Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer
- University of Washington, United States
- University of Illinois, United States
- Fred Hutchinson Cancer Research Center, United States
- LaTrobe Institute for Molecular Science, Australia
- Olivia Newton-John Cancer and Wellness Centre, Australia
- La Trobe University, Australia
- The Walter and Eliza Hall Institute of Medical Research, Australia
- University of Melbourne, Australia
Figures
Figure 1 with 1 supplement
Schematic of BCL2 family interactions.
BCL2 proteins are categorized by their net effect on cell fate and the presence of shared structural domains. BH3-only proteins (BOPs) are sequestered by pro-survival homologs (labels 1 and 2), and …
Figure 1—figure supplement 1
Design strategy.
BINDI, a de novo three-helix bundle inhibitor of BHRF1, was employed as a scaffold protein to engineer altered specificities toward each of six human pro-survival proteins. BINDI was first …
Figure 2 with 1 supplement
Design of specific inhibitors for each of the six human pro-survival BCL2 homologs.
On and off rates were determined by BLI with multiple-concentration binding titrations for each computationally designed protein (A–F) and optimized variants (G–K; mean ± SD; n = 3). On-target …
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Figure 2—source data 1
Source data relating to Figure 2A–M and Figure 2—figure supplement 1H.
Biolayer interferometry data fit to a 1:1 binding model using ForteBio analysis software. Three separate binding titrations (n = 3) were completed for each binding pair, except when negligible signal was detected at high analyte concentrations (indicated in file where applicable). Figure 2—source data 1. Source data relating to Figure 2—figure supplement 1I. CD data collected for chemical denaturation experiments in guanidinium hydrochloride from 0 to 6 M.
- https://doi.org/10.7554/eLife.20352.005
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Figure 2—source data 2
Source data relating to Figure 2—figure supplement 1I.
CD data collected for chemical denaturation experiments in guanidinium hydrochloride from 0 to 6 M.
- https://doi.org/10.7554/eLife.20352.006
Figure 2—figure supplement 1
Computational design and screening methods.
(A) BINDI (gray) was docked to the hydrophobic binding groove of Bcl-xL (red) by alignment to a bound BH3 peptide (not shown). The docked pose undergoes successive rounds of design with ROSETTA, in …
Figure 3 with 1 supplement
The crystal structure of αMCL1•Mcl-1 is very close to the design model.
(A) Alignment of the computational design model M-CDP04 (gray cartoon) and crystal structure (Mcl-1, purple surface; αMCL1, pink cartoon). (B) Buried contact surfaces on Mcl-1 bound to a BH3-like …
Figure 3—figure supplement 1
Structural analysis of the αMCL1•Mcl-1 complex via lysine-specific chemical cross-linking.
Cross-linking studies of αMCL1 bound to Mcl-1 were consistent with the computational design model and crystal structure. The protein complex was incubated with three different cross-linking agents; …
Figure 4
Comparison of design sequences with BH3-mimetic peptides and natural BH3 motifs.
(A) Sequences of optimized inhibitors are aligned, excluding αBCLW, which binds to Bcl-w using a shifted interaction surface. The BH3-mimetic region of designed inhibitors is compared to natural BH3 …
Figure 5 with 3 supplements
Analysis of computational design success.
(A) Deep sequencing analysis of the naïve and sorted 2-CDP06 SSM library enabled quantitative analysis of the fitness of each single amino acid substitution for specificity and affinity toward …
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Figure 5—source data 1
Source data relating to Figure 5 and Figure 5—figure supplement 1.
Enrichment ratios of all SSM mutants, calculated from deep sequencing of naïve and sorted populations of SSM libraries based on the indicated CDP. Raw data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus repository with accession number GSE80194.
- https://doi.org/10.7554/eLife.20352.012
Figure 5—figure supplement 1
Sequence analysis of SSM libraries.
(A) SSM libraries were generated based on the indicated CDPs and sorted for high affinity and specificity to each target BCL2 homolog. Sequence fitness landscapes show enriched mutations (blue) that …
Figure 5—figure supplement 2
Computational docking calculations: CDPs.
CDPs were computationally docked into the canonical binding groove of each pro-survival BCL2 homolog, sampling both local (close to the input bound conformation) and global (entire protein surface) …
Figure 5—figure supplement 3
Computational docking calculations: optimized inhibitors.
