1. Biochemistry and Chemical Biology
  2. Neuroscience
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Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models

  1. M Catarina Silva
  2. Fleur M Ferguson
  3. Quan Cai
  4. Katherine A Donovan
  5. Ghata Nandi
  6. Debasis Patnaik
  7. Tinghu Zhang
  8. Hai-Tsang Huang
  9. Diane E Lucente
  10. Bradford C Dickerson
  11. Timothy J Mitchison
  12. Eric S Fischer
  13. Nathanael S Gray  Is a corresponding author
  14. Stephen J Haggarty  Is a corresponding author
  1. Massachusetts General Hospital, Harvard Medical School, United States
  2. Dana-Farber Cancer Institute, United States
  3. Harvard Medical School, United States
Research Article
Cite this article as: eLife 2019;8:e45457 doi: 10.7554/eLife.45457
16 figures, 1 table and 5 additional files

Figures

Figure 1 with 2 supplements
Design and working model for a new hetero-bifunctional tau degrader.

(A) A degrader molecule was designed to preferentially recognize disease-associated forms of tau (Module 1), and simultaneously engage with CRBN in the CRL4CRBN E3 ubiquitin ligase complex (Module 3). (B) Degrader-mediated association of tau with CRL4CRBN and formation of a ternary complex is predicted to mediate tau ubiquitination and degradation by the proteasome. (C) QC-01–175 (I) was synthesized based on the T807 core scaffold for tau recognition, a thalidomide analog E3 ligand (pomalidomide) for CRBN engagement, and a linker designed for maximum target clearance efficiency (II). QC-03–075 is the inactive analog (III). (D) BLI Streptavidin (SA) biosensor assay to measure recombinant tau protein affinity to small molecules (e.g. T807, QC-01–175). (E) BLI results indicate that, in vitro, QC-01–175 binds to variant forms of tau within the same order of magnitude as T807. Bars represent mean KD (μM)±SD (n ≥ 3). Figure 1—figure supplement 1 shows representative BLI sensograms and steady-state graphs for tau WT and each variant affinity to QC-01–175 and control compounds, with respective KD values. Figure 1—figure supplement 2 shows QC-01–175 effect on monoamine oxidase (MAO) activity. The following figure supplements are available for Figure 1.

https://doi.org/10.7554/eLife.45457.002
Figure 1—figure supplement 1
In vitro characterization of tau-binding affinity to QC-01–175 and control compounds.

(A) Association-dissociation curves for BLI biosensor measurements (64 μM compound is shown, except for PE859 at 50 μM) and (B) BLI steady-state graphs, representing human recombinant biotinylated-tau WT (I-III), A152T (IV-VI) and P301L (VII-IX) in vitro binding affinity to compounds PE859 (I, IV, VII), T807 (II, V, VIII) and QC-01–175 (III, VI, IX). Representative curves of n ≥ 3 are shown.

https://doi.org/10.7554/eLife.45457.003
Figure 1—figure supplement 2
QC-01–175 effect on MAO activity.

(A) Monoamine oxidase activity assay shows that the inhibitory effect of T807 is greater than that of QC-01–175. Parnate is a known MAO inhibitor (positive control) and pomalidomide is used as a negative control. Data points represent mean values of % of MAO inhibition ± SD (n = 3). (B) IC50 values extrapolated from A.

https://doi.org/10.7554/eLife.45457.004
Figure 2 with 2 supplements
Concentration effect of QC-01–175 

