Enhancing Specific Disruption of Intracellular Protein Complexes by Hydrocarbon Stapled Peptides Using Lipid Based Delivery

Linear peptides can mimic and disrupt protein-protein interactions involved in critical cell signaling pathways. Such peptides however are usually protease sensitive and unable to engage with intracellular targets due to lack of membrane permeability. Peptide stapling has been proposed to circumvent these limitations but recent data has suggested that this method does not universally solve the problem of cell entry and can lead to molecules with off target cell lytic properties. To address these issues a library of stapled peptides was synthesized and screened to identify compounds that bound Mdm2 and activated cellular p53. A lead peptide was identified that activated intracellular p53 with negligible nonspecific cytotoxicity, however it still bound serum avidly and only showed a marginal improvement in cellular potency. These hurdles were overcome by successfully identifying a pyridinium-based cationic lipid formulation, which significantly improved the activity of the stapled peptide in a p53 reporter cell line, principally through increased vesicular escape. These studies underscore that stapled peptides, which are cell permeable and target specific, can be identified with rigorous experimental design and that these properties can be improved through use with lipid based formulations. This work should facilitate the clinical translation of stapled peptides.

: ATSP-7041, sMTIDE-02, VIP-82 and their scrambled analogues were screened for their ability to induce LDH release in HEK293 cells after a 2 hour treatment period in A) the absence of FCS and B) the presence of 10% FCS. Compounds were tested at concentrations of 12.5 µM, 25 µM and 50 µM. Results were normalized to maximal LDH release as measured by treatment with 0.1% Triton X100.  Ramage Chemmatrix resin was obtained from PCAS-Biomatrix (Quebec, Canada). L-amino acids were obtained from Advanced Chemtech (Louisville, KY). Fmoc-threonine, serine, glutamic acid and tyrosine were t-butyl protected and Fmoc-tryptophan was not Boc protected. Unnatural alkenyl amino acids were purchased from OKeanos (Beijing, China). All other solvents and reagents were obtained from Sigma-Aldrich. 1,2-dichloroethane (DCE) was dried overnight over activated molecular sieves and purged with Argon for 30 min prior to use. All other reagents were used as received.
Ring-closing metathesis of resin-bound, N-acetylated peptides was performed manually using a 5 mg/mL solution of Grubbs I catalyst (20 mol%) in dry DCE at room temperature under an atmosphere of inert argon (3 x 2 h treatments). After the reaction, the solution was drained, the resin washed with DCE (3 x 1 min), DMSO (1 x 2 h) and methanol (3 x 1 min) then dried in vacuo overnight. Cleavage of the peptide from the resin was achieved using 8 mL of TFA cocktail consisting trifluoroacetic acid/triisopropylsilane/water (95/2.5/2.5) for 2 h followed by filtration and precipitation with diethyl ether. The precipitate was collected by centrifugation, dried and redissolved in a 3:2 mixture of acetonitrile and water.
The pure peptides (>90% purity) were obtained by purification using a preparative HPLC system (Agilent) on a Jupiter C12 reversed-phase preparative column (Phenomenex, 4 μm, Proteo 90 Å, 250 x 10 mm). The peptides were characterized by LC-MS. Mass spectra were obtained by electrospray in positive or negative ion mode.

