Nerve Targeting via Myelin Protein Zero and the Impact of Dimerization on Binding Affinity

Abstract

Background: Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin protein zero (P0), we intend to support fluorescence-guided nerve-sparing surgery. To that end, we aimed to develop a dimeric tracer that shows a superior affinity for P0. Methods: Following truncation of homotypic P0 protein-based peptide sequences and fluorescence labeling, the lead compound Cy5-P0101–125 was selected. Using a bifunctional fluorescent dye, the dimeric Cy5-(P0101–125)2 was created. Assessment of the performance of the mono- and bi-labeled compounds was based on (photo)physical evaluation. This was followed by in vitro assessment in P0 expressing Schwannoma cell cultures by means of fluorescence confocal imaging (specificity, location of binding) and flow cytometry (binding affinity; KD). Results: Dimerization resulted in a 1.5-fold increase in affinity compared to the mono-labeled counterpart (70.3 +/− 10.0 nM vs. 104.9 +/− 16.7 nM; p = 0.003) which resulted in a 4-fold increase in staining efficiency in P0 expressing Schwannoma cells. Presence of two targeting vectors also improves a pharmacokinetics of labeled compounds by lowering serum binding and optical stability by preventing dye stacking. Conclusions: Dimerization of the nerve-targeting peptide P0101–125 proves a valid strategy to improve P0 targeting.

A Truncated 14-Amino-Acid Myelin Protein-Zero-Targeting Peptide for Fluorescence-Guided Nerve-Preserving Surgery

“…Image acquisition and processing for the assessment of P0 staining were performed using LASX software (Leica Application Software Suite 4.8). Image analysis (3D) was performed using Image J [ 26 ]. For the affinity assessment, fluorescence was measured using a FACSCanto II flow cytometry device (BD Biosciences, Franklin Lakes, NJ, USA) in the APC-A channel, and the accompanying FlowJo TM v5 software (BD Biosciences, Franklin Lakes, NJ, USA) was used for further analysis.…”

“…The sequences of P0 peptides, Sulfonate-(SO 3 )Cy5(SO 3 )-Maleimide, and its precursors were synthesized according to earlier reported procedures [ 26 , 31 , 32 ]. For synthesis of the Cy7 analogue, the precursor Sulfonate-(SO 3 )Cy7(SO 3 )-COOH [ 33 ] was dissolved in dry DMSO (5 mL), followed by the addition of PyBOP (2.5 eq), N-(2-aminoethyl)maleimide (1.2 eq), and N-methyl morpholine (4 eq).…”

“…Image acquisition and processing were performed using LASX software (Leica Application Software Suite 4.8). Image analysis, including (3D) surface plot and co-localization analysis, was performed using Image J [ 26 ].…”

“…Over the last years, a number of such tracers have been developed [ 18 ], including non-conjugated fluorescent dyes [ 19 , 20 , 21 , 22 ], peptides with either unknown nerve-related or intracellular targets such as myelin basic protein (MBP; [ 23 , 24 , 25 ]), or peptides that target myelin protein zero (MPZ, P0). The latter of these is the most abundant protein present in myelin [ 26 , 27 , 28 ]. The pursuit of the targeting of nerve-specific proteins is in line with conventional tumor receptor-targeted molecular imaging strategies [ 29 ].…”

“…This yielded the P0-specific lead compound Cy5-P0 101–125, consisting of 26 amino acids that exhibited a binding affinity of 104 ± 17 nM [ 27 ]. Dimerization of this peptide (Cy5-(P0 101–125 ) 2 ; [ 26 ]) yielded a 1.5-fold increase in affinity but almost doubled the molecular weight. In vitro and in vivo evaluation using fluorescence immunohistochemistry of stained nerve tissue, blocking and FLIZA studies, and in vivo and ex vivo imaging after local intravenous and intraneural administration showed the robustness of this P0-taregting concept [ 26 ].…”