Tripeptides as Integrin-Linked Kinase Modulating Agents Based on a Protein–Protein Interaction with α-Parvin

Integrin-linked kinase (ILK) has emerged as a controversial pseudokinase protein that plays a crucial role in the signaling process initiated by integrin-mediated signaling. However, ILK also exhibits a scaffolding protein function inside cells, controlling cytoskeletal dynamics, and has been related to non-neoplastic diseases such as chronic kidney disease (CKD). Although this protein always acts as a heterotrimeric complex bound to PINCH and parvin adaptor proteins, the role of parvin proteins is currently not well understood. Using in silico approaches for the design, we have generated and prepared a set of new tripeptides mimicking an α-parvin segment. These derivatives exhibit activity in phenotypic assays in an ILK-dependent manner without altering kinase activity, thus allowing the generation of new chemical probes and drug candidates with interesting ILK-modulating activities.


Hot spot calculations
For the in silico identification of hot spots two different approaches were used. First, ILK kinase domain (PDB 3KMW, Chain A), was subjected to FTMap server (http://fmap.bu.edu/) in PDB format. Results were retrieved in PyMOl session format and analysed. 1 FTMap algorithm docks small organic probe molecules with different size, shape and polarity on the surface of the protein submitted. Then, it finds the most favourable positions for each probe type, clusters them, and ranks the clusters based on its energy. Those regions that bind several different probe clusters are selected as consensus clusters or consensus sites and they represent putative binding hot spots.
On the other hand, HotPoint method was also used for the hot spot identification. 2 This approach determines computationally hot spots based on pair potentials and solvent accessibility of interface residues. 3 The accession code 3KMW was introduced into the server and then, results were analysed by means of post-scripts retrieved from web.

Cell culture
Human mesangial cells (HMC) were cultured according to previously described procedures 7 . Briefly, portions of macroscopically normal cortical tissue were obtained from human kidneys immediately after nephrectomy. Isolated glomeruli were treated with collagenase type IA and plated in plastic culture dishes. They were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, l-glutamine(1 mM), penicillin (0.66 g/mL), streptomycin sulfate (60 g/mL),and buffered with HEPES and bicarbonate, pH 7.4, in a 5% CO 2 atmosphere. All Reagents were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) The identity of the cells was confirmed by morphologic and functional criteria. Culture media were changed every 2 days. When the cells reached confluence, they were subcultured at a ratio of 1:4 using the same incubation medium. The cells were serum-deprived for 24 h before the treatments.

MTT viability test
Toxicity tests were achieved in HMC as previously described. 8 Briefly, cells were seeded in 24well plates and, once they reached confluency, they were deprived and treated with the compounds for 24 h. RPMI was supplemented with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Merck KGaA, Darmstadt, Germany) (0.5 mg/mL final concentration) and incubated at 37°C. After 4 h, The medium was removed, and the purple formazan crystals formed were then dissolved by adding 500 μL of dimethyl sulfoxide (DMSO, Merck KGaA, Darmstadt, Germany) and mixed effectively by pipetting up and down. Spectrophotometric absorbance of the purple blue formazan dye was measured using Multimode Plate Reader (Perkin Elmer) at 570 nm. Optical density of each sample was compared with control optical density.

Transfection with siRNA
HMC were lipofectamine-transfected with a mixture of three specific siRNAs against ILK or unspecific, scramble siRNAs) as a control as previously described. 9 siRNAs were purchased at Santa Cruz Biotechnologies, Dallas, TX. A mixture of the three specific oligonucleotides or scramble siRNAs at 20 nM concentration were mixed with 3 μL of Lipofectamine 2000, in 200 μL of Opti-MEM (both reagents from Thermo Fisher Scientific Waltham, MA, USA) and incubated at 37 °C for 20 min. Then, the mixture was added to HMC at 70% of confluence in 800 μL of Opti-MEM and incubated overnight at 37 °C. 72 hours after transfection, cells were treated with the compounds for 24 additional hours.

Immunofluorescence microscopy
For intracellular localisation and quantitative counting of filamentous actin (F-actin), immunofluorescence determination of F-actin was achieved in HMC after the treatments, as previously described 11 . Briefly, HMC were plated on coverslips. After the treatment with the compounds or forskolin, cells were fixed with 4% paraformaldehyde for 30 min and permeabilised with 0.05% Triton-X-100 in phosphate buffered saline (PBS) for 10 min. Cells were incubated with 2.5% bovine serum albumin (BSA) on PBS at room temperature for 1 h and washed afterwards. Reagents from Merck KGaA, Darmstadt, Germany. For intracellular localisation and quantitative counting of filamentous actin (F-actin), cells were incubated with 0.1 µg/mL Alexa 568-phalloidin (Molecular Probes, Thermo Fisher Scientific Waltham, MA, USA) for 45 min at room temperature. Coverslips were washed with PBS and distilled H2O before to ad Vectashield mounting medium that includes nuclear colouring (Vector Laboratories, UK). Confocal images of the coverslips were obtained with a Zeiss LSM 510 Meta confocal laser scanning head attached to a Zeiss. Images were exported and F-actin was quantified with ImageJ software (NIH, USA). S14 2 Experiental procedures for the synthesis of 1 to 8.
All reagents were acquired from the following commercial sources and used without further purifications: Merck, ACROS, Novabiochem, Fluorochem and Alfa Aesar. Solvents were purchased from Scharlab. 1 H and 13 C-NMR spectra were recorded on a either a Varian Mercury VX-300, Varian Unity 300 or Varian Unity 500 MHz spectrometer at room temperature in the deuterated solvent stated. Chemical shifts (δ) are quoted in parts per million (ppm) and referenced using the water peak or solvent residual peak as an internal reference (δH = 3.31 ppm and δC = 79.0 ppm for CD3OD). Multiplicities are denoted as singlet (s), doublet (d), triplet (t), quartet (q), apparent (ap) and multiplet as (m). The abbreviation br denotes a broad resonance peak. Coupling constants (J) were recorded as Hert (Hz).

