Towards optimizing peptide-based inhibitors of protein–protein interactions: predictive saturation variation scanning (PreSaVS)

A simple-to-implement and experimentally validated computational workflow for sequence modification of peptide inhibitors of protein–protein interactions (PPIs) is described.


Supplementary Figures
. Analysis of the relationship between helicities determined from CD data and MCL-1 binding affinity for NOXA 75-93 variants (values in kJ/mol). Squares are L78 variants, circles denote V85 variants, numbers in brackets denote fractional helical propensities of amino acid at the stated position). 3 The data suggest that small decreases in helicity areobserved for all variant peptides regardless of the helical propensity of the variant amino acid, reinforcing the conclusion that the amino acid is tolerated in terms of MCL-1 recognition.

Computational Methods
Input for PreSaVS is simply a PDB file of the complex in question. We used the first models of the NMR structures of the NOXA-B/MCL-1 complex (2JM6) and the SIM/SUMO complex (2LAS). A program, saturation_mutagenesis.py, was written in Python3 to perform PreSaVS, leveraging functions and methods from BudeAlaScan. 4 The data presented here were generated with the two commands: saturation_mutagenesis.py full -v -p 2jm6.pdb -l A > 2jm6_full.log saturation_mutagenesis.py full -v -p 2las.pdb -l B > 2las_full.log By default, the method requires a PDB file with two protein chains and identifies the interfacial residues between them. Each interfacial residue position is replaced with one of 16 proteinogenic amino acids (i.e., standard residues except Ala, Gly, Pro and Cys, noting Ala is covered under BudeAlaScan/BAlaS) 4, 5 and scored with the BUDE forcefield with the rotamer correction previously described. 4 Each substitution gives an interaction energy and subtracting this from the interaction energy of the native residue gives a value in kJ/mol, such that a residue stabilising the interface with respect to the native residue will give a positive value The software is available on demand via github see: https://github.com/richardbsessions/BUDE_SM

Solid Phase Peptide Synthesis
General Remarks: All amino acids and resins were purchased from either Novabiochem (Merck) or Sigma-Aldrich. All amino acids were N-Fmoc protected and side chains protected with Boc (Lys); O t Bu (Asp, Ser, Thr); Trt (Asn, Gln); Pbf (Arg). Synthesis of all peptides was performed using a microwave assisted automated peptide synthesiser (CEM, Liberty or Liberty Blue). Coupling of 6-aminohexanoic acid, γ-aminobutyric acid and N-terminal labelling were performed manually. DMF used in peptide synthesis was of ACS grade and from Sigma Aldrich. Peptides were synthesised on an 0.1 mmol scale and split before acetylation and fluorescent ligation. Lyophilisation was performed using a BenchTop Pro with Omnitronics TM from VirTis SP Scientific. Preparative HPLC was performed on an Agilent Technologies 1260 infinity controller in conjunction with a diode array detector. Analytical HPLC was performed on an Agilent Technologies 1260 infinity controller in conjunction with a diode array detector. Mass spectrometry data were obtained on a Bruker Daltonics micrOTOF using electrospray ionisation (ES)MS.
Cycles for Automated Peptide Synthesis: Peptides that were prepared on a microwave assisted Liberty CEM peptide synthesiser followed this cycle: Resin Loading -Clean reaction vessel; wash with DMF, wash with CH 2 Cl 2 ; transfer resin to reaction vessel; wash with DMF, wash with CH 2 Cl 2 ; vessel draining.
For methods that did not use microwave assistance, the reaction cycle was the same, expect the microwave method for deprotection and coupling was replaced by agitation of the resin at rt for 10 min and 90 min respectively.
After the final residue, the resin was ejected from the reaction vessel and linker coupling, capping, cleavage and deprotection was performed manually.
Peptides that were prepared on the microwave assisted Liberty Blue CEM peptide synthesiser followed this cycle: After the final residue, the resin was ejected from the reaction vessel and linker coupling, capping, cleavage and deprotection was performed manually using methods A to F For the microwave methods used, the temperature and total time is shown below:

N-terminal acetylation
Acetic anhydride (10 equiv.) and DIPEA (10 equiv.) were dissolved in DMF (1 mL) and the solution was transferred to the resin. After 2 h, the resin was drained, washed with DMF (3 × 2 mL × 2 min) and successful capping determined by a negative Kaiser test.
Cleavage and deprotection of Rink amide MBHA resin After elongation and N-terminal capping was complete, the resin was washed with CH 2 Cl 2 (5 × 2 mL × 2 min), Et 2 O (5 × 2 mL × 2 min) and dried under vacuum for ca. 2 h. Peptides were simultaneously cleaved and side-chain deprotected using 'Reagent K' (TFA:EDT:Thioanisole:Phenol:H 2 O 82:3:5:5:5; 3 × 2 mL × 2 h). The solution was precipitated in ice-cold Et 2 O (25 mL) and placed in a centrifuge (3000 rpm × 10 min), the supernatant removed and the precipitate resuspended in ice-cold Et 2 O and placed in a centrifuge again. This process was repeated 3-4 times and the precipitate was dried under a stream of nitrogen overnight, before being dissolved in H 2 O and lyophilised.