(A) on tau protein levels of A152T and control neurons. Analysis of total tau (TAU5) and phospho-tau (S396 P-tau) levels upon treatment by western blot (B) and ELISA (C). Analysis of total tau (TAU5) and phospho-tau (S396 P-tau) levels upon treatment with the negative control QC-03–075 (D), by western blot (E) and ELISA (F). Representative western blots are shown (B, E) with mean densitometry quantification (bands corresponding to brackets)±SEM (n = 3). (C, F) For ELISA, data points represent mean tau levels (μg of total protein) normalized to vehicle-treated ± SEM (n = 4). Both assays show QC-01–175 dose-dependent effect on tau levels, with QC-03–075 minimal effect (~10%). (G) IF of A152T neurons treated with vehicle or 10 μM compound, immuno-probed for total tau (K9JA, red), P-tau (PHF-1, red) and the neuronal marker MAP2 (green), scale bar 50 μm. (H–J) Tau ELISA of control neurons treated with QC-01–175, which did not show a dose-dependent effect. (H) 8330–8-RC1 line; (I) MGH2069-RC1 line; (J) CTR2-L17-RC2 line. Data points represent mean tau levels (μg of total protein) normalized to vehicle-treated ± SEM (n = 3). All neurons were differentiated for 6 weeks and treated with compound for 24 hr. Student T-test between each concentration and vehicle-treated tau levels nsp> 0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 2—figure supplement 1 depicts the variability of degrader effect across biological replicates, by western blot, with an overall 60% to 90% efficacy. Figure 2—figure supplement 2 shows degrader effect in P301L neurons and compares concentration effect across all lines. Figure 2—source data 1 summarizes the information pertaining to each cell line included in this study. Figure 2—source data 2 includes all ELISA data. The following figure supplements are available for Figure 2.

https://doi.org/10.7554/eLife.45457.005
Figure 2—source data 1

Human neural cell lines derived from tauopathy-affected (progressive supranuclear palsy, PSP or behavioral variant of FTD, bvFTD) and age-matched unaffected individuals, and MAPT KO line employed in this study.

± refers to heterozygous variant carriers.

https://doi.org/10.7554/eLife.45457.008
Figure 2—source data 2

Numerical description and statistics for data presented in Figure 2 and respective supplement 2 ELISAs.

https://doi.org/10.7554/eLife.45457.009
Figure 2—figure supplement 1
Variability of the effect of QC-01–175 across biological replicates.

(A– I) Western blot analysis of total tau (TAU5) and S396 P-Tau protein, upon 24 hr treatment with QC-01–175 or the negative controls lenalidomide, T807 and QC-03–075. Assessment of the variability in tau clearance across multiple concentrations and nine biological replicates. Red brackets indicate protein bands that were quantified by densitometry. Experiments done with A152T neurons differentiated for 6 weeks, except for (C, E) where age-matched non-mutant controls (1, 8330–8-RC1 and 2, MGH2069-RC1) were included for comparison. (J) Scatter plot of western blot densitometry, for A152T neurons treated with QC-01–175 (n = 9) or the respective negative controls (n ≥ 5), for 24 hr. Lines represent mean tau densitometry ± SD, relative to vehicle-treated. Student T-test for each concentration/antibody relative to vehicle-treated, nsp>0.05, *p≤0.01, **p≤0.001, ***p≤0.0001. Some blot images were cropped for the purpose of this figure only, to exclude samples not included in this manuscript (Silva and Ferguson, Manuscript in preparation, 2019), but all samples in each set were run in the same gel.

https://doi.org/10.7554/eLife.45457.006
Figure 2—figure supplement 2
Demonstration of QC-01–175 effect in tau-P301L neurons.

(A-B) Concentration effect of QC-01–175 and QC-03–075 on total tau (TAU5) and S396 P-Tau by ELISA, in P301L neurons at 6 weeks of differentiation. Data points represent mean values of tau normalized to total μg of protein, relative to vehicle-treated ± SD (n = 3). Student T-test between each concentration and vehicle-treated tau levels nsp> 0.05, *p<0.05, ***p<0.001, ****p<0.0001. (C-E) Overview of concentration effect of QC-01–175 24 hr treatment on (C) total tau and (D) S396 P-Tau across all genotypes. Control (1) 8330–8-RC1, control (2) MGH2069-RC1, control (3) CTR2-L17-RC2. Data points represent mean tau levels by ELISA, normalized to total μg of protein and relative to vehicle-treated ± SD (n ≥ 3). (E) Two-Way ANOVA statistical analysis for (C) and (D).

https://doi.org/10.7554/eLife.45457.007
Figure 3 with 1 supplement
Mechanism of QC-01–175 clearance of tau is CRL4CRBN and UPS-dependent.