Kaiser Test
The Kaiser or ninhydrin test for the detection of primary amines was carried out by taking a small amount of dried resin in a small glass tube. To the dried resin were added 2 drops of 5% (w/v) ninhydrin in ethanol, 2 drops of 80% (w/v) phenol in ethanol, and 2 drops of 20 μM potassium cyanide (KCN) in pyridine (0.01M). The tube was then heated for a few seconds at ∽ 120 °C. The test is used routinely to monitor the presence of free amine after deprotection (dark blue color, positive) and the completeness of the amino acid coupling step (yellow color, negative test).

Chloranil Test
The Chloranil test for secondary amine was conducted by taking a small amount of dried resin in a small glass tube. Four drops of 2% p-chloranil (w/v) in toluene were added to the dried resin and next, 10 drops of acetone. The tube was then agitated for few seconds at room temperature. No coloration develops in the beads supporting an Fmoc-protected peptide, while dark green coloration was observed after Fmoc removal.

General Methods for Solid Phase Peptide Synthesis
All peptides were synthesized using standard Fmoc-based solid phase peptide synthesis (SPPS) procedures on 2-chlorotrityl chloride resin (1.55 -1.27 mmol/g) and using commercially available Fmoc-protected amino acids. The synthesis was performed in a 5 mL plastic syringe provided with caps and frit (25 μm pore size, Multi Syn Tech GmbH). Reactors were shaken using IKA RW 20 digital mixer at 73 rpm and set horizontally in a Heidolph Reax 2 overhead shaker. Polypropylene reactors were filtered using a VacMaster multiport vacuum manifold coupled to a water-jet pump.

Resin Swelling
2-Chlorotrityl chloride resin (100 mg) was placed in a clean, dry plastic syringe with DCM (30 mL/g) and capped. The reactor was shaken with DCM (20 mL/g) for 5 min and then, DCM was removed with the vacuum manifold.

Initial Resin Loading
Fmoc-aa-OH (1.5 equiv) was dissolved in dry DMF (30 mL/g of resin) and DIPEA (3 equiv) was added. The final solution was mixed with 2-chlorotrytl chloride resin (100 mg). The suspension was shaken for 1.5 h at room temperature. After this, the reaction was quenched with MeOH (0.8 mL/g of resin) and shaken for further 30 min. Then, the mixture was filtered off and subsequently washed with DMF, MeOH, DCM (30 mL/g, 3 x 3 min).

Fmoc Removal
The Fmoc-aa-resin or Fmoc-peptide-resin (100 mg) of was treated with 20% piperidine in DMF (3 mL) and stirred in a rotational shaker for 30 min at room temperature. Then, the mixture was filtered off and subsequently washed with DMF, MeOH, DCM (30 mL/g, 3 x 3 min). After this, a Kaiser test was carried out to determine the completion of the reaction (blue, positive).

Loading Test
Resin loading was verified by measuring the absorbance of the piperidine-dibenzofulvene adduct formed after Fmoc removal of the first supported amino acid. A 100 µL aliquot of the Fmoccleavage cocktail was taken prior reactor filtration. This was diluted into 10 mL of DCM and then, the absorbance of the mixture at 301 nm was measured by using a UV-Vis spectrophotometer to estimate the level of Fmoc removal, which correlates to the yield of the anchoring step and to the loading of the new resin. The first residue attachment was estimated from the following equation: (mmol/g) = 7800 3000 Where Abs is the absorbance at 301 nm, 7800 is the extinction coefficient (ε) of the piperidinedivenzofluorene adduct and 3000 is a diluting factor.

General Resin Elongation (Standard Fmoc/tBu SPPS)
Fmoc-aa-OH (3 equiv) was dissolved in dry DMF (30 mL/g of resin) then, the corresponding coupling agent (3 equiv) and DIPEA (6.5 equiv) were added and the solution stirred for a few seconds. The final solution was mixed with preswelled NH2-aa-O-resin or NH2-peptide-O-resin in a rotational shaker for 3 h at room temperature.
Then, the mixture was filtered off and subsequently washed with DMF, MeOH, DCM (30 mL/g, 3 x 3 min). Then, a Kaiser test was performed to determine the completion of the reaction (yellow, negative).

N-Terminus acetylation
Peptide-NH2 resin was mixed with DCM/acetic anhydride solution (1:1, 30 mL/g of resin) and shaked for 30 min at room temperature. Then, the mixture was filtered off and subsequently washed with DMF, MeOH, DCM (30 mL/g, 3 x 3 min). After this, a Kaiser test was carried out to determine the completion of the reaction (yellow, negative).

Resin cleavage and lateral chain deprotection
A) N-acetil-peptide resin was mixed with a TFA/TIPS/H2O 9:0.5:0.5 solution (3 mL, 30 mL/g of resin) and shaken for 3 h at room temperature. Subsequently, the mixture was filtered, solvents were removed under vacuum and the residue was precipitated in cold ether and sonicated to afford a white solid that was sonicated, centrifuged and washed twice with diethyl ether to yield the final product.
B) N-acetil-peptide resin was mixed with 4M HCl in 1,4-dioxane/TIPS/MeOH solution 9:0.5:0.5 (30 mL/g of resin) and shaked for 5 h at room temperature. Subsequently, the mixture was filtered, solvents were removed under vacuum and the residue was precipitated in diethyl ether to afford a white solid that was sonicated, centrifuged and washed twice with diethyl ether to yield final product.