Tailored chondroitin sulfate glycomimetics via a tunable multivalent scaffold for potentiating NGF/TrkA-induced neurogenesis

We have engineered structurally well-defined tunable chondroitin sulfate glycopeptides using a polyproline scaffold to selectively modulate the NGF-mediated neuronal signaling pathway.


2-Propargyl O-(sodium-β-D-glucopyranosyluronate)-(1→3)-2-deoxy-2-acetamido-β-D-galactopyranoside 12:
Compound      (1)   FTIR was used to monitor the azide vibrational band (~2100 cm -1 ) for the completion of the click coupling reaction. Fourier transform infrared spectroscopy (FTIR) was carried out using a Perkin Elmer FTIR Spectrum 100 between 4000 and 800 cm −1 at a spectral resolution of 4 cm −1 , and the number of scans was 4. Samples were placed on a germanium stage and pressed before every measurement. The disappearance of the azide band in the spectra of the glycopeptides indicated the completion of the coupling reactions ( Figure S7). S13 Figure S7. FT-IR spectra of polyprolines and glycopeptides. S14 Circular Dichroism analysis: CD spectra were recorded on an Aviv 410 circular dichroism spectrometer equipped with temperature controller. Glycopeptide solutions at concentrations of 200 μM (2.4 mM for compound 12 and 13)

2-Propargyl
were used. All sample solutions made in 10 mM sodium phosphate-dibasic buffer, pH 7.0, were equilibrated for 24 hr at 4 °C and then 1 hr at room temperature before CD measurements. Cells of 1 mm path length were used.
Spectra were recorded from 260 to 190 nm at 25 °C. Mean residue ellipticity [θ] was calculated as follows; [θ] = θ/(10·N·c·l) θ represents the ellipticity in millidegrees, N the number of amino acid residues, c the molar concentration in mol·L -1 , and l the cell path length in cm.   Maestro 9.6 (www.schrodinger.com). The disaccharide units were built using Maestro and a short polyproline chain was built based on X-ray crystallography structure in literature. 3 It was then lengthened by connecting multiple copies of the short fragment. Disaccharide units were then added at the appropriate locations along the polyproline chain. The chain geometry was fixed, and the disaccharide side chain conformation was energy minimized using OPLS_2005 until the rms deviation was less than 0.1 Å individually.
To determine the binding site, the NGF/TrkA complex was first loaded from the indicated PDB file 4 and the water molecules removed for simplicity. Coarse binding sites were first identified by examining positively charged residues on the NGF/TrkA complex. Rigid body docking of the glycopeptide was then performed manually and at least Arg314 or Arg342 on TrkA was included in the binding site. The glycopeptide was brought close to the positively charged residues such that the sugar pendants were within 3Å of the positive residue while avoiding steric clashes. Bonds in the disaccharide units and interacting residues on NGF/TrkA were rotated to maximized potential interactions and the energy was briefly minimized as described above. Potential hydrogen bonding partners on NGF/TrkA were then identified. This was done by first identifying residues in the vicinity of the sugar pendants that can participate in hydrogen bonding (Ser, Asp, Glu, Thr, Tyr). The side groups of these residues and the sugar pendants were then rotated to bring them into optimal hydrogen bonding distance (2 -4 Å) and bond angle (~170 to 180° between O-H-O). If these manipulations failed, hydrogen bonding between identified residue and sugar pendant was deemed unlikely and any structural changes were undone. Large manipulations to sugar pendants and participating residues were eschewed as much as possible to avoid potential disruptions to electrostatic interactions identified above. Energies of the interacting residues and interacting sugar pendants were then minimized. Western blot. PC12 cells were seeded at a density of 300 cells/mm 2 and one coverslip was used for each treatment (~40,000 cells). Cells were starved in differentiation medium for 12 -18 hr before use. For glycopeptide treatment, cells were incubated with fresh differentiation medium containing 10 μM glycopeptide for 1 hr at 37 °C. To induce TrkA phosphorylation, cells were treated with 4 ng/mL NGF for 5 minutes at 37°C. Cells were washed once with ice cold PBS and lysed using RIPA lysis buffer (Santa Cruz Biotechnology SC-24948) containing protease and phosphatase inhibitors (Thermo Scientific 88668). The lysates were concentrated and purified by overnight ethanol precipitation at -20 °C. Protein pellets were obtained by centrifuging at 14,000 g for 30 minutes in a 4 °C pre-cooled centrifuge. The pellets were dissolved in 1x LDS sample buffer (Life Technologies NP0008), separated on 4 -12 % SDS-PAGE gel (Life Technologies NP0321BOX) and transferred to nitrocellulose membrane. Membranes were blocked with 5% milk in TBST (for total TrkA) or 5% BSA in TBST (for pTrkA) for 1 hr at room temperature and probed with anti-TrkA (763) rabbit antibody (1:1500; Santa Cruz Biotechnology SC-118) and anti-pTrkA (Tyr490) rabbit antibody (1:1500; Cell Signaling 9141L) overnight at 4 °C, followed by 2 hr incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Promega W4011).
Enhanced chemiluminescence system (GE Healthcare RPN 2232) was used for detection and images were captured using Bio-Rad ChemiDoc TM MP imaging system. The bands were quantified using ImageJ