Neurons were pre-treated for 6 hr with (A) either CRBN ligand excess lenalidomide or tau ligand excess T807, (B) the NAE inhibitor MLN4924, the autophagy inhibitor Baf.A1, or (C) the proteasome inhibitor carfilzomib; followed by 18 hr treatment with QC-01–175 (or negative control QC-03–075), for a total of 24 hr. Total (TAU5) and P-tau S396 levels were analyzed by western blotting. (A–C) Representative blots are shown. (D–F) Densitometry bars represent tau mean intensity values ± SD (n = 3), relative to vehicle-treated samples. Student T-test of QC-01–175 samples relative to vehicle treated, and the remainder bars show p-value of each pre-treatment relative to QC-01–175 to assess rescue of clearance effect (***p<0.001, **p<0.01, *p<0.05, nsp>0.05). A152T neurons were differentiated for 6 weeks. Figure 3—figure supplement 1 includes additional specificity controls for A152T, P301L and control neuronal models. The following figure supplement is available for Figure 3.

https://doi.org/10.7554/eLife.45457.010
Figure 3—figure supplement 1
Additional specificity controls for QC-01–175-mediated tau clearance.

(A–C) Western blot and densitometry analysis of total tau, S396 P-tau and CRBN upon 24 hr treatment with QC-01–175 or the negative controls QC-03–075, T807 and a thalidomide analog, lenalidomide (1 μM), in (A) A152T, (B) P301L or (C) control-1 8330–8-RC1 neurons. Cropped lanes correspond to samples not included in this manuscript (Silva and Ferguson, Manuscript in preparation, 2019), but all samples in each set were run in the same gel. Densitometry graph bars represent tau or CRBN mean intensity levels ± SD (n = 3), relative to vehicle-treated samples. (D) QC-01–175 had minimal effect on CRBN upon 24 hr treatment. Student T-test of compound-treated samples relative to vehicle-treated ***p<0.001, **p<0.01, *p<0.05, nsp>0.05.

https://doi.org/10.7554/eLife.45457.011
Demonstration of ternary complex formation in A152T neurons upon QC-01–175 treatment, by co-IP and western blot analysis.

Neurons (6-week differentiated) were treated for 4 hr with 1 μM QC-01–175 ± 30 min pre-treatment with proteasome inhibitors (carfilzomib or bortezomib at 5 μM), with the goal of capturing maximum molecular interactions at 4 hr and halting tau clearance. QC-03–075 is a negative control for CRBN binding. (A) Co-IP by DDB1 pulldown and detection of tau in the complex by probing for total tau (TAU5), P-tauS396/S404 (PHF-1), and ubiquitinated proteins (Ubi-1). (B) Co-IP by tau pulldown (TAU5) and detection of CRL4CRBN subunits CRBN, CUL4A and DDB1. Western blot of total tau (K9JA) was used as a control. (C) Control western blot analysis with 3% (10 μg) of IP input confirms the effect of QC-01–175 ± proteasome inhibitors on tau and P-tau S396. Red arrows and brackets indicate the predicted bands for each immunoprobed protein (n = 3).

https://doi.org/10.7554/eLife.45457.012
Figure 5 with 1 supplement
Comparative analysis of the effect of QC-01–175 at (A)

