Structural characterization and in vivo evaluation of β-Hairpin peptidomimetics as specific CXCR4 imaging agents

Abstract

The CXCR4 chemokine receptor is integral to several biological functions and plays a pivotal role in the pathophysiology of many diseases. As such, CXCR4 is an enticing target for the development of imaging and therapeutic agents. Here we report the evaluation of the POL3026 peptidomimetic template for the development of imaging agents that target CXCR4. Structural and conformational analyses of POL3026 and two of its conjugates, DOTA (POL-D) and PEG12-DOTA (POL-PD), by circular dichroism, two-dimensional NMR spectroscopy and molecular dynamics calculations are reported. In silico observations were experimentally verified with in vitro affinity assays and rationalized using crystal structure-based molecular modeling studies. [(111)In]-labeled DOTA conjugates were assessed in vivo for target specificity in CXCR4 expressing subcutaneous U87 tumors (U87-stb-CXCR4) through single photon emission computed tomography (SPECT/CT) imaging and biodistribution studies. In silico and in vitro studies show that POL3026 and its conjugates demonstrate similar interactions with different micelles that mimic cellular membrane and that the ε-NH2 of lysine(7) is critical to maintain high affinity to CXCR4. Modification of this group with DOTA or PEG12-DOTA led to the decrease of IC50 value from 0.087 nM for POL3026 to 0.47 nM and 1.42 nM for POL-D and POL-PD, respectively. In spite of the decreased affinity toward CXCR4, [(111)In]POL-D and [(111)In]POL-PD demonstrated high and significant uptake in U87-stb-CXCR4 tumors compared to the control U87 tumors at 90 min and 24 h post injection. Uptake in U87-stb-CXCR4 tumors could be blocked by unlabeled POL3026, indicating specificity of the agents in vivo. These results suggest POL3026 as a promising template to develop new imaging agents that target CXCR4.

Keywords: CXCR4; SPECT/CT; chemokine; chemokine receptor; molecular imaging.

Figure 1

CD spectroscopy of POL, POL-D, and POL-PD. (A) Schematic depiction of POL3026 and the derivatives. The CD spectra of (B) POL, (C) POL-D, and (D) POL-PD at pH 7.4 in detergent-free phosphate buffer (black) and the following micellar solutions DPC (red), SDS (green), DPC:SDS 9:1 (blue), and DPC:SDS 5:1 (cyan).

Figure 2

NMR structural analysis of POL. (A) The fingerprint region of the ¹H–¹H TOCSY spectrum recorded for POL in DPC micellar solution at 310 K and pH 5.5. (B) Graphical representation of NOE effects and the vicinal coupling constants, ³J_HNHα [Hz], observed for POL in DPC micellar solution at 310 K and pH 5.5. The thickness of the lines corresponds to NOE intensities. Disulfide bridge and head-to-tail linkage are shown by a solid line. Asterisk in 2B corresponds to d_αδ(i,i+1) connectivity.

Figure 3

Molecular dynamic simulations of POL, POL-D, and POL-PD. (A) Diffusion of POL from the hydrophobic core of the DPC micelle to the interface over molecular dynamic time. (B) Stereoview of 20 conformers of POL obtained in the last steps of 24 ns molecular dynamics simulations with time-averaged distance constraints and φ dihedral angle constraints as well as POL structure obtained after 24 ns of MD simulations. The side chains of Arg², 2-Nal³, Tyr⁵, and Arg¹⁴ that crucial for binding to CXCR4 are marked in red. (C) Stereoview of a comparison of the 3D structure of POL obtained in this study (cyan) with the 3D crystal structure of CVX15 (orange) extracted from the CXCR4-CVX15 complex taken from PDB database (3OE0). (D) Surface electrostatic potential of POL. Electrostatic potential is presented as blue for positive and white for neutral potentials. Figure was prepared with MOLMOL. Position of (E) POL-D and (F) POL-PD in the DPC micelle after 24 ns of MD simulations. (G) Stereoview of a comparison of the 3D structures of POL (blue), POL-D (green), and POL-PD (firebrick) obtained after 24 ns of MD simulations.

Figure 4

POL peptidomimetics affinity. (A) Representative normalized curves of in vitro inhibition of CXCL12-red binding to CXCR4, illustrating decrease of IC₅₀ upon transition from CVX15 to POL and conjugation of DOTA or PEG₁₂-DOTA. (B) Superimposed binding modes of CVX15 (red), POL (blue), and POL-D (green) to CXCR4 as predicted by an in situ ligand minimization protocol (Discovery Studio 3.1 client). The in situ experiments used 3OE0 X-ray coordinates of CXCR4. The protein is presented as a light gray line ribbon. (C) In vitro binding of [¹¹¹In]POL-D and POL-PD to U87 and U87-stb-CXCR4 cells; ***, P < 0.001.

Figure 5

Specificity of POL-D and POL-PD binding to CXCR4. Ex vivo biodistribution of [¹¹¹In]POL-D and [¹¹¹In]POL-PD in NOD/SCID mice bearing U87 and U87-stb-CXCR4 glioblastoma xenografts brain tumor (n = 3), presented as percent of injected dose per gram tissue (% ID/g) at 90 min post injection (A). (B) Blocking experiments, in which mice were injected with different doses (5, 15, and 45 µg) of POL3026 30 min prior to the administration of [¹¹¹In]POL-D. (C) Ex vivo biodistribution 24 h after administration of [¹¹¹In]POL-D and [¹¹¹In]POL-PD. **, P < 0.01; ***, P < 0.001.

Figure 6

Imaging CXCR4 expression with POL peptidomimetics: Representative whole body SPECT/CT images of NOD/SCID mice bearing U87 and U87-stb-CXCR4 glioblastoma xenografts on left (unfilled arrow) and right (solid arrow) flanks, respectively, recorded (A) 2 h and (B) 24 h after tail vein injection of ~350 µCi of [¹¹¹In]POL-D (left panel) and [¹¹¹In]POL-PD (right panel). All images were scaled to the same maximum threshold value. K, kidney; L, liver; B, bladder. Images clearly indicate that CXCR4 expression can be detected using both radiotracers due the high and specific accumulation in U87-stb-CXCR4 tumors.