Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119

Abstract The acyl‐binding UNC119 proteins mediate the activation and transport of various N‐myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α‐helical peptides that target UNC119a. By using the hydrocarbon peptide‐stapling approach, cell‐permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.


Chemicals and instrumentation
Unless otherwise noted, chemicals were purchased from Sigma Aldrich, Merck, Okeanos, Roth or Alfa Aesar and were used without further purification. Protected Fmoc-amino acids and coupling reagents were purchased from Novabiochem and Iris Biotech GmbH. Building blocks S 5 and R 5 for hydrocarbon peptide stapling was purchased from Okeanos Tech. Co. LTD. All solvents were purchased from commercial suppliers and used without further purification. Analytical HPLC was performed using an Agilent 1100 Series with either a C18 HPLC column 3 µm (Macherey Nagel) or a C18 HPLC column 1.8 µm (Macherey Nagel).
The system was run at a flow rate of 1.0 mL/min over 30 min using H 2 O (0.1% TFA) and MeCN (0.1% TFA) as solvents. Linear gradients were run over varying periods of time. HPLC-MS analyses were performed with an Agilent 1100 Series connected to a Thermo LCQ Advantage mass spectrometer using a C18 HPLC column 3 µm (Macherey Nagel). The system was run at a flow rate of 1 mL/min over 15 min using H 2 O (0.1% formic acid) and MeCN (0.1% formic acid) as eluents. Semi-preparative HPLC was carried out on a Agilent 1100 Series using a SP125/10 Nuclear C18 Gravity 5 µm column (Macherey Nagel) at a flow rate of 6 mL/min. Linear gradients using H 2 O (0.1% TFA) and MeCN (0.1% TFA) were run over varying periods of time. High resolution mass spectra were recorded on a QLT Orbitrap mass spectrometer coupled to an Acceka HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1mm particle size 1.9 µm, ionization method: Electrospray Ionization). Automated peptide synthesis was performed using a CEM-Discover microwave and a CEM-Liberty peptide synthesizer. Fluorescence polarization was measured with a Tecan Safire 2 . Absorbance measurements were performed on a Tecan infinite M200 and Thermo scientific Nanodrop 2000c. For ITC measurements an iTC 200 microcalorimeter by MicroCal was used. Data was analyzed using the software Microcal LLC ITC provided by the manufacturer. FACS analysis was performed on a BD LSRII analyzer (Becton Dickinson, USA). Microscope images were obtained on an Axiovert Observer Z1 (Carl Zeiss, Germany).

General
Peptides were synthesized on solid-phase using the Fmoc-strategy and Rink Amide (MBHA) resin, Rink Amide NovaSyn TGR resin or ChemMatrix Rink Amide resin as solid support. Solvents and soluble reagents were removed by suction. Washings between coupling and deprotection were carried out in DMF and DCM using 1 mL solvent per 100 mg resin. Coupling efficiency was monitored by ESI-MS and/or HPLC analyses.

Fmoc deprotection
The resin was swollen in DMF and treated with a solution of piperidine/DMF (20/80, v/v) for 2x 5 min.
Subsequently, this mixture was added to the resin and shaken for 30 min at room temperature. For coupling of the alkyne building blocks 1 -5, the building blocks 12 -14 and the subsequent amino acid: Fmoc-Xaa-OH (4 eq.) was dissolved in DMF in the presence of COMU (3.9 eq.), Oxyma (3.9 eq.) and DIEA (8 eq.), added to the resin and shaken for 1 h at room temperature. Except coupling of the alkyne building blocks 1 -5 and the alkene building blocks 12 -14, all couplings were performed as double couplings. All equivalents are calculated based on theoretical loading of the resin as provided by the vendor.

N-terminal acetylation
For preparation of N-acetylated peptides and whenever a quantitative yield even after recoupling treatments was not achieved, the free N-terminal amino group was acetylated using a solution of Ac 2 O/DIEA/DMF (1/1/8, v/v/v) for 2x 10 min at room temperature.