0.01 μM, (B) 0.1 μM, (C) 1 μM, and (D) 10 μM after 4 hr, 8 hr or 24 hr of treatment. Graph bars represent mean levels of total tau (TAU5) and S396 P-tau protein measured by ELISA, normalized to total μg of protein and to vehicle-treated samples ± SEM (n = 3), in A152T 6-week differentiated neurons. Student T-test for each dose/time is relative to vehicle-treated tau levels nsp> 0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 5—figure supplement 1 shows concentration effect curves for QC-01–175 at 4 hr and 8 hr, for all doses tested in A152T and P301L neurons; as well as the 4 hr effect seen by western blot. Figure 5—source data 1 includes all values plotted in the main Figure and supplement. The following figure supplement is available for Figure 5.

https://doi.org/10.7554/eLife.45457.013
Figure 5—source data 1

Numerical description and statistics for data presented in Figure 5 and respective supplement 1.

https://doi.org/10.7554/eLife.45457.014
Figure 5—figure supplement 1
Degrader concentration and time effect on tau, in A152T and P301L neurons.

(A–C) Concentration and time effect of QC-01–175 on total tau (TAU5) and S396 P-tau levels, in A152T 6-week differentiated neurons, treated for (A) a short 4 hr interval, (B) an intermediate 8 hr interval, (C) compared to 24 hr treatment. (D) Concentration effect of QC-01–175 4 hr treatment in A152T neurons, by western blot analysis of total tau (TAU5) and P-tau S396 (representative blot is shown) with mean densitometry quantification (bands corresponding to brackets)±SEM (n = 3). (F–H) Concentration and time effect of QC-01–175 on total tau (TAU5) and S396 P-tau levels, in P301L 6-week differentiated neurons treated for (F) 4 hr, (G) 8 hr and (H) 24 hr. (A-C, F-H) Data points represent mean levels of tau protein measured by ELISA, normalized to total μg of protein and to vehicle-treated ± SEM (n = 3). Student T-test between each dose and vehicle-treated tau levels nsp>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.45457.015
Mass spectrometry-based proteomics to quantify the effect of QC-01–175 treatment on the proteome of A152T neurons.

6-week differentiated neurons were treated for 4 hr with (A) 1 μM of QC-01–175, (B) 1 μM of the negative control QC-03–075, or (C) 10 μM MLN4924 (NAE inhibitor, 30 min pre-treatment) and 1 μM of QC-01–175. Upon degrader QC-01–175 treatment (A), three off-targets were detected within statistical significance, which all belong to known IMiD targets, an effect rescued by the negative control (B) or inhibition of neddylation by MLN4924 (C). Significant hits were assessed by moderated t-test as implemented in the limma package (Ritchie et al., 2015), with the log2 fold change shown on the y-axis, and negative log10 P values on the x-axis (n = 3 for treatment with DMSO, QC-01–175, and QC-03–075, and n = 2 for QC-01–175 + MLN4924).

https://doi.org/10.7554/eLife.45457.016
QC-01–175 treatment rescued stress vulnerability of A152T neurons.

(A) Aβ(1-42) proteotoxicity causes concentration- and genotype-dependent loss of neuronal vulnerability, affecting preferentially A152T and P301L neurons, with a rescue by MAPT KO. Data points represent mean viability relative to vehicle-treated neurons (100%)±SEM (n ≥ 3); two-way ANOVA statistical analysis relative to non-mutant control-1 neurons (black curve, 8330–8-RC1), ****p<0.0001, nsP > 0.05. (B) Assay overview to measure effect of the stressor Aβ(1-42) on neuronal viability (c) and potential rescue by pre-treatment with QC-01–175 (b). Effect of 24 hr treatment with QC-01–175 alone was also tested (a). (C) QC-01–175 (light blue-stripe bar) but not the negative control QC-03–075 (dark blue-stripe bar), rescued viability loss caused by Aβ(1-42) (white-stripe bar) in A152T neurons differentiated for 8 weeks, in a comparable manner to genetic MAPT knockout (black-stripe bar). Graph bars represent mean % viability ± SD, relative to vehicle-treated (white) neurons. T-test ***p≤0.001; nsp>0.05 (n = 3). Figure 7—source data 1 includes all values plotted in main Figure 7A and C.

