molecular dynamics calculations indicate decreased structural flexibility of MCL-1ΔNΔC after MAIM1 derivatization and C286W mutagenesis.

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We thank E. Smith for graphics support; J. Opferman (St. Jude Children's Research Hospital) for WT and Mcl1−/− MEFs; and W. Massefski (Dana-Farber Cancer Institute) for technical assistance with MAIM1 characterization by NMR. This research was supported by NIH grant 1R35CA197583, a Leukemia and Lymphoma Society (LLS) Marshall A. Lichtman Specialized Center of Research project grant, the Todd J. Schwartz Memorial Fund, the Wolpoff Family Foundation and an LLS Scholar Award (L.D.W.); NIH grant R01GM101135 and a Waters Corporation research collaboration (J.R.E.); and NIH grant T32GM007753 (J.L.).

Author information




S.L., T.E.W., N.A.C., J.R.E. and L.D.W. designed the study; G.H.B. generated stapled peptides; D.T.C. synthesized and characterized MAIM1; C.G.G., D.T.C., S.E., A.J.H. and N.A.C. performed biochemical experiments; S.L. and T.E.W. conducted the HXMS experiments under the guidance of J.R.E.; S.E. and D.T.C. performed the cellular experiments; J.L. conducted molecular dynamics simulations; and S.L., T.E.W., S.E., D.T.C., J.L., J.R.E. and L.D.W. analyzed the data and wrote the manuscript, which was reviewed by all coauthors.

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Correspondence to Loren D Walensky.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Small-molecule covalent inhibition of MCL-1ΔNΔC.

(a) Fluorescence polarization dilutional binding assay of FITC−BID BH3 and MCL-1ΔNΔC in the presence and absence of N-(4-hydroxy-1-naphthalenyl)-benzenesulfonamide (CAS# 36942-42-4), a small molecule screening hit that exhibited irreversible inhibition of the BH3-binding activity of MCL-1ΔNΔC. (b) Chemical structures of MAIM1 analogs that emerged from our competitive MCL-1 SAHB A −MCL-1ΔNΔC screen. Competitive potency corresponded to compound reactivity, based on the nature of the leaving group (R1) and electron withdrawing capacity (R2). Data are mean ± s.e.m. (n = 3 technical replicates). (c) Chemical synthesis of MAIM1. (d-e) Trypsin digestion and LC-MS/MS of unmodified (d) and MAIM1-conjugated (e) MCL-1ΔNΔC. Full scan spectra (left) demonstrate the peptide containing unmodified C286 (mass: 2329.18 Da) and the MAIM1 conjugate (mass: 2483.19 Da), reflecting the added mass of cleaved MAIM1 adduct (i.e. minus tosyl). MS/MS spectra (right) further confirm MAIM1 conjugation at position C286. Whereas the masses of ions y3, y4, y5, y6, and y12 are shared between the peptides, there is a discrete mass shift for ion y15, which contains C286. For clarity, only the y ions are labeled.

Supplementary Figure 2 C286S mutagenesis abrogates the MAIM1 effect on deuterium exchange of MCL-1ΔNΔC in the presence of BID BH3.

Relative difference plots of the relative deuterium incorporation of MCL-1ΔNΔC C286S in the presence of BID BH3 minus the relative deuterium incorporation of MCL-1ΔNΔC C286S (brown), and the relative deuterium incorporation of MCL-1ΔNΔC C286S in the presence of MAIM1 and BID BH3 minus the relative deuterium incorporation of MCL-1ΔNΔC C286S in the presence of MAIM1 (orange). Changes in deuterium exchange above a stringent significant threshold of 0.8 Da are shaded light purple (0.8 – 2 Da), purple (2 – 3.5 Da), and dark purple (> 3.5 Da). HXMS data are for deuterium labeling at 10 seconds and represent the average of two independent experiments using distinct preparations of protein, protein conjugates, and peptide and protein mixtures.

Supplementary Figure 3 C286W mutation impairs MCL-1ΔNΔC protection of BID BH3 from deuterium exchange.

(a-d) MS spectra of BID BH3 peptide at (a) 0, (b) 10, (c) 20, and (d) 40 seconds for peptide alone (black) or the peptide combined with MCL-1ΔNΔC (red) or MCL-1ΔNΔC C286W (purple).

Supplementary Figure 4 Microscopic images of MEFs subjected to serum withdrawal.

The indicated MEFs were maintained in (a) full media or (b) serum-free media for 16 h, followed by phase contrast microscopic imaging. Exemplary apoptotic bodies are indicated by the black arrows. Bar, 25 μm.

Supplementary Figure 5 C286S mutagenesis does not affect the cell-viability response to serum withdrawal of MCL-1-reconstituted MEFs.

(a) Western blot of lysates from Mcl-1-/- MEFs reconstituted with comparable levels of human, full-length, wild-type or C16S,C286S mutant MCL-1. See also Supplementary Data Set 1. (b) Cell viability (48 h) for the indicated MEFs subjected to serum withdrawal. Data are normalized to the corresponding MEFs maintained in complete media. Error bars are mean ± s.d. (n = 3 technical replicates). *, p <0.01 by two-tailed student’s t test; n.s., not statistically significant.

Supplementary Figure 6 Comparison of human and mouse structures of MCL-1 in the C286 and F267 region.

(a-c) An overlay of the unliganded human (grey, PDB ID 2MHS) and mouse (blue, PDB ID 1WSX) forms of MCL-1 demonstrate a similar structural disposition of C286 and F267 (a), with C286 (orange) pointing toward the surface (b) and F267 (green) juxtaposed to a confluence of internal hydrophobic residues (yellow) (c). (d-f) In contrast, an overlay of BH3-liganded forms of human (grey, PDB ID 2PQK) and mouse (blue, PDB ID 2ROC) MCL-1 demonstrate differential orientations of C286 and F267 (d), with the location of C286 (orange) shifted within a relatively more unfolded region of the protein but still pointing toward the surface (e), and F267 (green) slightly torqued but with similar orientation and adjacency to internal hydrophobic residues (yellow) (f).

Supplementary Figure 7 Comparative BH3-based pulldown of MCL-1 and MCL-1 C286W from reconstituted Mcl1–/– MEFs.

(a-b) Western blots of streptavidin pull-downs from lysates of the indicated MEFs treated with biotinylated MCL-1 SAHB D , a selective stapled BH3 inhibitor of MCL-1 (left). See also Supplementary Data Set 1. The relative amount of precipitated MCL-1 vs. MCL-1 C286W was quantitated by densitometry of immunoreactive MCL-1 bands in the pull-down as ratioed to input control (right). Two independent streptavidin pull-down (SAP) experiments from treated lysates of the MEF cultures are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1377 kb)

Supplementary Data Set 1

MCL-1 and actin western blot analyses (PDF 194 kb)


Molecular dynamics simulation of wild-type MCL-1 (MOV 13053 kb)


Molecular dynamics simulation of MAIM1-derivatized MCL-1 (MOV 12245 kb)

MCL-1 C286W

Molecular dynamics simulation of C286W-mutant MCL-1 (MOV 11396 kb)

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Lee, S., Wales, T., Escudero, S. et al. Allosteric inhibition of antiapoptotic MCL-1. Nat Struct Mol Biol 23, 600–607 (2016).

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