1. Volume 23, Issue 2, Pages 257-266 (February 2016)
Natural Product Kongensin A is a Non-Canonical HSP90 Inhibitor that Blocks RIP3- dependent Necroptosis 
Dianrong Li, Chao Li, Lin Li, She Chen, Lei Wang, Qiang Li, Xiaodong Wang, Xiaoguang Lei, Zhirong Shen 
Cell Chemical Biology 
Volume 23, Issue 2, Pages (February 2016)
DOI: /j.chembiol
Copyright © 2016 Elsevier Ltd Terms and Conditions

2. Cell Chemical Biology 2016 23, 257-266DOI: (10. 1016/j. chembiol. 2015
Copyright © 2016 Elsevier Ltd Terms and Conditions

3. Figure 1 Identification of KA as Necroptosis Inhibitor
(A) Assay for screening necroptosis inhibitors. Necrosis was induced by adding the final concentrations of 20 ng/ml TNF-α (T), 100 nM Smac mimetic (S), and 20 μM z-VAD (Z) to the cell culture wells. The number of surviving cells was normalized to control cells that were treated with DMSO. Identical concentrations of these necrosis-inducing agents were used in subsequent experiments unless otherwise stated. Necrostatin-1 was used as a positive control.
(B) Chemical structure of KA.
(C) Dose-dependent inhibition of necrosis by KA in HT-29 cell. The cells were treated with T/S/Z plus the indicated concentrations of the KA. Cell viability was determined by measuring ATP levels. The data are represented as the mean ± SD of triplicate wells.
(D) KA blocks T/S/Z induced phosphorylation of RIP3 and MLKL. HT29 cells were treated with the indicated stimuli for 6 hr and then harvested. 20-μg aliquots of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting analysis of the indicated proteins.
(E) KA blocks T/S/Z induced form of necrosome. HT29-Flag-RIP3 cells were treated with the indicated stimuli for 6 hr. The cell extracts were then prepared and used for immunoprecipitation with an anti-Flag antibody as described in the Experimental Procedures. The immunocomplexes were then analyzed by immunoblotting using antibodies as indicated. 20-μg aliquots of whole-cell lysates (Input) were subjected to SDS-PAGE.
(F) KA blocks the formation of RIP3 punctae. KA (2 μM) was used in the treatment. HT29-Flag-RIP3 cells were treated with the indicated stimuli for 12 hr. The distribution of RIP3 (green) was detected by immunofluorescence as described in the Experimental Procedures. The scale bars represent 2.5 μm.
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions

4. Figure 2 Identification of HSP90 as KA's Cellular Target
(A) Chemical structure of the KA negative (KA-1).
(B) KA-1 cannot block T/S/Z-induced cell death. HT29 cells were treated with T/S/Z plus the indicated concentrations of the KA and KA-1. Cell viability was determined by measuring ATP levels. The data are represented as the mean ± SD of triplicate wells.
(C) KA-1 cannot block the formation of RIP3 punctae; 2 μM KA was used. HT29-RIP3 cells were treated with the indicated stimuli for 12 hr. The distribution of RIP3 (green) was detected by immunofluorescence as described in the Experimental Procedures. The scale bars represent 2.5 μm.
(D) The inhibitory effect of KA-P on necroptosis was compared with KA. The cells were treated with T/S/Z plus the indicated concentration of the compound. Cell viability was determined by measuring ATP levels. The data are represented as the mean ± SD of triplicate wells.
(E) Identification of HSP90 as the cellular target of KA. HT29 cells were harvested, and cell lysates were used for biotin-KA pull-down as described in the Experimental Procedures. The samples were then analyzed by SDS-PAGE followed by silver staining. The indicated protein bands were identified by MS.
(F) Protein binding to biotin-KA (2.5 μM) with or without KA competition (2.5 μM) was subject to pull-down experiments and immunoblotting for HSP90. The asterisk(*) indicates non-specific bands.
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions

5. Figure 3 KA Targets Cys420 of HSP90
(A) KA covalently binds toHSP90β. A 1-mg aliquot of HSP90β was incubated with 2 nmol KA-P at 4°C. After 6 hr, the reaction mixture was incubated with 2 nmol biotin-modified oOQM precursor at 37°C for 16 hr to form a linker between oOQM and thiol-vinyl ether. The mixture samples were subjected to SDS-PAGE, followed by immunoblotting using an anti-biotin antibody. An equal aliquot of input HSP90β was measured by immunoblotting analysis.
(B) KA interacts with HSP90β through covalent binding. A 1-mg aliquot of HSP90β was incubated with 2 nmol KA-P at 4°C. After 6 hr, the reaction mixture was incubated with 2 nmol biotin-modified oOQM precursor at 37°C for 16 hr to form a linker between oOQM and thiol-vinyl ether. The reaction mixture was prepared and used for immunoprecipitation with anti-streptavidin beads as described in the Experimental Procedures. The protein samples were subjected to SDS-PAGE followed by immunoblotting using an anti-GST antibody. An equal aliquot of input HSP90β was measured by immunoblotting analysis.
(C) Effect of KA on the ATPase activity of HSP90β. After incubation with HSP90β plus DMSO, HSP90β plus KA (5 μM), or incubation with the HSP90β plus GA (2 μM), ATP was measured by Cell Titer Glo and normalized. The data are represented as the mean ± SD of triplicate wells.
(D) The MS fragmentation spectrum of the KA modified peptide corresponding to residues 420–435 of HSP90β, together with the assignation of the fragmentation series to the sequence of the modified peptide. Recombinant human HSP90β was either incubated with DMSO, KA, or KA-1 in PBS for 6 hr. Samples were then trypsin digested and analyzed by LC-MS by using a linear ion trap detector as described in the Experimental Procedures.
(E) Binding of recombinant HSP90β or HSP90β(C420A) to KA-P. 1-mg aliquots of HSP90β or HSP90β(C420A) were incubated with 2 nmol biotin-KA at 4°C. After 6 hr, the reaction mixture was incubated with 2 nmol biotin-modified oOQM precursor at 37°C for 16 hr to form a linker between oOQM and thiol-vinyl ether. The reaction mixtures were prepared and used for immunoprecipitation with anti-streptavidin beads as described in the Experimental Procedures. The protein samples were subjected to SDS-PAGE followed by immunoblotting analysis using an anti-GST antibody. Equal aliquots of input HSP90β or HSP90β(C420A) were also measured by immunoblotting analysis.
(F) Diagram of C420 relative to HSP90 domain architecture (upper panel). The lower panel shows the sequence alignment around C420 from different organisms. C420 or corresponding residues in other organisms/isoforms are marked with asterisk.
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions

6. Figure 4 KA is a Novel HSP90 Inhibitor
(A) Effect of KA on HT29 cells was compared with GA. HT29 cells were exposed to DMSO, GA, or KA for 24 hr, and then whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting analysis of specified proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. The data represent results from three independent experiments.
(B) KA induced caspase 3 activation. HT29 cells were treated with the indicated stimuli for 6 hr and then harvested. 20-μg aliquots of whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting analysis of the indicated protein.
(C) Effect of KA or KA-1 on apoptosis. HT29 or A549 cells were exposed to DMSO, KA, or KA-1 at indicated concentration for 48 hr. The number of surviving cells was determined by measuring ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells.
(D) HeLa cells were transfected with vector control or Myc-HSP90 for 12 hr, and then cells were treated with KA for12 hr. The cells were then harvested, and the whole-cell extracts were immunoprecipitated with anti-Myc antibody. The immunocomplexes were analyzed by immunoblotting analysis using the indicated antibodies. 20-μg aliquots of whole-cell lysates (Input) were subjected to SDS-PAGE followed by immunoblotting analysis of specified proteins. Tubulin is shown as a loading control.
(E) HeLa cells were transfected with vector control, Myc-HSP90, or Myc-HSP90(C420A) for 12 hr, and the cells were treated with KA for12 hr. The cells were then harvested, and the whole-cell extracts were immunoprecipitated with anti-Myc antibody. The immunocomplexes were analyzed by immunoblotting analysis using the indicated antibodies. 20-μg aliquots of whole-cell lysates (Input) were subjected to SDS-PAGE followed by immunoblotting analysis of specified proteins. Tubulin is shown as a loading control.
(F) KA blocks LPS/Smac mimetic/z-VAD induced necrosis in THP-1 cells. The cells were treated with 10 ng/ml LPS, 100 nM Smac mimetic, and 20 μM z-VAD (LPS/S/Z) plus the indicated concentrations of the GA, KA, or KA-1. Cell viability was determined by measuring ATP levels. The data are represented as the mean ± SD of triplicate wells.
(G) KA blocks polymerized RIP3 induced necrosis. Briefly, RIP3 polymerization was induced in U2OS Tet-On cells with tet-inducible expression of flag-tagged RIP3 fusion with two AP20187-binding (FKBPv) domains (U2OS-diRIP3). Expression of RIP3 was induced by doxycycline overnight, and then treated with different compounds for 2 hr before the dimerizer was used to induce RIP3 polymerization-dependent cell death. After 10 hr, the number of surviving cells was determined by measuring ATP levels. The data are represented as the mean ± SD of triplicate wells.
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions

7. Figure 5 KA Binds to Cys420 of HSP90β and Blocks Its Function
Schematic model of binding of KA to C420 of HSP90β. Binding of KA dissociates cochaperone CDC37 from HSP90β, leading to dysfunction of client kinases and subsequent degradation. The red star represents the double bond of KA, which will mediate covalent binding to Hsp90.
Cell Chemical Biology  , DOI: ( /j.chembiol )
Copyright © 2016 Elsevier Ltd Terms and Conditions