https://doi.org/10.7554/eLife.45457.017
Figure 7—source data 1

Numerical description and statistics for data presented.

https://doi.org/10.7554/eLife.45457.018
Synthesis route for the tau degrader QC-01-175.
https://doi.org/10.7554/eLife.45457.019
Chemical structure 1
3-(4-(4-nitropyridin-3-yl)phenyl)propan-1-ol (3)
https://doi.org/10.7554/eLife.45457.020
Chemical structure 2
3-(4-(3-((tert-butyldiphenylsilyl)oxy)propyl)phenyl)-4-nitropyridine (4)
https://doi.org/10.7554/eLife.45457.021
Chemical structure 3
7-(3-((tert-butyldiphenylsilyl)oxy)propyl)-5H-pyrido[4,3-b]indole (5)
https://doi.org/10.7554/eLife.45457.022
Chemical structure 4
tert-butyl 7-(3-((tert-butyldiphenylsilyl)oxy)propyl)-5H-pyrido[4,3-b]indole-5-carboxylate (6)
https://doi.org/10.7554/eLife.45457.023
Chemical structure 5
tert-butyl 7-(3-hydroxypropyl)-5H-pyrido[4,3-b]indole-5-carboxylate (7)
https://doi.org/10.7554/eLife.45457.024
Chemical structure 6
3-(5H-pyrido[4,3-b]indol-7-yl)propanoic acid (10)
https://doi.org/10.7554/eLife.45457.025
Chemical structure 8
QC-03-075: Was prepared according to Scheme 1.
https://doi.org/10.7554/eLife.45457.027

