Fluorescent vinblastine probes for live cell imaging

Abstract

Herein we describe the synthesis of several fluorescent analogues of the clinically approved microtubule destabilizing agent vinblastine. The evaluated probes are the most potent described and provides the first example of uptake, distribution and live cell imaging using this well known antimitotic agent.

Figure 1

(a) Vinblastine bound between two α/β tubulin heterodimers (Protein Data Bank ID code 1Z2B). (b) Structure of vinblastine. (c) A portion of the vindoline subgroup is solvent exposed, providing a point for fluorophore attachment.

Figure 2

(a) IC₅0 curves of vinblastine, vinblastine-fluorophores 2a/2b, 3a/3b, 4a/4b, and commercially available vinblastine-BODIPY (VBLS) in OVCA429 cells using a fluorometric assay. (b) Plot of tubulin polymerization assay. K_d represents the time it takes to reach half maximal microtubule density with 2 μM of agonist (see experimental). * Non-significant compared to vinblastine.

Figure 3

High-resolution live cell imaging of the most potent vinblastine conjugate 2a in HT1080 cells expressing tubulin RFP. Cells were incubated for 3 h at (a) 100 μM (b) 10 μM, and (c) 1 h at 1 μM.

Figure 4

Live cell imaging of vinblastine-fluorophore conjugates in OVCA429 cells transfected with CellLight^® RFP tubulin. The cells were incubated with 100 μM of probe for 3.5 h in media followed by a single wash and then imaged. Average paracrystal length is displayed at the top right. Bar = 10 μm.

Scheme 1

(a) Synthesis of vinblastine-fluorophore conjugates. (b) Excitation and emission spectra of vinblastine conjugates in phosphate buffered saline (PBS).