Development of “clickable” albumin binders and application to theranostic radioligands for somatostatin receptor subtype 2

Despite given high affinity and selectivity towards their distinct biological targets, theranostic radioligands can suffer strongly from fast blood clearance and metabolism, which limits the enrichment of the compounds in the tumour tissue. A viable strategy to increase the bioavailability of substances is the conjugation to human serum albumin (HSA). In this context, Nε-4-(4-iodophenyl)butanoyllysine (Nε-IPB-lysine) was recently discovered as potent albumin binder.[1] However, conjugation of Nε-IPB-lysine via its α-amino group to proteins, peptides or small molecules is challenging, as a free α-carboxyl group is required to maintain good binding to HSA.
To allow the broad applicability and late-stage introduction of Nε-IPB-lysine we present a novel approach of Cu-catalyzed azide-alkyne cycloaddition (CuAAC) using Nε-IPB-lysine derivatives with azide/alkyne bearing moieties at the α-amino group. For small scales, a solid phase synthesis strategy starting from Fmoc-Lys(Alloc)-OH was developed. For upscaling, a 3-step synthesis starting from Boc-Lys-OH has been established. The HSA binding affinities were determined using microscale thermophoresis and a fluorescence-based competition assay. A library of azide/alkyne containing compounds with a binding affinity to HSA ranging from 0.4 µM to 100 µM could be obtained.
To demonstrate the suitability of the novel albumin binders, Nα-5-azidopentanoyl-Nε-IPB-D-lysine (Kd = 8 µM) was coupled to the somatostatin receptor subtype 2 ligand NODAGA-Pra-O2Oc-TATE 1 by on-resin CuAAC. For radiolabelling, copper-64 was used. The resulting conjugate [64Cu]Cu-2 showed significantly improved binding to HSA compared to [⁶⁴Cu]Cu-1, [⁶⁴Cu]Cu-NODAGA-TATE, and [⁶⁴Cu]Cu-DOTA-TATE. All TATE derivatives were studied by PET imaging using a mouse phaechromocytoma (MPC)-allograft model highlighting an increased blood circulation time of [⁶⁴Cu]Cu-2 and higher tumor uptake at late time points (24 h) compared to the other radiotracers. Current studies aim to further optimise the biodistribution by selecting HSA binders of higher or lower binding affinity.

Literature:

[1] Dumelin et al. Angew. Chem. Int. Ed. Engl. 2008, 47(17), 3196-3201.