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (H. sapiens)8330–8-RC1Silva et al. (2016)
Stem Cell Reports.
Figure 2—source data 1.
Human iPSC-derived NPC
line, non-mutant tau.
Original fibroblasts GM08330
from Coriell Institute for
Medical Research.
Cell line (H. sapiens)MGH2069-RC1Seo et al. (2017) J.
Neuroscience. Manuscript
in preparation.
Figure 2—source data 1.
Human iPSC-derived NPC
line, non-mutant tau. Original
fibroblasts MGH-2069 from
Massachusetts General Hospital
Frontotemporal Dementia Clinic,
Massachusetts General
Hospital Neurodegeneration
Repository.
Cell line (H. sapiens)CTR2-L17-RC2Almeida et al., 2012 Cell
Reports. Silva et al. (2016)
Stem Cell Reports
Figure 2—source data 1. Human
iPSC-derived NPC line,
non-mutant tau.
Cell line (H. sapiens)FTD19-L5-RC6Silva et al. (2016)
Stem Cell Reports
Figure 2—source data 1.
Human iPSC-derived NPC
line, tau-A152T (NCBI
RefSeq NM_001123066;
rs143624519).
Cell line (H. sapiens)FTD19-L5-RC6;MAPT-KOSilva et al. (2016)
Stem Cell Reports
Figure 2—source data 1.
Human iPSC-derived NPC
FTD19-L5-RC6 line,
CRISPR/Cas9-engineered
MAPT knockout.
Cell line (H. sapiens)MGH2046-RC1Seo et al. (2017) J.
Neuroscience. Manuscript
in preparation.
Figure 2—source data 1.
Human iPSC-derived NPC
line, tau-P301L (NCBI
RefSeq NM_001123066;
rs63751273). Original
fibroblasts MGH-2046 from
Massachusetts General
Hospital Frontotemporal
Dementia Clinic, Massachusetts
General Hospital
Neurodegeneration
Repository.
AntibodyTAU5InvitrogenCat. AHB0042
RRID:AB_2536235
WB 1:1000
AntibodyTAU5AbCamCat. ab80579
RRID:AB_1603723
Co-IP
AntibodyTau K9JADAKO, AgilentCat. A002401-2IF 1:1000, WB 1:10,000
AntibodyP-Tau S396InvitrogenCat. 44752G
RRID:AB_1502108
WB 1:1000
AntibodyP-Tau PHF-1Dr. Peter DaviesAlbert Einstein
College of Medicine, NY
IF 1:400
AntibodyMAP2Chemicon,
Millipore
Cat. AB5543
RRID:AB_571049
IF 1:1000
AntibodyDDB1AbCamCat. ab109027
RRID:AB_10859111
WB 1:50,000/Co-IP
AntibodyCUL4ACell Signaling
Technology
Cat. 2699
RRID:AB_2086563
WB 1:1000
AntibodyCRBNProteinTechCat. 11435–1-AP
RRID:AB_2085739
WB 1:500
AntibodyUbiquitin, Ubi-1MilliporeCat. MAB1510
RRID:AB_2180556
WB 1:500
Antibodyβ-ActinSigma-AldrichCat. A1978
RRID:AB_476692
WB 1:10,000
AntibodyGAPDHAbCamCat. ab8245
RRID:AB_2107448
WB 1:5000
AntibodyAlexaFluor-488 2°
antibody
Life TechnologiesCat. A11039
RRID:AB_142924
IF 1:500
AntibodyAlexaFluor-594 2°
antibody
Life TechnologiesCat. A11012
RRID:AB_141359
IF 1:500
AntibodyAlexaFluor-594 2°
antibody
Life TechnologiesCat. A11032
RRID:AB_141672
IF 1:500
AntibodyAnti-mouse IgG,
HRP-linked
Cell Signaling
Technology
Cat. 7076S
RRID:AB_330924
Western blotting, 1:4000
AntibodyAnti-rabbit IgG,
HRP-linked
Cell Signaling
Technology
Cat. 7074S
RRID:AB_2099233
Western blotting, 1:4000
AntibodyHoechst 33342InvitrogenCat. H3570IF Nuclear stain, 1:1000
Peptide, Recombinant
Protein
Tau-441(WT),
Biotinylated
SignalChemCat. T08-54BN
Lot. H2681-10
Human recombinant
protein expressed in
E. coli cells.
Peptide, Recombinant
Protein
Tau-441(A152T) ProteinSignalChemCat. T08-56VN
Lot. B2157-7
Human recombinant
protein expressed in
E. coli cells, tag-free.
Accession no. P10636-8.
Peptide, Recombinant
Protein
Tau-441(P301L) ProteinSignalChemCat. T08-56FN
Lot. O917-2
Human recombinant
protein expressed in
E. coli cells, tag-free.
Accession no. P10636-8.
Peptide, Recombinant
Protein
Aβ(1-42)Enzo LifesciencesCat. ALX-151–002CAS No. 107761-42-2
Commercial Assay, KitEZ-Link
NHS-PEG4-Biotinylation Kit
Thermo Fisher
Scientific
Cat. 21455
Commercial Assay, KitMAO-Glo Assay KitPromegaCat. V1401
Commercial Assay, KitPierce BCA Protein
Assay Kit
Thermo Fisher
Scientific
