Supplemental Information Validation of the Hsp70 …...2015/01/07 · Supplemental Information...
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Supplemental Information Validation of the Hsp70-Bag3 Protein-Protein Interaction as a Potential Therapeutic Target in Cancer Xiaokai Li 1 , Teresa Colvin 2 , Jennifer N. Rauch 1 , Martin Kampmann 3 , Diego Acosta-Alvear 3 , Bryan Dunyak 1 , Byron Hann 4 , Blake T. Aftab 5 , Min Cho 3 , Peter Walter 3 , Jonathan S. Weissman 3 , Michael Y. Sherman 2 , Jason E. Gestwicki 1 Supplemental Figure 1. Chemical structures of the compounds used in this study and characterization of JG98-biotin by NMR and MS/MS. Characterization of JG98-biotin 1 H NMR (400 MHz, DMSO) δ 8.32 (t, J = 6.0 Hz, 1H), 8.26 (d, J = 4.1 Hz, 1H), 8.07 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 4.0 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.56 (dd, J = 8.9, 2.2 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 6.55 (s, 1H), 6.39 (s, 1H), 6.33 (s, 1H), 5.73 (s, 2H), 4.33 – 4.27 (m, 1H), 4.22 (d, J = 5.9 Hz, 2H), 4.14 – 4.03 (m, 6H), 3.11 – 3.04 (m, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.11 (t, J = 7.4 Hz, 2H), 1.66 – 1.38 (m, 4H), 1.37 – 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 3H). ESI-MS: Calculated for C 35 H 38 ClN 6 O 3 S 4 + 753.16, found 753.11.
Transcript of Supplemental Information Validation of the Hsp70 …...2015/01/07 · Supplemental Information...
Supplemental Information
Validation of the Hsp70-Bag3 Protein-Protein Interaction as a Potential Therapeutic Target in Cancer
Xiaokai Li1, Teresa Colvin2, Jennifer N. Rauch1, Martin Kampmann3, Diego Acosta-Alvear3, Bryan Dunyak1, Byron Hann4, Blake T. Aftab5, Min Cho3, Peter Walter3, Jonathan S. Weissman3, Michael Y. Sherman2, Jason E. Gestwicki1
Supplemental Figure 1. Chemical structures of the compounds used in this study and characterization of JG98-biotin by NMR and MS/MS.
Characterization of JG98-biotin
1H NMR (400 MHz, DMSO) δ 8.32 (t, J = 6.0 Hz, 1H), 8.26 (d, J = 4.1 Hz, 1H), 8.07 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 4.0 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.56 (dd, J = 8.9, 2.2 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 6.55 (s, 1H), 6.39 (s, 1H), 6.33 (s, 1H), 5.73 (s, 2H), 4.33 – 4.27 (m, 1H), 4.22 (d, J = 5.9 Hz, 2H), 4.14 – 4.03 (m, 6H), 3.11 – 3.04 (m, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.11 (t, J = 7.4 Hz, 2H), 1.66 – 1.38 (m, 4H), 1.37 – 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 3H). ESI-MS: Calculated for C35H38ClN6O3S4
+
753.16, found 753.11.
Supplemental Figure 2. JG-98 disrupts the Hsp70-Bag interaction in HeLa cells. (A) The fullwestern blot corresponding to Figure 2B. The blot was stripped and re-probed for Hsp70. (B) Twoadditional replicates of the co-immunoprecipitation study (see Figure 2B) are shown. Note that in replicate 1, the levels of Hsp70 in the pulldowns were not equivalent. The cellular levels of Bag1were too low to confidently determine any compound effects.