Optimized, specific inhibitors were computationally docked into the canonical binding groove of each pro-survival BCL2 homolog, sampling both local (close to the input bound conformation) and global …
Figure 6 with 1 supplement
Determinants of binding specificity.
αMCL1•Mcl-1 and αBCL2•Bcl-2 crystal structures (upper panels, high complementarity) and non-cognate binding pairs modeled in Rosetta (lower panels, poor complementarity) were aligned. For select …
Figure 6—figure supplement 1
The crystal structure of the αBCL2•Bcl-2 complex.
(A) Alignment of the two non-crystallographic symmetry (NCS)-related Bcl-2 molecules show the binding modes of the two NCS-related αBCL2 molecules differ slightly (1.9 Å RMSD). (B) Alignment of …
Figure 7 with 1 supplement
Designed inhibitors induce apoptosis in vitro by engaging the BH3-binding grooves of specific pro-survival homologs.
(A) Western blot for cytochrome c in pelleted (P) and soluble (S) fractions of engineered MEFs after permeabilization and treatment with 10 mM BCL2 inhibitors. Bim-BH3, which binds all pro-survival …
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Figure 7—source data 1
Source data relating to Figure 7B and Figure 7—figure supplement 1A.
(A) Survival assays for WT and modified HeLa cell lines after treatment with the indicated inhibitors or inhibitor combinations. Raw data have been normalized to the negative control (empty virus). (B) Long-term survival assays for engineered MEFs with indicated BCL2 dependency, after inducing expression of αMCL1 or αBFL1. All values have been normalized to uninduced controls.
- https://doi.org/10.7554/eLife.20352.019
Figure 7—figure supplement 1
Long-term MEF survival and HeLa co-immunoprecipitation studies.
(A) Long-term survival of engineered MEFs (pro-survival protein dependence as indicated) was assayed by counting colonies after seven to ten days of doxycycline-induced expression of αMCL1 or αBFL1 …
Figure 8
Determination of functional BCL2 profiles in melanoma and glioblastoma cell lines.
(A) Melanoma and (B) glioblastoma cell lines were transduced with constructs for designed inhibitor expression and viability was assayed after 72 hr (mean ± SD; for melanoma, n = 2 to 4; for …
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Figure 8—source data 1
Source data relating to Figure 8.
Survival assays for melanoma and glioblastoma. For each experiment, three technical replicates were averaged and normalized to the negative control (empty virus). The average and standard deviation were calculated using these values from independent experiments (experimental replicates).
- https://doi.org/10.7554/eLife.20352.022
Figure 9 with 1 supplement
Determination of functional BCL2 profiles in colon cancer cell lines.
(A) Colon cancers were treated with small molecule drugs (2 µM) and/or doxycycline to induce expression of designed inhibitors, as indicated, and viability was assayed after 24 hr (mean ± SD; n = …
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Figure 9—source data 1
Source data relating to Figure 9 and Figure 9—figure supplement 1.
Short- and long-term survival assay for colon cancers. For short-term assays, all values have been normalized to the negative control (DMSO in media, equivalent to DMSO concentration diluted from small molecule stock solution). For long-term survival assays, all values have been normalized to uninduced (no doxycycline) negative control.
- https://doi.org/10.7554/eLife.20352.024
Figure 9—figure supplement 1
Drug titrations and long-term survival assays in colon cancers.
(A) Drug titrations for EC50 determination of ABT-263 and A-1331852 in colon cancer lines, with (dotted lines) and without (solid lines) expression of αMCL1 (mean ± SD, n = 3). (B) Long-term …
Additional files
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Supplementary file 1
Data tables.
(A) Summary of computational designs selected for protein production and biochemical analysis. (B) Sequences of computational designs and optimized variants. (C) Crystallographic data collection and refinement statistics. (D) Protein cross-linking of the αMCL1-Mcl-1 complex. (E) Sort conditions for all in vitro evolution experiments. (F) Mutation summary for evolved variants.
- https://doi.org/10.7554/eLife.20352.026
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Supplementary file 2
CDP design models.
PDB models of all computationally designed proteins (CDPs). Please see Supplementary file 1A for descriptions and computational statistics.
- https://doi.org/10.7554/eLife.20352.027