Cat. 23227
Commercial Assay, KitHuman Total Tau ELISAInvitrogenCat. KHB0041
Commercial Assay, KitP-Tau[pS396] Human
ELISA
InvitrogenCat. KHB7031
Commercial Assay, KitELISA Compatible
Lysis Buffer
InvitrogenCat. FNN0011
Commercial Assay, KitImmunoprecipitation
Kit Dynabeads Protein G
Novex, Life
Technologies
Cat. 10007D
Commercial Assay, KitPierce IP Lysis BufferThermo Fisher
Scientific
Cat. 87787
Commercial Assay, KitTandem mass tag (TMT) reagentsThermo Fisher
Scientific
Cat. A34807
Commercial Assay, KitAlamarBlue Cell
Viability Reagent
Thermo Fisher
Scientific
Cat. DAL1025
Chemical Compound, DrugT807 (AV-1451)MedChem ExpressCat. HY-101184CAS No. 1415379-56-4
Chemical Compound, DrugT807 core scaffoldThis paper(Intermediate 10)Methods, Synthetic
methods general
protocols. Figure 1C.
Chemical Compound, DrugPomalidomideSigma AldrichCat. P0018CAS No. 19171-19-8
Chemical Compound, DrugLenalidomideSigma AldrichCat. 901558CAS No. 191732-72-6
Chemical Compound, DrugQC-01–175This paperMethods, Synthetic
methods general
protocols. Figure 1C.
Chemical Compound, DrugQC-03–075This paperMethods, Synthetic
methods general
protocols. Figure 1C.
Chemical Compound, DrugMLN4924MedChem ExpressCat. HY-70062CAS No. 905579-51-3
Chemical Compound, DrugBafilomycin A1Enzo LifeSciencesCat. BML-CM110CAS No. 88899-55-2
Chemical Compound, DrugCarfilzomibMedChem ExpressCat. HY-10455CAS No. 868540-17-4
Chemical Compound, DrugBortezomibSelleckchemCat. S1013CAS No. 179324-69-7
Chemical Compound, DrugPE859MedChem ExpressCat. HY-12662CAS No. 1402727-29-0
Chemical Compound, DrugParnate
(Tranylcypromine)
Sigma-AldrichCat. P8511CAS No. 1986-47-6
Chemical Compound, DrugProtease inhibitor
cocktail
RocheCat. 04 693 124 001
Chemical Compound, DrugPhosphatase
inhibitor cocktail 2
Sigma-AldrichCat. P5726
Software, AlgorithmData Acquisition HT 11.0ForteBio
(www.fortebio.com/octet-software.html)
Version 11
(BLI Analysis and
KD calculation)
Software, AlgorithmAdobe Photoshop CS5Adobe Photoshop
(www.adobe.com/Photoshop)
Version 12.0.4
(Histogram function,
western blots densitometry)
Software, AlgorithmGraphPad PrismGraphPad Prism
(www.graphpad.com)
Version 8
Software, AlgorithmProteome Discoverer 2.2Thermo Fisher ScientificRRID:SCR_014477Version 2.2
Software, AlgorithmR frameworkTeam RCR: A Language
and Environment for
Statistical Computing
http://www.R-project.org/;
accessed Nov. 1, 2017
R Version 3.5.1
– Feather Spray
Software, AlgorithmStatistical Analysis
Limma Package
(R framework)
BioconductorRitchie et al. (2015)
Nucleic Acids Res.
OtherOctet Red384 InstrumentForteBiohttps://www.fortebio.com/octet-red384.html
OtherIN Cell Analyzer 6000 Cell
Imaging System
GE Healthcare Life
Sciences
OtherEnVision Multilabel
Plate Reader
Perkin Elmer
OtherHPLCWaters 2489/2545
OtherUPLCWaters Aquity I UPLC
OtherHPLCAgilent 1260 Infinity II
LC System
OtherOrbitrap Fusion
Lumos mass spectrometer
Thermo Fisher ScientificIQLAAEGAAPFADBMBHQ
OtherProxeon EASY-nLC 1200
LC pump
Thermo Fisher ScientificLC140
OtherEasySpray ES803 75 μm
inner diameter
microcapillary column
Thermo Fisher ScientificES803

Additional files

Supplementary file 1

1H NMR Spectra of QC-01-175.

https://doi.org/10.7554/eLife.45457.028
Supplementary file 2

1H NMR Spectra of QC-03-075.

https://doi.org/10.7554/eLife.45457.029
Supplementary file 3

UPLC chromatogram and mass spectra of QC-01-175.

https://doi.org/10.7554/eLife.45457.030
Supplementary file 4

UPLC chromatogram and mass spectra of QC-03-075.

https://doi.org/10.7554/eLife.45457.031
Transparent reporting form
https://doi.org/10.7554/eLife.45457.032

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