Tubulin derived peptides as optical imaging probes targeting RHAMM

Objectives The receptor for hyaluronan-mediated motility (RHAMM) is a hyaluronan (HA) binding protein expressed in breast cancer lines that exhibit progenitor capability and thus development of an imaging probe for this biomarker could aid in breast cancer imaging. The carboxy terminal tail (CTT) region of tubulins has been shown to interact with kinesins, which have structural homology to RHAMM. We hypothesize that peptide fragments derived from tubulin CTTs may have affinity for RHAMM and may be useful compounds for developing a molecular imaging probe.

Methods 12-mer peptides corresponding to the CTT of different tubulin subtypes were synthesized, biotinylated and their affinities for RHAMM-CT were assessed using ELISA. Candidate peptides were conjugated to FITC and their specificities for MDA-MB-231 cells were evaluated using a cellular fluorescence assay. Peptides were also conjugated with cysteine for immobilization to a gold-plated sensor and their binding affinities determined using surface plasmon resonance (SPR) spectroscopy.

Results Tubulin CTTs corresponding to the α- and β-subtypes showed affinity for RHAMM as indicated by ELISA studies, with α-CTT having the highest affinity. The breast cancer cell line MDA-MB-231 showed intra-cellular fluorescence when incubated with the dye-conjugated peptides. Cells blocked with anti-RHAMM showed 25-45% reduction in cellular fluorescence with the α-CTT variant demonstrating the highest specificity. Using SPR, the binding affinities (K_D) of the cysteine-conjugated peptides were evaluated, with α-CTT having a K_D of 24±2 nM and β-CTT a K_D of 32±3 nM. In addition, peptide sequences directly adjacent to CTTs with reported affinity for kinesins were evaluated.

Conclusions These experiments indicate that α- and β-CTT were able to specifically target RHAMM expressing tumour cells. Further assessment of these optical-based probes may facilitate translation into isotope-based imaging probes.

Research Support OIC