Mdm2 (1-125) protein Expression and Purification
Mdm2 (1-125)) was ligated into the GST fusion expression vector pGEX-6P-1 (GE Lifesciences) via a BAMH1 and NDE1 double digest. BL21 DE3 competent bacteria were then transformed with the GST tagged (1-125) Mdm2 fusion construct. A single colony was picked and transformed cells were grown in LB medium at 37°C to an OD600 of ~0.6 and induction was carried out with 1 mM IPTG at room temperature. Cells were harvested by centrifugation and the cell pellets were resuspended in 50 mM Tris pH 8.0, 10% sucrose and then sonicated. The sonicated sample was centrifuged for 60 mins at 17,000 g at 4°C. The supernatant was applied to a 5 ml FF GST column (Amersham) pre-equilibrated in wash buffer (Phosphate Buffered Saline, 2.7 mM KCL and 137 mM NaCL, pH 7.4) with 1mM DTT. The column was then further washed by 6 volumes of wash buffer. Mdm2 was then purified from the column by cleavage with Precission (GE Lifesciences) protease. 10 units of precission protease, in one column volume of PBS with 1mM DTT buffer, were injected onto the column. The cleavage reaction was allowed to proceed overnight at 4°C.
The cleaved protein was then eluted off the column with wash buffer. Protein fractions were analyzed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator, Millipore. Mdm2 protein samples were then dialyzed into a buffer solution containing 20mM Bis-Tris, pH 6.5, 0.05M NaCl with 1mM DTT and loaded onto a monoS column pre-equilibrated in buffer A (20mM Bis-Tris, pH 6.5, 1mM DTT). The column was then washed in 6 column volumes of buffer A and bound protein was eluted with a linear gradient of 1M NaCL over 25 column volumes. Protein fractions were analyzed with SDS page gel and concentrated using a Centricon (3.5 kDa MWCO) concentrator, Millipore. The cleaved Mdm2(1-125) construct was then purified to 90% purity. Protein concentration was determined using A280 using an extinction coefficients of 10430 M-1cm-for Mdm2 (1-125). To validate the fitting of a 1:1 binding model we carefully determined that the anisotropy value at the beginning of the direct titrations between MDM2 and the FAM-labeled peptide did not differ significantly from the anisotropy value observed for the free fluorescently labeled peptide.

Mdm2 Competitive Fluorescence Anisotropy Assay and Kd Determination
Negative control titrations of the ligands under investigation were also carried out with the fluorescently labeled peptide (in the absence of MDM2) to ensure no interactions were occurring between the ligands and FAM-labeled peptide. In addition we ensured that the final baseline in the competitive titrations did not fall below the anisotropy value for the free FAM-labeled peptide, which would otherwise indicate an unintended interaction between the ligand and the FAMlabeled peptide to be displaced from the MDM2 binding site.

Nanobret Mdm2:p53 Cell Based Assay
HEK293 cells were seeded at a cell density of 250,000 cells per well into a 6 well plate and incubated overnight at 37 ˚C and 5% CO2 in DMEM with 0.3 mg/ml glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum. Each well was transfected with a 1µg:1µg DNA mixture of NanoLuc-MDM2 fusion vector and p53-HaloTag fusion vector using Fugene HD following the manufacturer's instructions (PROMEGA). After a 20 hour overnight incubation period cell media was removed and the cells were washed with PBS saline. Cells were then trypsinised and re-suspended in Opti-MEM media with 10% FCS. Cells were then spun down at 1000 rpm for 5 minutes at room temperature. Supernatant was then discarded and cells resuspended to a density of to 2.

NanoCargo (Tecrea Ltd, UK)
Working peptide solutions were prepared by serially diluting a 10 mM stapled peptide stock solution (100% DMSO v/v) in DMSO to prepare peptide concentrations from 5 mM to 1.5 nM, in addition to the starting 10mM concentration. Stapled peptides were then formulated with NanoCargo by dilution of 1 µl of peptide working solution (100% DMSO v/v) with 18 µl of phosphate buffered saline (PBS), followed by mixing with 1 µl of NanoCargo solution (provided by manufacturer). The peptide:NanoCargo mixture was then further diluted by addition of 380 µl of cell media (with or without 10% FCS). For addition to 96 well cell based assays, cell media was aspirated, followed by addition of 100 µl of peptide:Nanocargo mixture. Negative control treatments were prepared by omitting addition of the NanoCargo mixture and replacing with PBS.
A negative control NanoCargo treatment was also constituted by replace of the peptide component with PBS.