A compound-based proteomic approach discloses 15-ketoatractyligenin. methyl ester as a new PPARγ partial agonist with anti-proliferative ability

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1 upplementary Information A compound-based proteomic approach discloses 15-ketoatractyligenin methyl ester as a new PPARγ partial agonist with anti-proliferative ability Michele Vasaturo 1, Lorenzo Fiengo 1,6, unziatina De Tommasi 1, Lina abatino 2, Pamela Ziccardi 2, Vittorio Colantuoni 2 Maurizio Bruno 3, Carmen Cerchia, 4 Ettore ovellino 4, Angelo Lupo 2*, Antonio Lavecchia 4*, Fabrizio Dal Piaz 1,5 1 Department of Pharmacy, University of alerno, Via Giovanni Paolo II, 132, Fisciano, Italy 2 Department of ciences and Technologies, University of annio, Via port Arsa, Benevento, Italy 3 Department of rganic Chemistry, University of Palermo, Viale delle cienze - Parco d'rleans II, Palermo, Italy 4 Department of Pharmacy, "Drug Discovery" Laboratory, University of apoli Federico II, Via D. Montesano, 49, apoli, Italy 5 chool of Medicine and urgery, University of alerno, via alvatore Allende, 18, Baronissi (A) 6 PhD Program in Drug Discovery and Development, University of alerno, Via Giovanni Paolo II 132, I Fisciano (A), Italy *Correspondence: lupo@unisannio.it (A.L.), antonio.lavecchia@unina.it (A.L.) upplemental Figures and Tables Figure 1. Chemical modification of compound 1 for chemical proteomics experiments. Figure 2. Western blot analysis of proteins eluted in the chemical proteomics experiments. Figure 3. PR sensorgrams obtained for compounds 2-8 and rosiglitazone.

2 Figure 4. PR sensorgrams obtained injecting 1 on PPARγ previously incubated with the same compound. Figure 5. PPARγ transactivation activity of compound 1 in the presence of Tween 20. Figure 6. tructures of known PPARγ partial agonists selected as the active compound set. Figure 7. RC curve for the virtual screening performed on PDB entry 3B3K. Table 1. M analysis of peptide fragments generated in the trypsin digestion of the 1/PPARγ LBD complex. Table 2. M analysis data of Hsp60 incubated with 1 and digested with trypsin. Table 3. Cognate docking and virtual screening results for six PPARγ crystal structures.

3 Figure 1. Chemical modification of compound 1 aimed to its immobilization for chemical proteomics experiments. Reaction conditions were optimized to preserve compound 1 biological activity.

4 C 1 Hsp60 PPARγ Figure 2. Western blot validation of chemical-proteomics results. Proteins eluted from control (C) and 1-modified (1) beads were revealed using anti-hsp60 and anti-pparγ antibodies. Blots are representative of three separate experiments with similar results.

5 Figure 3. PR sensorgrams achieved injecting different concentrations (from to 1 µm) of compound 2-7 and of the positive control rosiglitazone on immobilized PPARγ LBD.

6 Figure 4. PR sensorgrams obtained injecting 1 on PPARγ previously incubated with the same compound.

7 Figure 5: PPARγ transactivation activity of compound 1 in presence of Tween 20. Human HEK293 cells stably expressing an exogenous Flag-tagged wild type PPARγ1 were transiently transfected with the PPRE-luciferase reporter gene and treated for 24 hs with rosiglitazone or compound 1, respectively, at the indicated doses in presence or absence of a detergent such as Tween 20 at 0.001% in the medium. Luciferase activity is reported as foldinduction after normalization to β-galactosidase activity used as control for transfection efficiency. The results are further normalized to those from cells treated with vehicle only (e. g. DM in the absence of compound 1 or Rosiglitazone). Data are the mean ± D of three independent experiments performed in duplicate.

8 2FVJ F F H 2G0G F 3 C CF 3F H 2G0H 2I4P 2I4Z H F 3 C 2P4Y H F 3 C H H H H H H H H 2Q5P 2Q5 2Q61 2Q6R 2Q6 2YFE H H H H 3B1M H H Br 3I H Br Br 3W 3B3K H Br 3R8A H 3FUR H H H CF 3 3K8 39 Br H H 3LMP 3T03 H H H 3V9T H H H 3V9V H 3V2 H H 3V H H H H H F H H H H 4A4V 4A4W 4F9M 4HEE H Figure 6. tructures of known PPARγ partial agonists selected as the active compound set. 4PRG

9 Figure 7. RC curve for the virtual screening performed on PDB entry 3B3K.

10 Table 1. Mass spectrometry analysis data of the PPARγ LBD/1 digested with trypsin. bserved m/z Measured MW Peptide 1 Theoretical MW [M+2H] [M+H] [M+H] [M+H] [M+2H] [M+2H] [M+H] [M+H] [M+2H] ( ) [M+2H] [M+2H] [M+2H] [M+2H] [M+2H] [M+H] [M+4H] [M+2H] [M+H] [M+H] [M+2H] [M+2H] umbers correspond to those of the first and the last AA in the human PPARγ (isoform 1) sequence.

11 Table 2. Mass spectrometry analysis data of Hsp60 incubated with 1 and digested with trypsin. bserved m/z Measured MW Peptide 1 Theoretical MW 1057,079 [M+2H] , , ,865 [M+2H] , , ,0884 [M+3H] , , ,473 [M+H] + 854, , ,418 [M+3H] , , ,886 [M+2H] , , ,358 [M+2H] , , ,877 [M+2H] , , ,044 [M+2H] , , ,174 [M+2H] , , ,595 [M+2H] , , ,179 [M+3H] , , ,517 [M+H] + 843, , ,519 [M+2H] , , ,540 [M+H] + 900, , ,305 [M+2H] , , ,517 [M+H] + 959, , ,448 [M+2H] , , ,921 [M+2H] , , ,338 [M+2H] , , ,710 [M+3H] , , ,816 [M+2H] , ,613 1 : umbers correspond to the number of the first and the last AA in human Hsp60 sequence

12 Table 3. Cognate docking and virtual screening results for six PPARγ crystal structures. PDB Resolution Cognate Ligand RC EF 2% a EF 5% b EF 10% c (Å) RMD (Å) 2G0G G0H I4P I4Z B3K PRG a Maximum enrichment factor at 2 % = b Maximum enrichment factor at 5 % = c Maximum enrichment factor at 10 % = 10.0.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI:.38/ncb97 P ( μm, hours) 1 2 4 P DMSO Figure S1 unningham et al. E 97 65 27 MFN1 GFP-Parkin Opa1 ctin GPDH HEK293 GFP-Parkin 19 115 97 65 27 Mitochondrial Fraction SH-SY5Y GFP-Parkin Mito DMSO Mito