The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvβ6-integrin targeted radiotherapeutics

Abstract

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvβ6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvβ6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

Fig. 1. Structures of compounds discussed in the text.

Fig. 2. Synthesis of tetrameric conjugates of αvβ6-integrin binding peptides. Synthesis of Y0, Y4, and Y8 was done by reacting DOTPI-tetraazide with the peptides FF, YF, and YY, respectively. The asymetrical conjugate Y3 was isolated from the CuAAC reaction of DOTPI-tetraazide with a mixture of FF and YF by means of preparative HPLC. Functional groups related to the CuAAC are highlighted in red.

Fig. 3. Structure of the tetramer GFK-Y4 obtained by CuAAC of DOTPI-tetraazide with GFK-FY, a peptide building block comprising a mono-tyrosine cyclic peptide as featured in FY and the brush-border cleavable linker GFK (for a full synthesis scheme, see Experimental section). Functional groups related to the CuAAC are highlighted in red.

Fig. 4. Biodistribution data for ¹⁷⁷Lu-labeled peptide tetramers in H2009 (human lung adenocarcinoma) xenografted mice. For details and numerical data in tabular form, see ESI.

Fig. 5. Biodistribution in H2009 tumor mice, and resulting tumor-to-organ ratios for the ¹⁷⁷Lu-labeled tetramer Y8 at different time points after injection. For details and numerical data in tabular form, see ESI.

Fig. 6. Single-photon emission computed tomography (SPECT) of a SCID mouse bearing a human H2009 tumor xenograft on the right shoulder, recorded at 4 time points (1 h, 3 h, 24 h, and 72 h) after i.v. injection of 36.5 MBq of ¹⁷⁷Lu-Y8, scan time 1 h. Tumor position is indicated by white dotted circle. Adjacent figures denote ROI-based tumor uptakes in % IA mL⁻¹.

Fig. 7. Comparison of trends in polarity proxies and liver uptakes, 3 d p.i. Connecting lines between data points are intended only to visualize trends and do not indicate a functional correlation.

Fig. 8. Plots of liver uptakes against octanol–water distribution coefficients (log D) for a) peptide tetramers (this work) and b) Trivehexin-related trimers reported earlier (RSC Med. Chem. 2023, 14, 2429). Large-size circles in b highlight the conjugates of the trimer series (Y0, Y2F, Y3F, and Y6, see ref. 34, Fig. 1) which, in terms of percentage and distribution of Phe/Tyr units, are most equivalent to the four tetramers in this work. Color codes of circles correspond to the ones used for the respective compound series; for trimers see ref. , Fig. 2a; for the tetramers, see this work, Fig. 4. Note that similar colors in a and b do not necessarily indicate a structural match.

Fig. 9. Synthesis of the building block GFK-FY, a derivative of FY featuring the brush border cleavable linker GFK.