A Structure-Activity Relationship Study of Bimodal BODIPY-Labeled PSMA-Targeting Bioconjugates

Abstract

The aim of this study was to identify a high-affinity BODIPY peptidomimetic that targets the prostate-specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure-activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu-CO-Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA-positive LNCaP cells. One compound was identified with comparable affinity (IC₅₀ =21.5±0.1 nM) to Glu-CO-Lys-Ahx-HBED-CC (PSMA-11) (IC₅₀ =18.4±0.2 nM). Radiolabeling was achieved by Lewis-acid-mediated ¹⁹ F/¹⁸ F exchange in moderate molar activities (∼0.7 MBq nmol^-1 ) and high radiochemical purities (>99 %) with mean radiochemical yields of 20-30 %. Cell internalization of the ¹⁸ F-labeled high-affinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMA-mediated internalization over time. By fluorescence microscopy, localization of the high-affinity BODIPY-PSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a high-affinity BODIPY-PSMA conjugate has been identified as a suitable candidate for the development of PSMA-specific dual-imaging agents.

Keywords: BODIPY; PET; PSMA; bimodal imaging; fluorescence imaging.

Scheme 1

A) difluoroboron dipyrromethene (BODIPY) scaffold. B) BODIPY dyes 1 with a carboxylic acid functionality for bioconjugation and its corresponding NHS ester 2 used in this study.

Scheme 2

Synthetic procedure for the preparation of BODIPY‐PSMA bioconjugates 3–6 using a combination of solid‐phase and solution‐based chemistry.

Figure 1

ORTEP representation of 2 with thermal ellipsoids drawn at 50 % probability level.

Figure 2

Normalized A) absorption and B) emission spectra (λ_exc=460 nm) of 1 in 95 % TFA at different time points. Normalized C) absorption and D) emission spectra (λ_exc=460 nm) of 1 in 1 M SnCl₄ in acetonitrile at different time points.

Scheme 3

Representative radiolabeling of BODIPY‐PSMA conjugate 4 by Lewis acid mediated isotopic ¹⁹F/¹⁸F exchange.

Figure 3

A) Representative UV/vis HPLC chromatogram of 4 (insert: MS spectrum of 4). B) Radio‐HPLC chromatogram of [¹⁸F]F‐4. C) Representative UV/vis HPLC chromatogram of 4 without BF₂ (insert: MS spectrum of 4 ‐BF₂). D) UV/vis HPLC chromatogram of [¹⁸F]F‐4 showing loss of the BF₂ entity during radiolabeling by Lewis acid mediated isotopic ¹⁹F/¹⁸F isotopic exchange.

Figure 4

PSMA‐specific surface bound and internalized fractions of [¹⁸F]F‐4 in LNCaP cells. Values are given as mean±SD. Results are from two independent experiments in triplicate.

Figure 5

PSMA‐mediated uptake of 4 into LNCaP cells, confirmed by SIM‐fluorescence microscopy over time at 25 °C at A) 15 min, B) 30 min, C) 1 h, D) 1 h at 4 °C, E) 1 h+2‐PMPA, F) DAPI control.