Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVβ3 receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of αVβ3 integrin expression: human glioblastoma U87 (αVβ3+), human lung carcinoma A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468 (αVβ3−). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αVβ3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (αVβ3+, αVβ5+, αVβ6−, α5β1+) and MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, α5β1−) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αVβ3, but also αVβ5, αVβ6, and α5β1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αVβ3 (e.g., αVβ5).


Equal contributors
Experimental details, characterization data and copies of spectra

Biological assays S3
Solid phase receptor binding assays S3 Determination of integrin α V β 3 expression by flow cytometry S4 Cell culture S4 Cell therapy and viability assay S5 Competition experiment of compounds 10 and 11 S5

Determination of integrin α V β 3 expression by flow cytometry
The expression of integrin α V β 3 in U87-MG, A549 and MDA-MB 468 cells was determined by flow cytometry on a FACSCalibur device (Becton Dickinson). Before staining, cells were fixed with fixation solution (0.5% PFA in PBS). 5 × 10 5 cells per sample were stained in staining medium (PBS, 25 mM HEPES, 3% FCS, 0.02% Na-azide) with an anti-human integrin α V β 3 antibody conjugated to Alexa Fluor 488 (R&D Systems) or isotype control conjugated to Alexa Fluor 488 (Thermo Fischer) at a concentration of 4 µg/mL for 45 min at room temperature. The cells were washed with PBS and the mean fluorescence intensity was measured for 10.000 gated events. The data were analyzed using flow cytometry and associated software (BD Biosciences) ( Figure S1). Figure S1. Flow cytometry analysis of integrin α V β 3 expression in cancer cell lines. U87-MG: integrin α V β 3 overexpressed; A549 and MDA-MB 468: integrin α V β 3 negative.

Cell culture
All cell culture reagents were purchased at PAN-Biotech GmbH unless otherwise stated. Cell lines were obtained from CLS (U87-MG, MDA-MB 468 and A549). Cell lines were authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently.
[S1] The SNP profiles matched known profiles or were unique. The purity of the cell lines was validated using the Multiplex cell Contamination Test by Multiplexion (Heidelberg, Germany) as described recently. [S2] No mycoplasma, SMRV or interspecies contamination was detected. U87-MG, MDA-MB 468 and A549 cells were cultivated continuously for not more than 3 months in MEM Eagle´s, DMEM or Ham´s F12 medium, respectively, supplemented with 10% heat inactivated fetus calf serum, L-glutamine and antibiotics. Cell lines were maintained at 37 ºC and 5% CO 2 in a high humidity atmosphere.
Glutarate NHS ester-aminohexyl-α-amanitin (13a) S12 Compound 12a (11 mg, 10.9 µmol, 1 equiv) was dissolved in 150 µL of dry DMF under nitrogen atmosphere. The solution was cooled to 0 °C, then di-N-succinimidyl glutarate (4 mg, 12 µmol. 1.1 equiv) and DIPEA (2 µL, 12 µmol, 1.1 equiv) were added and the mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC (CHCl 3 /MeOH/water 65:25:4, cinnamaldehyde staining). The crude was poured into 10 mL precooled MTBE placed in a 10 mL centrifugal tube (the reaction flask was rinsed with 3 × 50 µL of DMF and each rinsing solution was transferred to the MTBE tube). The tube was sealed, vortexed and placed in ice for 10 min. The tube was spun for 3 min at 4500 RPM in a precooled centrifuge. The supernatant was transferred to a 50 mL flask. The pellet was suspended again into 10 mL of MTBE by vortexing and sonication. The tube was placed in ice for 10 mins and the centrifugation was repeated, then the pellet was dried in vacuo. The combined MTBE phases were concentrated under reduced pressure and checked for remaining product by TLC and HPLC. The dried pellet was used in the next step without further purification (12 mg, 90% yield). (8) A solution of 4 (5.0 mg, 5.8 µmol, 1 equiv) in 150 µL of PBS was added to a solution of 13a (12 mg, 10 µmol, 1.7 equiv) in 150 µL of DMF at 0 °C. The pH was adjusted to 7.3-7.6 by adding small aliquots of aqueous NaOH (0.2 M) during the first hours of reaction, until a stable value was observed, and then the reaction mixture was stirred overnight at room temperature.

