Fig 2: 6-SH targeted HSP60 and inhibited its stability. (A) The modification process of AL-6-SH-MMs and the technology roadmap for target fishing method. (B) SDS-PAGE and Western blotting analysis of captured protein by 6-SH probe (Lane 1: A549 lysate; Lane 2: a negative control without the 6-SH probe; Lane 3: 6-SH probe-captured proteins). (C) The sequence of HSP60 protein and the identified peptide (red). (D,E) 6-SH treatment (20 µM) inhibited the thermal stability of HSP60 measured by CETSA; the statistics show the normalized density (n = 3). Values represent the means ± SD. * p < 0.05, ** p < 0.01 versus “Ctrl” group. (E,F) Cells were pre-treated with nonactin (10 or 20 µM) for 4 h, and then, they were treated with 6-SH for 72 h. The inhibition and IC50 value of 6-SH were assayed. (G) A549 cells were treated with MG-132 (5 or 10 µM) for 1 h before the addition of 40 µM 6-SH for another 5 h, and then, the HSP60 was examined; the statistics show the normalized density (n = 3). Values represent the means ± SD. * p < 0.05, ** p < 0.01 versus corresponding group.

Fig 3: The effects of WNQP on 35 types of cytokines in nude mice tumors, detected using an Apoptosis Array Kit (n = 3). The arrows indicate factors marked for further detection: 1. Bax, 2. Bcl-2, 3. Bcl-xL, 4. cleaved caspase-3, 5. cIAP, 6. clusterin, 7. TRAIL R2/DR5, 8. FADD, 9. Fas, 10. HIF-1a, 11. HO-1, 12. HSP 60, 13. HtrA2, 14. livin, 15. SMAC, 16. survivn, 17. XIAP. (A, B) Scatter diagram of 35 cytokines. The relative density is the ratio of the absolute value and reference spot value. The red dots indicate factors with a change of >20% relative to control mice. (C, D) WNQP strongly enhanced the expression levels of Bax and suppressed the expression levels of Bcl-xL, HSP27, HSP60, and HSP70 in tumor tissues. Quantitative protein expression data were normalized to the corresponding GAPDH levels, and the average fold changes in band intensity are marked (E, F) (n = 3).

Fig 4: MD analysis. (A) RMSD of the complex over time. (B) RMSF was calculated, and the green line represents the location of the amino acids of the small molecule binding site. (C) Analysis of binding free energy. (D) Molecular docking analysis of HSP60 and 6-SH (blue: HSP60, orange: 6-SH).