https://openworks.mdanderson.org/sumexp21/1167/thumbnail.jp

Engel, Brian J

Gray, Joshua P

Millward, Steven W

Vucic, Iva

English

OpenWorks @ MD Anderson

Autophagy is a Cell Survival Mechanism that Leads to Treatment Resistance4B1W is a Cyclic PeptideInhibitor of LC3aReferences1) White et al. Clin. Cancer Res. 2015;21(22);5037-50462) Nieto-Torres et al. Trends Biochem. Sci. 2021;46(8);673-6863) Gray et al. Chem. Sci., 2021;12;3526–35434Synthesis and Purification of Cyclic Peptide Autophagy Inhibitor 4B1W and Its Target LC3aVucic I1, Engel BJ1, Gray JP1, Millward SW1.1Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center LC3a Loads Cargo into AutophagosomesExpression & Purification of Recombinant LC3a ProteinSolid Phase Peptide Synthesis of 4B1W 4B1W Purification & Final YieldsFigure 1. General Mechanism of Autophagy. Autophagy is a cellular processto recycle old or misfolded proteins and organelles for reuse in cellularbiogenesis. First, cargo is enveloped around a double-membrane structurecalled the phagopore, which fully closes into a vesicle called theautophagosome. This then fuses with the lysosome to form anautophagolysosome. Cargo is broken down into substituent biomolecules to bereused. Induction of autophagy occurs after treatment in some cancers as anescape mechanism. Inhibition of autophagy, therefore, would be a suitabletarget for combating treatment resistance. Figure adapted from 1.Starvation, oxidative stress, accumulation of misfolded proteinsPhagoporeLC3a organelle protein aggregateAutophagolysosomePhagoporeFigure 2. Cargo is loaded into autophagosomes by LC3a. LC3a aids in theelongation of the phagophore and formation of a fully closed autophagosome.As the phagophore grows, LC3a molecules bind to cargo (e.g. organelles andprotein aggregates) through adapter proteins. These are sequestered to theinner phagophore membrane and are enclosed inside the autophagosome anddegraded upon fusion with the lysosome. Figure adapted from 2.Figure 5. SDS-PAGE and BCA assay of recombinant GST-LC3a. (A) SDS-PAGE of initial purification with 10 mM glutathione did not elute GST-LC3a,however expression was detected as a protein band near 49 kDa. (B) A follow-up SDS-PAGE of the resin showed GST-LC3a was still bound. (C) The resinwas incubated with 50 mM glutathione to elute GST-LC3a from the resin. SDS-PAGE analysis shows characteristic 49 kDa bands in each elution fraction,indicating GST-LC3a was properly eluted. (D) BCA assay standard curve (bluedots) and pooled, dialyzed elution fractions (red dot) revealed an undilutedconcentration of 1.16 mg/mL with final yield of 11.6 mg GST-LC3a/L E. coli.Figure 5. General Protocol for GST-LC3a expression and purification. (1)BL21 E. coli are transformed with GST-LC3a expression plasmid. (2) Selecttransformed colonies with ampicillin agar plates. (3) Inoculate 500 mL culturewith colony and induce protein expression with IPTG. (4) Lyse cells withlysozyme and tip-sonication then centrifuge. (5) Filter lysate for affinitypurification. (6) Add glutathione agarose resin to lysates to bind GST-LC3a andpurify by gravity column. (7) Recover flow-through containing unboundproteins. (8) Wash resin with buffer. (9) Elute GST-LC3a glutathione buffersolution. (10) Run SDS-PAGE of all steps to analyze expression and purity.A B CFinal Yield: 11.6 mg/LDFigure 6. General solid phase peptide synthesis protocol. (1) Deprotectresin using 25% piperidine to remove Fmoc protecting groups. (2) Wash resin5x with DMF to remove released Fmoc groups. (3) Couple Fmoc-amino acid toresin. HBTU stabilizes carboxylic acid groups and DIEA initiates the couplingreaction. (4) Wash 5x with DMF to remove excess Fmoc-amino acid andreagents. (5) Repeat steps 1-4 for each amino acid to build a peptide chain. (6)After synthesis is complete, deprotect with 25% piperidine and cleave peptideoff the resin with TFA. (7) Cyclize the peptide using DSG.Figure 7. Purification, identification, and final yields of linear 4B1W. (A) HPLC of a gradient from 10%-95% acetonitrile+0.1% TFA over 35 minutes revealed a large peak near 8.4 min for each sample. Mass spectrometry (inset) confirmed product identity as linear 4B1W. (B) Summary of fourteen 126 µmol scale linear 4B1W syntheses yielded of 507.11 mg peptide (average percent yield 32.01%). Successful syntheses have mass spectra similar to the inset of (A), only the [M+2H]2+ adduct is shown for simplicity.Conclusion & Future Work• Linear 4B1W peptide and GST-LC3a produced at high yield and purity• Cyclization of 4B1W is ongoing• Cyclic 4B1W will be used in in vitro cell assays & mouse models to evaluate the efficacy of autophagy inhibition in different contexts• Recombinant GST-LC3a will be used as a target for further improvement of peptide inhibitors of autophagyA BFigure 3. Cyclic 4B1W structure and computational model of 4B1W:LC3ainteraction. (A) Chemical structure of cyclic 4B1W. Cyclization occurs betweenthe lysine sidechain and the N-terminus of methionine. (B) Cyclic 4B1W bindsstrongly to the LC3a interacting motif, blocking interaction with other autophagyproteins. As a result, cyclic 4B1W is an inhibitor of cellular autophagy whichsensitizes cancer cells to chemotherapeutics. Figure adapted from 3.Introduction• Autophagy is an energy producing mechanism that helps somecancer cells build resistance to chemotherapeutics.• Inhibiting autophagy leads to decreased treatment resistance.• The goals of this work are to:1) Purify a key autophagy protein to be used to develop futurepeptide inhibitors2) Produce a cyclic peptide inhibitor of the protein target thatwill be used to inhibit autophagy in vitro and in vivoTotal Yield: 505.81 mgAverage Percent Yield:31.94%AB8.4minadapter protein

Synthesis and Purification of Cyclic Peptide Autophagy Inhibitor 4B1W and Its Target LC3a

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1167&context=sumexp21

Synthesis and Purification of Cyclic Peptide Autophagy Inhibitor 4B1W and Its Target LC3a

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