Glutarate NHS ester-Val-Ala-α-amanitin (13b)
Compound 12b (10 mg, 8.3 µmol, 1 equiv) was dissolved in 150 µL of dry DMF under nitrogen atmosphere. The solution was cooled to 0 °C, then di-N-succinimidyl glutarate (3.2 mg, 9.9 µmol, 1.2 equiv) and DIPEA (1.6 µL, 9.1 µmol, 1.1 equiv) were added and the mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC (CHCl 3 /MeOH/water 65:25:4, cinnamaldehyde staining). The crude was poured into 10 mL precooled MTBE placed in a 10 mL centrifugal tube (the reaction flask was rinsed with 3 × 50 µL of DMF and each rinsing solution was transferred to the MTBE tube). The tube was sealed, vortexed and placed in ice for 10 min. The tube was spun for 3 min at 4500 RPM in a precooled centrifuge. The supernatant was transferred to a 50 mL flask. The pellet was suspended again into 10 mL of MTBE by vortexing and sonication. The tube was placed on ice for 10 mins and the centrifugation was repeated. Then the pellet was dried in vacuo. The combined MTBE S14 phases were concentrated under reduced pressure and checked for remaining product by TLC and HPLC. The dried pellet was used in the next step without further purification (8.3 mg, 71% yield). (10) A solution of 4 (3.4 mg, 3.9 µmol, 1 equiv) in 150 µL of PBS was added to a solution of 13b (8.3 mg, 5.9 µmol, 1.5 equiv) in 150 µL of DMF at 0 °C. The pH was adjusted to 7.3-7.6 by adding small aliquots of aqueous NaOH (0.2 M) during the first hours of reaction (until a stable value was observed), then the reaction mixture was stirred overnight at room temperature. The solution was directly filtered into a 3 mL vial and purified by preparative HPLC (gradient: from 95% (H 2 O + 0.05 % CF 3 COOH)/5% CH 3 CN to 60% (H 2 O + 0.05% CF 3 COOH)/40% CH 3 CN in 14.5 min), t R (product): 9.2 min. The purified product was freeze-dried to give the final product as a white solid (4.8 mg, 62% yield). MS (ESI+): m/z calculated for [C 86 H 118 (17) 4-Pentynoic acid (10 mg, 0.100 mmol, 1 equiv) was dissolved in 2 mL of dry DCM under argon and cooled to 0 °C. EDC·HCl (23 mg, 0.120 mmol, 1.2 equiv) and NHS (14 mg, 0.120 mmol, 1.2 equiv) were sequentially added to the solution and the reaction mixture was stirred overnight at room temperature. The mixture was diluted in 20 mL of DCM and the organic phase was washed with water (3 × 7 mL), dried over MgSO 4 , filtrated and concentrated under reduced pressure to give the product as a yellowish oil that was used in next step without further modifications.

4-Pentynoic acid NHS ester
were added to a solution of compound 12b (5.0 mg, 4.2 µmol, 1 equiv) in 200 µL of DMF kept at 0 °C. The reaction mixture was stirred overnight at room temperature. Volatiles were evaporated under reduced pressure and the crude was dissolved in 500 µL of MeOH and then purified by preparative HPLC (gradient: from 95% (H 2 O + 0.05% CF 3 COOH) / 5% (CH 3 CN) to 0% (H 2 O + 0.05% CF 3 COOH)/100% (CH 3 CN) in 15 mins), t R product = 7.97 min. The collected fraction was concentrated under reduced pressure and freeze-dried from water/ACN 1/1 to afford the product as a white solid (4.7 mg, 88% yield 14- 6,9, 100 µL (0.05 mmol, 1 equiv) of a 0.5 M solution of 14-azido-3,6,9,12-tetraoxatetradecanoic acid (~0.5 M in tert-butyl methyl ether) were diluted in 100 µL of dry DCM under argon and cooled to 0 °C. EDC·HCl (12,5 mg, 0.065 mmol, 1.3 equiv) and NHS (7.5 mg, 0.065 mmol, 1.3 equiv) were sequentially added to the solution and the reaction mixture was stirred overnight at room temperature. The mixture was diluted in 10 mL of DCM and the organic phase was washed with water (3 × 3 mL), dried over MgSO 4 , filtrated and concentrated under reduced pressure to give the product as a transparent oil (18.2 mg, 97% yield). The crude product was used in the next step without further modifications.