Fluorescence labelling with FITC
Prior to fluorescence labelling with FITC the Mtt protecting group of the C-terminal Lys was removed with DCM/TFA/TIPS/Water (994/1/2.5/2.5 v/v/v/v). Afterwards the resin was treated with FITC (5 eq.) and DIEA (10 eq.) for 16 h at room temperature under exclusion of light and the resin was washed with DMF (3x), DCM (3x) and dried to constant weight in vacuo.

Ring-closing olefin metathesis
The dried resin was swollen in DCE for 15 min. A solution of Grubbs 1 st generation catalyst (2 mg•mL -1 ) in DCE was added to the resin and reacted for 2 h at room temperature. During the reaction time argon was bubbled through the reaction mixture to remove ethene. The procedure was repeated twice and the resin was washed with DCE (3x), DCM (3x), DMF (3x).

Cleavage from the resin
The dry resin was treated with a solution of TFA/EDT/TIS/H 2 O (94/1/2.5/2.5, v/v/v/v) 100 µL for 10 mg resin for 2x 1 h and 1x 5 min. The solvents were evaporated and the crude peptide was precipitated by the addition of cold diethyl ether. After centrifugation (10 min, 16.100 x g, 4°C) the supernatant was removed.
The crude product was dissolved in H 2 O/MeCN (2/1, v/v) and lyophilized. The crude peptides were purified by semi-preparative HPLC.

Fmoc quantification
A defined amount of dry resin was transferred into an Eppendorf cap and treated with 0.5 mL deprotection solution for 15 min. The UV absorption of the supernatant was determined at 305 nm and the occupation density calculated using Beer-Lambert law (ԑ = 7800 cm -1 •M -1 ).

Peptide quantification
The concentration of fluorescein labelled peptides was determined by UV absorption in 20 mM phosphate buffer (pH 8.5) at 496 nm (ԑ = 77.000 cm -1 •M -1 ). The concentration of acetylated peptides was determined gravimetrically or via UV absorption at 280 nm.

Fluorescence anisotropy assay
UNC119a/b was serially diluted in a buffer containing 30 mM Tris (pH 7.5), 100 mM NaCl, 1 mM TCEP and 0.01% Chaps (assay buffer), treated with 5 nM fluorescein-labeled peptides and incubated for 4 h at room temperature. Binding assays were performed in 384-well, small volume, black flat-bottom, non-binding plates (Greiner). Fluorescence polarization values (λ ex = 470 nm, λ em = 525 nm) were determined using a Safire II plate reader (Tecan) at room temperature. All measurements were performed in triplicates. After correction for changes in fluorescence intensity upon binding, the fluorescence anisotropy data were converted into fraction bound (equation 1) of the FITC-labeled peptide and fitted via non-linear regression in Prism 5.0 (Graphpad). [1] fraction bound = A: observed anisotropy; A free : anisotropy of free fluorophore A bound : anisotropy of bound fluorophore Q: change in fluorescence intensity between free and bound state

