Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 7;3(1):69-78.
doi: 10.1039/d1cb00036e. eCollection 2022 Jan 5.

Synthesis and preliminary evaluation of octreotate conjugates of bioactive synthetic amatoxins for targeting somatostatin receptor (sstr2) expressing cells

Alla Pryyma  1 , Kaveh Matinkhoo  1 , Yong Jia Bu  1 , Helen Merkens  2 , Zhengxing Zhang  2 , Francois Bénard  2 , David M Perrin  1
Affiliations

Synthesis and preliminary evaluation of octreotate conjugates of bioactive synthetic amatoxins for targeting somatostatin receptor (sstr2) expressing cells

Alla Pryyma et al. RSC Chem Biol. .

Abstract

Targeted cancer therapy represents a paradigm-shifting approach that aims to deliver a toxic payload selectively to target-expressing cells thereby sparing normal tissues the off-target effects associated with traditional chemotherapeutics. Since most targeted constructs rely on standard microtubule inhibitors or DNA-reactive molecules as payloads, new toxins that inhibit other intracellular targets are needed to realize the full potential of targeted therapy. Among these new payloads, α-amanitin has gained attraction as a payload in targeted therapy. Here, we conjugate two synthetic amanitins at different sites to demonstrate their utility as payloads in peptide drug conjugates (PDCs). As an exemplary targeting agent, we chose octreotate, a well-studied somatostatin receptor (sstr2) peptide agonist for the conjugation to synthetic amatoxins via three tailor-built linkers. The linker chemistry permitted the evaluation of one non-cleavable and two cleavable self-immolative conjugates. The immolating linkers were chosen to take advantage of either the reducing potential of the intracellular environment or the high levels of lysosomal proteases in tumor cells to trigger toxin release. Cell-based assays on target-positive Ar42J cells revealed target-specific reduction in viability with up to 1000-fold enhancement in bioactivity compared to the untargeted amatoxins. Altogether, this preliminary study enabled the development of a highly modular synthetic platform for the construction of amanitin-based conjugates that can be readily extended to various targeting moieties.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Chemical structure of α/β-amanitin with commonly used conjugation sites highlighted with green.
Fig. 2
Fig. 2. (A) Chemical structures of sstr2 targeting agent octreotate (TATE–N3, 2); (B) synthetic bioactive amatoxins 3 and 4; (C) three bioconjugates 5–7 synthesized in this study. Highlighted in blue and red are conjugation sites; (a) and (b) show two proposed pathways for self-immolative drug release of bio-reducible linker conjugate 6.
Scheme 1
Scheme 1. Synthetic approach towards a modular assembly of conjugates 5, 6, and 7 employing linkers with suitable orthogonal protecting groups; (A) PEG-linked and disulfide-containing bio-reducible conjugate 5 and 6 of amatoxin 4; (B) Cathepsin B cleavable self-immolative conjugate of amatoxin 3.
Fig. 3
Fig. 3. Synthesis of bifunctional linkers 10, 16 and 18: (A) PEG-based linker containing TES protected alkyne and azide handles; (B) linker 16 containing a bio-reducible disulfide, a TES protected alkyne; (C) Cathepsin B cleavable linker 18 functionalized with an alkyne and a p-nitrophenyl carbonate ester; py – pyridyl.
Scheme 2
Scheme 2. (A) Final steps of the synthesis of non-cleavable and disulfide containing bioconjugates 5 an 6 through CuAAC reaction; (B) final steps of the synthesis of Cathepsin B cleavable bioconjugate 7; astarting material (3) was partially recovered.
Fig. 4
Fig. 4. Cytotoxicity of amanitin-based conjugates 5, 6 and 7 to Ar42J cells expressing sstr2 in cell proliferation MTS assay; cells were incubated with TATE–PEG–Ama 5, TATE–SS–Ama 6, TATE–VCit–Ama 7, TATE–N32 and amatoxins 1, 3 and 4 for 72 h at 37 °C (n = 3), EC50 (effective concentration giving half-maximal response), data represented as mean value ± S.D., n/d – not determined.
Fig. 5
Fig. 5. Structure of the toxin liberated inside of the targeted cells as a result of the disulfide reduction and self-immolation of bioconjugate TATE–SS–Ama 6.
Fig. 6
Fig. 6. FACS scan of Ar42J cells that were untreated (gray) show a time-dependent increase in an sstr2-negative population from 48–96 h. Cells treated with 6 (30 nM) show a similar effect (black), however, the sstr2-positive population is depleted in comparison to the sstr2-negative population. Similar results were also observed when cells were treated with 6 at 60 nM (see ESI†).
Fig. 7
Fig. 7. Fluorescence imaging of Ar42J cancer cells (sstr2-positive) using TATE–SS–Rhod 23; (a) cells were incubated with 5 nM TATE–SS–Rhod 23 for 30 min – control; (b) blocking – cells were pre-treated with 250 nM TATE–SS–Ama 6 (30 min) followed by incubation with 5 nM TATE–SS–Rhod 23 (30 min).
Fig. 8
Fig. 8. Cell death kinetics of bioconjugate TATE–SS–Ama 6 and α-amanitin 1 evaluated on Ar42J cells following 24 and 48 hours of incubation (MTS, n = 3), data reported as mean ± S.D.; *P < 0.01; control experiments are cells grown under the same conditions and treated with 0.5% DMSO (vehicle) culture media.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Chari R. V. J. Acc. Chem. Res. 2008;41:98–107. doi: 10.1021/ar700108g. - DOI - PubMed
    1. Ghose T. Nigam S. P. Cancer. 1972;29:1398–1400. doi: 10.1002/1097-0142(197205)29:5<1398::AID-CNCR2820290542>3.0.CO;2-D. - DOI - PubMed
    1. Laguzza B. C. Nichols C. L. Briggs S. L. Cullinan G. J. Johnson D. A. Starling J. J. Baker A. L. Bumol T. F. Corvalan J. R. F. J. Med. Chem. 1989;32:548–555. doi: 10.1021/jm00123a007. - DOI - PubMed
    1. Nicolaou K. C. Rigol S. Angew. Chem., Int. Ed. 2019;58:11206–11241. doi: 10.1002/anie.201903498. - DOI - PubMed
    1. Strebhardt K. Ullrich A. Nat. Rev. Cancer. 2008;8:473–480. doi: 10.1038/nrc2394. - DOI - PubMed