Peptide Purification
In general, peptides were purified by automated RP column chromatography on a Biotage Isolera 1.3.3., using a RediSep ® Rf gold reversed phase C18 column by Teledyne Isco on an increasing gradient of acetonitrile (5-50%) in water + 0.1% TFA (v/v) at a flow rate of 12 mL min -1 . Crude peptides were suspended in H 2 O as concentrated as possible, fractions were checked by LCMS, concentrated in vacuo and lyophilised. Peptides were purified further by preparative UV-or MD-HPLC using a Jupiter Proteo preparative column (reversed phase) on an increasing gradient of acetonitrile in water + 0.1% formic acid (v/v) at a flow rate of 10 mL min -1 . Crude peptides were suspended in H 2 O at an approximate concentration of 20 mg mL -1 . Purification runs injected a maximum of 0.9 mL of crude peptide solution and were allowed to run for 30 min, with acetonitrile increasing at a stated gradient. In regards to UV-HPLC, the eluent was scanned with a diode array at 220, 210 and 280 nm. For MD-HPLC, the mass directed chromatography software Masshunter by ChemStation (Agilent) was used to allow the collection of the desired peptide by mass, with the eluent split into an Agilent 6120 Quadropole LCMS which triggers collection of eluent at a programmed m/z. Fractions containing purified peptide were combined, concentrated in vacuo and lyophilised.

Peptide Characterization Data
Tabulated HRMS data of synthesised peptides are shown below. Peptide identity was confirmed by the inspection of multiple charge states and are quoted as the monoisotopic peak for the Expected (Exp d ) and Observed (Obs d ) masses. hSUMO-1 18-97 was expressed and purified as previously described from a pet19b His-TEV-SUMO-1 (18-97) construct with cleavage of the tag using TEV protease. 4

Fluorescence Anisotropy
Fluorescence anisotropy assays were performed in 384-well plates (Greiner Bio-one). Each experiment was run in triplicate and the fluorescence anisotropy measured using a Perkin Elmer EnVisionTM 2103 MultiLabel plate reader, with excitation at 480 nm (30 nm bandwidth), polarised dichroic mirror at 505 nm and emission at 535 nm (40 nm bandwidth, S and P polarised). All assays were performed in 20 mM Tris, 100 mM NaCl, 0.1 mM DTT, pH 7.5 unless otherwise stated and data analysed following previously published methods.
Fluorescence anisotropy data was processed as described previously. [2] Briefly. the data from both the P (perpendicular intensity) and S (parallel intensity) channels, resulting from this measurement and corrected by subtracting the corresponding control wells, were used to calculate the intensity and anisotropy for each well following Equations 1 and 2: For direct titration the average anisotropy (across three experimental replicates) and the standard deviation of these values were then calculated and fit to a logistic model using Direct binding assays: Fluorescence anisotropy direct titration assays were performed with protein concentration diluted over 16-24 points using 2-fold dilutions. Followed by addition of tracer peptide was added to the wells. For control wells, the tracer peptide was replaced with an identical volume of assay buffer. Plates were read after 45 minutes.
Competition binding assays: For control wells, the tracer peptide was replaced with an identical volume of assay buffer. The total volume in each well was 60 μL. Plates were read after 45 minutes of incubation at room temperature.

Isothermal Calorimetry
ITC experiments were carried out using a Microcal ITC200i instrument (Malvern) at 25°C in 20 mM Tris, 150 mM NaCl, pH 7.5 buffer. The protein of interest was dialysed against the buffer prior to experiment, and lyophilized peptides were dissolved in the same buffer. Protein was present in the cell and titrated with peptide solutions loaded into the syringe using 20 x 2 uL injections with 120 s spacing between the injections. Heats of peptide dilution were subtracted from each measurement raw data. Data was analysed using Microcal Origin 8 and fitted to a one-binding site model

Circular Dichroism
Spectra were recorded on a Chirascan circular dichroism spectropolarimeter (Applied Photophysics), at 20°C, using 1 mm cells and a scan speed of 5 nm/min. The spectra were averaged over 3 repeats with a buffer baseline subtracted. Peptide concentrations of approximately 0.1 mg/mL were used (although the exact concentration was used to allow determination of MRE). The solvent signal was subtracted to the raw circular dichroism data obtained for the peptides before conversion to the mean residue ellipticity (MRE).