Protein expression and purification
UNC119a and UNC119b were expressed in E. coli according to the previously reported procedures. [2] Cell culture Mouse L cells and human cervical carcinoma cell line HeLa were obtained from ATCC, USA (ATCC CRL-2468, ATCC CRM-CCL-2). Cells were grown in Dulbecco's Modified Eagle's medium DMEM (PAN Biotech, Germany) supplemented with 10% fetal bovine serum (FBS, Gibc,), 1 mM sodium pyruvate and nonessential amino acids at 37°C and 5% CO 2 in a humidified incubator. Additionally, HeLa cells were supplemented with penicillin and streptomycin. All cultures were routinely tested and found to be free of mycoplasma contamination.
Flow cytometry 3 x10 4 L or Hela cells were seeded per well in 6-well plates and grown overnight. The following day, cells were treated with the indicated compound concentrations for 72 h or with nocodazole as a control for 24 h). Cells were washed, detached using trypsin and re-suspended in phosphate-buffered saline (PBS).
Cells were centrifuged at 200xg for 5 min at room temperature (RT) and washed again. Cells were then fixed with 70% ethanol and stained with propidium iodide solution (12 µg/mL propidium iodide (Sigma, Germany), 0.1% (v/v) Triton X-100 and 100 µg/mL DNase-free RNase A (Sigma, Germany) in PBS) for 30 min at room temperature. Cell suspensions were filtered to FACS tubes through a nylon mesh before analysis.
10,000 cells for each sample were sorted by the BD LSRII analyzer (Becton Dickinson, USA). FlowJo 6.2.1 software was used for quantification and analysis of all data. For every analysis, FSC and SSC (forward and side scatter, respectively) gating was performed to exclude doublets and debris. In addition, cells were gated for singlets (PI-W vs. PI-A).
Fluorescence Microscopy 2x10 4 Hela cells were seeded per well in a 24-well plate on cover slips and incubated overnight. Next day, cells were treated with different concentrations of the peptides or DMSO, nocodazole and cytochalasin D as controls and incubated at 37°C and 5% CO 2 for the indicated times (72 h or 24 h). Cells were then fixed using 3.7% paraformaldehyde for 15 min at room temperature followed by permeabilization using 0.1% Triton X-100 in PBS for 15 min. Blocking was performed in 2% BSA in PBS containing 0.1% Tween 20 for 1 h at room temperature. Samples were incubated with the indicated primary antibodies (anti-tubulin primary antibody conjugated to FITC) in the blocking solution overnight at 4°C. After washing, cells were incubated with 1 µg/mL DAPI in blocking solution for 1 h at room temperature, followed by three washes with PBS.
Coverslips were then mounted on glass slides and stored at 4°C. Images were acquired on an Axiovert Observer Z1 (Carl Zeiss, Germany) using 63X oil objective and analyzed using ZEN 2011 software (Carl Zeiss, Germany).
When 80% confluency cell culture media was removed from each flask and new media containing 25 µM of peptides was added and incubated with cells for 30 min and 72 h. Following peptide incubation, cells were washed twice with PBS, trypsinized and pelleted. Pellets where lysed in RIPA buffer (Perkin Elmer) for total lysis; or CelLytic NuCLEAR Extraction Kit (SIGMA) for subcellular fractionation. Total protein quantification BCA (Pierce) was performed prior to MS analysis to normalize sample data.
Total and sub-cellular quantities of peptides 4, 10 and R8 were quantified [3] in lysates by UPLC-MS utilising a Waters Xevo TQ-XS (WBA0259) and a Waters Acquity UPLC system consisting of, Sample Manager (M16UFL953M), Acquity PDA (F17UPD457A), Column Oven (E17CMP703G) and Binary Solvent Manager (E17BUR621G). For peptides 4 and 10 analytical UPLC separation was performed using a Waters CSH C18 1.7 µm, 2.1 x 50 mm column. A gradient was utilized of 97% A to 5% in 4mins, 0.5 min hold, 5% A to 97% in 0.5mins, where A = 0.1% HCOOH (aq) and B = 0.1% HCOOH in MeCN at a flow rate of 0.75 mL/min (Supporting Figure S7). Compound R8 was analyzed in HILIC mode using a Waters BEH Amide 1.7 µm, 2.1 x 100 mm column with a gradient of 30% A to 95% in 1.5 mins, 1 min hold, 95% A to 30% in 1.5 min, 1 min hold at 0.65 mL/min. The Waters TQ-XS was operated in +ve ion Electrospray (ESI) mode with the optimized transitions for PC658-AC, PC658-MYR and Ac-R8 (Supporting Table S3 and Figure S8). The chromatograms at each transition were extracted, smoothed and integrated to give the standard curve. Chromatograms of the samples were treated similarly and by linear regression (1/x) an in-cell concentration was established in the re-suspended cell lysate using the Waters MassLynx TargetLynx TM product (Supporting Tables S4 -S7).

Supporting Figures
Supporting Figure S1. Synthesis of stapled peptides. A detailed overview of all synthesized peptides (sequences, analytical data) is shown in Supporting Table S1 and Supporting Figures S9 and S10.

Supporting Tables
Supporting Table S1. Detailed overview of all synthesized peptides based on the non-modified Gnat1 sequence Entry N-Term mod. [a]