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Directed evolution of cyclic peptides for inhibition of autophagy†

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.


Expression and Purification of Recombinant LC3
pQTEV-MAP1LC3A vectors for expression and purification of N-terminally polyhistidine tagged recombinant LC3A were ordered through Addgene (Plasmid #34824) and transformed into BL21-DE3 pLysS E. coli (Promega). 2 Liters of LB media were inoculated from overnight cultures and grown to an OD600 of 0.6 before expression was induced with IPTG at a concentration of 0.6 mM for 3 hours. Cell pellets were resuspended in 15 mL equilibration buffer (20 mM HEPES pH 7.2, 200 mM NaCl, 10 mM imidazole) and lysed by sonication.
Clarified lysate was incubated with 1 mL Ni-NTA resin for one hour, loaded onto a fritted column, and flowthrough eluted. Resin was washed four times with equilibration buffer and the protein was eluted with 15 mL elution buffer (20 mM HEPES pH 7.2, 200 mM NaCl, 250 mM imidazole). Eluted protein was buffer exchanged against dialysis buffer (20 mM HEPES pH 7.2, 200 mM NaCl, 25% glycerol) using 3,000 MWCO dialysis tubing (Fisher) and concentrated by Amicon Ultracel 3k centrifugal filters (Millipore) to a final concentration of 1.4 mg/mL (7 mg/L total yield). His6-LC3A was biotinylated using 5x molar excess biotin-NHS (Sigma). The reaction was incubated on ice for 2 hours before excess biotin-NHS was removed by 7,000 MWCO Zeba spin column (Thermo). Biotinylation was confirmed by HABA-Avidin assay (Pierce) and the final concentration of biotinylated His6-LC3A was measured at 1.25 mg/mL by BCA assay.
pDEST15-LC3A and pDEST15-LC3B constructs for N-terminally tagged GST-LC3A and GST-LC3B were provided by Dr. Terje Johansen 1,2 . Transformation and expression of plasmids were performed as described above. Clarified lysate was incubated with 1 mL glutathione agarose (Goldbio) for one hour, loaded onto a fritted column, and flow-through eluted. Resin was washed four times with equilibration buffer and the protein was eluted with 15 mL elution buffer (20 mM HEPES pH 7.2, 200 mM NaCl, 10 mM reduced glutathione).
Synthesis of N-methyl Alanine amber codon suppressor tRNA NVOC-N-methyl-L-alanine cyanomethyl ester was synthesized according to previously described protocols for conjugation to the dinucleotide pdCpA and ligation to the amber suppressor THG73 tRNA 3 . NVOC-Cl (269 mg, 970 µmol, Sigma) was dissolved in 8 mL THF and added dropwise to N-methyl-L-alanine (100 mg, 969.7 µmol, Sigma) dissolved in 2 mL 100mM sodium carbonate. 10 N NaOH was added to bring the reaction mixture to pH > 10 and the reaction mixture was stirred at room temperature protected from light for 3 hours. Solvents were removed in vacuo and the dried compound was dissolved in 3 mL dry DMF to which 2 mL chloroacetonitrile and removed in vacuo and the remaining solid was purified by silica gel flash chromatography with EtOAc/hexanes (3:1) yielding 84.6 mg NVOC-N-methyl-L-alanine cyanomethyl ester (22.8% yield) ( Figure S8A).

.3 µmol, TriLink
Biotechnologies) were dissolved in 400 µL dry DMF under nitrogen and stirred at room temperature protected from light for 4 hours before quenching the reaction with 20 µL ammonium acetate (25 mM, pH 4.5). The crude product was purified by reverse phase preparative HPLC using a gradient of ammonium acetate (25 mM, pH 4.5) to acetonitrile and the fractions corresponding to the product were combined and lyophilized yielding 3 mg of NVOC-N-Methyl-L-Alanine-pdCpA (36.1% yield). Product was confirmed by observing the [M+H] + peak at MW = 961.54 (expected 960.21) using electrospray ionization mass spectrometry ( Figure S8B).

Sanger Sequencing
After library convergence, the final pool was TOPO-TA cloned into pCR™II-TOPO® vector (Thermo-Fisher), transformed into One Shot TOP10 chemically competent E. coli (Thermo-Fisher), and plated on ampicillin-agarose to isolate individual sequences from the final selection pool. Twelve colonies from each parallel selection were selected at random and plasmid minipreps (Life Technologies) were prepared from 5 mL overnight cultures. Amplified plasmids were submitted for Sanger sequencing at the MD Anderson Advanced Technology Genomics Core and analyzed using FinchTV software.