DMSO
Hsp70
IgG
________JG-9850 µM
Hsp70
IgG
________YM-0150 µM
Hsp70
IgG
________
Bag2
Bag3
Bag1
replicate 1 replicate 2
DMSO
Hsp70
IgG
________JG-9850 µM
Hsp70
IgG
________YM-0150 µM
Hsp70
IgG
________
Bag2 (lower exposure)
lysa
te (1
:10)
lysa
te (1
:10)
Lysa
te
(1:10
)DMSO
Hsp70
IgG
________JG-9850 µM
Hsp70
IgG
________YM-0150 µM
Hsp70
IgG
________
(A) Unedited western blot corresponding to Figure 2B
Bag3
Hsp70(strippedandre-probed)
(B) Replicates of Hsp70-Bag3 co-Immunoprecipitation study
Compound EC50/µM MCF-7
EC50/µM MDA-MB-231
EC50/µM A375
EC50/µM MeWo
EC50/µM HeLa
EC50/µM HT29
EC50/µM SKOV3
EC50/µM Jurkat
EC50/µM MEF
YM-01 5.2 ± 0.8 2.2 ± 0.2 >10 >10 4.5 ± 0.5 0.46 ± 0.08 7.6 ± 0.3 3.8 ± 0.5 >50
JG-98 0.71 ± 0.22 0.39 ± 0.03 0.87 ± 0.08 2.4 ± 0.3 0.85 ± 0.08 0.38 ± 0.03 0.85 ± 0.17 0.55 ± 0.10 4.5 ± 0.5
YM-01
1 10 100 1000 10000 1000000
40
80
120 SKOV3MeWoHT-29A375HeLaJurkatMEFMDA-MB-231MCF7
conc./nM
% G
row
th
JG-98
1 10 100 1000 10000 1000000
40
80
120 SKOV3MeWoHT-29A375HeLaJurkatMEFMDA-MB-231MCF7
conc./nM
% G
row
th
Supplemental Figure 3. Additional information on the viability of cells treated with YM-01 or JG-98, includign raw curves (A) and the originsof the cell lines tested (B). See Figure 3 and the materials and methods for additional details.
Cell Line OriginMCF7 adenocarcinomaMDA-MB-231 adenocarcinomaA375 malignant melanomaMeWo malignant melanomaHeLa adenocarcinomaHT-29 colorectal adenocarcinomaSKOV3 adenocarcinomaJurkat acute T cell leukemiaMM1.R multiple myelomaINA6 multiple myelomaRPMI-8226 multiple myelomaJJN-3 multiple myelomaU266 multiple myelomaNCI-H929 multiple myelomaL363 multiple myelomaMM1.S multiple myelomaKMS11 multiple myelomaLP-1 multiple myelomaAMO-1 multiple myelomaOPM1 multiple myelomaOPM2 multiple myelomaMEF mouse embryonic fibroblast
(A) Representative viability curves from MTT experiments.(B) Origin of cell lines tested.
Kidney test
BUN mg/dL
Creatinine mg/dL
Vehicle 26 ± 1 0.2 ± 0 0.3 mg/kg 26 ± 2 0.2 ± 0 1 mg/kg 26 ± 1 0.2 ± 0 3 mg/kg 23 ± 1 0.1 ± 0
Liver test
ALT IU/L
Bilirubinmg/dL
Vehicle 28 ± 4 0.1 ± 0 0.3 mg/kg 28 ± 2 0.1 ± 0 1 mg/kg 27 ± 0 0.1 ± 0 3 mg/kg 32 ± 3 0.1 ± 0
(B) Normal liver and kidney function in treated mice
pharmacokinetics parameters Dosage 0.3 mg/kg 1 mg/kg 3 mg/kg
Cmax / nM 6.3 17.0 73.5 Tmax / h 1 1 1 t1/2 / h 23.4 18.1 23.7
AUC0 - ∞ / nM × h 156 369 1667 MRT / h 33.7 25.8 33.9 CL / h × L 0.108 0.152 0.101
(A) Normal liver and kidney function in treated mice
Supplemental Figure 4. Pharmacokinetics parameters for JG-98 andresults of blood analysis for liver and renal function. (A) Values calculatedusing PKSolver. See Figure 4. (B) An analysis of plasma from treatedanimals showed normal levels of major kidney and liver markers.