Peptide Synthesis and Cyclization
Peptides were synthesized using a Prelude automated peptide synthesis instrument (Protein Technology, Inc.).
2-chlorotrityl resin (0.439 g, 0.5 mmol, Advanced Chemtech) was swollen in DMF for 1 hour and washed twice with 5 mL DMF. The initial C-terminal amino acid coupling to the resin was performed manually prior to automated synthesis of peptides. 2 equivalents Fmoc-propargylglycine (1 mmol, 335 mg) and 3 equivalents N,Ndiisopropylethylamine (DIEA, 1.5 mmol, 262 µL, Sigma) were added to the swelled resin in 6 mL DMF and rotated at room temperature for 1 hour. To synthesize each peptide, 100 mg of Fmoc-propargylglycine resin was loaded onto the automated synthesis instrument and synthesis was performed with 3 equivalents of Fmoc amino acid (Oakwood Products, Inc.), DIEA, and N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, Oakwood Products, Inc.) in N-methyl Pyrrolidone (NMP). A total of 3 coupling reactions (10 minute duration) were performed for each amino acid building block. Deprotection of Fmoc groups was achieved using 20% (v/v) piperidine in NMP. After synthesis and final deprotection, the resin was washed 5x with NMP (1.5 mL, 2.5 minutes), 5x with DCM (1.5 mL, 2.5 minutes), and dried under vacuum for 1 hour.
Peptides were cleaved from the resin with 95% (v/v) trifluoroacetic acid, 2.5% (v/v) water, 2.5% (v/v) triisopropylsilane at room temperature for 3 hours, precipitated with cold diethyl ether, and dried under vacuum. Purified products were confirmed by electrospray ionization mass spectrometry. HPLC chromatograms and mass spectra for each peptide may be found in the Supplemental Figures S9, S13, and S18.
Amine reactive cross-linker chemistry was utilized to cyclize peptides between the N-terminus of the peptide and the side-chain of a C-terminal lysine residue. Purified linear peptide was dissolved in DMF and mixed with 1.1 equivalents of disuccinimidyl glutarate (DSG) and 7 equivalents DIEA at room temperature for 3 hours. After 3 hours the reaction was quenched with hydroxylamine (10 mM final concentration) for 15 minutes and solvent was removed in vacuo. The crude product was dissolved in 1 mL 95:5 (v/v) dH2O:CH3CN + 0.1% (v/v) TFA and purified by reverse phase preparative HPLC using gradient elution (5-20% Buffer B over 5 minutes, 20-95% Buffer B over 60 minutes; Buffer A dH2O + 0.1% (v/v) TFA, Buffer B CH3CN + 0.1% (v/v) TFA). Appropriate fractions were combined and lyophilized yielding a white solid. Purified products were confirmed by electrospray ionization mass spectrometry. HPLC chromatograms and mass spectra for each peptide may be found in Supplemental Figures S11, S15, S20, and S23.

Fluorescence Polarization Assays
For fluorescence polarization assays, stock solutions of GST, GST-LC3A and GST-LC3B were adjusted Quantification of colocalization between SUPR4B1W-ROX and eGFP-LC3 puncta was performed in ImageJ. Three fields of images for both starved and unstarved treatments were selected and split into red and green channels. Cells that were mitotic or had lost eGFP-LC3 expression were removed from the analysis.
Manders M1 and M2 coefficients were calculated using the Just Another Colocalization (JACoP) 4 plugin using appropriate pixel intensity thresholding to filter background signal.

Synthesis of SUPR4B1W-ROX
Copper-catalyzed click chemistry was used to conjugate carboxy-X-rhodamine (ROX) azide to the side-chain alkyne of the C-terminal propargylglycine residue of SUPR4B1W. 3 µmol of SUPR4B1W was dissolved in DMSO and added to 1.2 eq ROX-azide (50 mg/mL, Lumiprobe), 50 µL click solution (16 mg/mL CuSO4·2H2O and 30 mg/mL L-ascorbic acid in dH2O), 5 µL of TBTA (100 mg/mL). Reaction mixture was brought to a final volume of 150 µL with dH2O and rotated at room temperature for 2 hours and purified by reverse phase preparative HPLC

Chloroalkane azide synthesis
Chloroalkane carboxylic acid was first synthesized as previously described 5 and chloroalkane azide was then synthesized from chloroalkane carboxylic acid and 3-Azido-1-propanamine (See Scheme below).
To synthesize chloroalkane azide, 200 mg of Chloroalkane carboxylic acid was dissolved in DMF (2 mL), 1.1 equivalents of DIEA, and 1.1 equivalents HBTU were added followed by the addition of 1.2 equivalents of freshly prepared 3-Azido-1-propanamine. The resulting mixture was rotated at room temperature for 2 hours and purified by reverse phase preparative HPLC (Luna Phenomenex, 5 µm C18(2), 250 mm x 21.2 mm LC column) using gradient elution (10-95% Buffer B over 35 minutes; Buffer A dH2O + 0.1% (v/v) TFA, Buffer B CH3CN + 0.1% (v/v) TFA). Appropriate fractions were combined and lyophilized yielding a white solid. Purified products were confirmed by electrospray ionization mass spectrometry. HPLC chromatograms and mass spectra for each peptide may be found in the Supplemental Figures S24.

Synthesis of chloroalkane-labeled SUPR peptides
Copper-catalyzed click chemistry was used to conjugate chloroalkane azide to the side-chain alkyne of the C-   Table S1: Statisical Parameters obtained from analysis of data from Figures 6 and 7. Results of two-way ANOVA with type I (cellular viability measurements) or type III (tumor weight measurements) sum of squares tests to determine the independent and interactive effects of platinum and anti-autophagy treatments.       Here we show that combined treatment with cisplatin and CQ ablates LC3II levels rather causing LC3II accumulation as was seen when SUPR4B1W was combined with cisplatin. This suggests that the mechanism of autophagy inhibition with SUPR4B1W differs from that of CQ. Treatment with SUPR peptides in the absence of cisplatin does not result in significant accumulation of LC3II although some accumulation of p62 is observed in the case of SUPR4B1W in HEY.