The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates

Ratjadone derivatives have been successfully introduced as suitable payloads with new mode of action for targeted drug conjugates.

The producing organism, the myxobacterium Sorangium cellulosum Soce1047, was isolated at the Helmholtz Centre of Infection Research, former GBF in 1989 from a soil sample collected at Cala Ratjada (Mallorca, Spain). Seed cultures on yeast agar were inoculated into 250-mL Erlenmeyer flasks containing 100 mL of medium.
The basic medium for growth and production had the following composition (in g/L distilled water): soybean flour (defatted) 4, glucose monohydrate 2, potato starch 8; MgSO4 7 H2O 1; CaCl2 2 H2O 1; ethylenediaminetetraacetic acid iron(III)-sodium salt 0.008. The pH of the medium was adjusted to 7.3 with KOH before autoclaving. In both media Soce1047 grew in small lumps, so that growth could not be measured optically or by counting the cell numbers.
A 20-L bioreactor (Giovanola Freres, Monthey, Switzerland) with 6 L of the production medium was inoculated with 1 L of a 4-day old preculture grown in the same medium in 1-L Erlenmeyer flasks with 500 mL medium under shaking (160 rpm, 30°C). For continuous adsorption of the produced ratjadone, 100 mL of the adsorber resin XAD-16 (Rohm and Haas, Frankfurt/M) was added before autoclaving. To prevent foam formation, 10 mL silicone antifoam (Tegosipon, Goldschmidt AG Essen) was added. The fermentation was run for 7 days at 30°C and the pH adjusted to 7.3-7.5 (2.5% H2SO4/2.5% KOH) with an aeration rate of 300 Lair per hour and a stirrer speed of 300 rpm. Ratjadon was excreted into the culture broth during the growth phase and became quantitatively adsorbed to the resin the end of the fermentation. The fermentation was done on 10L and 70L scale. The best yield was obtained on 10L scale with 32.4 mg of Ratjadone A/L. A total amount of 3.646 g of Ratjadone A was isolated from 150 L Fermentation culture.

General procedure A for the solid-supported synthesis of FA-N3
The solid-phase syntheses of the FA-N3-3-11 were carried out manually on a scale of 218 µmol on Rapp 2chlorotrityl resin (Rapp Polymere, Tübingen, Germany, 1.09 mmol/g) using fritted glas peptide synthesis vessels and Fmoc-protected amino acids. The side chain protections of the amino acids were as follows: Lys (

Synthesis of FA-SH-1
The

Synthesis of Folate-Fluorescein Conjugates
General procedure C for the synthesis of Folate-Fluorescein Conjugates via copper-free click reaction.

Cell proliferation assay
The corresponding cells were cultivated at 37 °C and 10 % CO2 in the medium given in table 1. 60 µL of serial dilutions of the test compound were given to 120 µL of suspended cells (50.000/mL) in wells of 96well plates. After 5 days of incubation growth inhibition (IC50) was determined using an MTT assay. 9      Cell proliferation assay under folate free conditions KB 3.1 (DSMZ ACC 158) cells were cultivated at 37 °C and 10 % CO2 in RMPI-medium without folic acid (Gibco) with 10% fetal calf serum (Gibco). 120 µL of suspended cells (100.000/mL) were seeded in in wells of 96-well plates and after 24 h at 37 °C and 10 % CO2 the medium was removed, the cells were washed with PBS and RPMI-medium without folic acid (Gibco) + 10% dialyzed fetal calf serum (Sigma) were added to the cells, before after additional 24 h at 37°C and 10 % CO2 60 µL of serial dilutions of the test compound were given to cells. After 1, 2 and 5 days of incubation growth inhibition (IC50) was determined using an MTT assay. 9

Monitoring export inhibitory activity
Export inhibitory ability of compounds was evaluated with the translocation biosensor system. This cellular assay depends on a recombinantly expressed fusion protein consisting of a nuclear localization signal (SV40-NLS), glutathione S-transferase (GST), green fluorescent protein (GFP) and a nuclear export signal (HIV1-RevNES). Due to the two transport signals (NLS/NES), the biosensor is permanently shuttling between nucleus and cytoplasm but resides prominently in the cytoplasm due to a comparatively stronger NES. Export inhibiting compound induce a nuclear accumulation of the GFP-signal 11,12 Cell lines were maintained as recommended by the American Type Culture Collection in DMEM containing 5% glutamine (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, USA). For quantifying the nuclear export inhibitory effect, HeLa cells stably expressing a fluorescent translocation biosensor (HeLaRevBio) 13 were seeded into black 96well µclear plates (Greiner, Germany). They next day compounds were added covering an appropriate concentration range. After 1h of incubation, cells were fixed in 4% PFA for 10 min and permeabilized with 0.1% Triton X 100 in PBS for 5min. After washing with PBS nuclei were labeled with Hoechst H33342 (30min 10µg/ml). The intracellular distribution of the biosensor-dependent GFP signal was quantitatively evaluated with the high content imaging system ImageXpress MicroXLS (Molecular Devices, Sunnyvale, USA). By using the translocation enhanced application module (Molecular Devices, Sunnyvale, USA) the GFP-intensity was quantified in the cytoplasm and nuclear region. As final readout the difference of Mean Inner and Mean Outer Intensity was calculated.
IC50s of nuclear export inhibition were calculated using Sigma Plot with four parameter logistics curve regression (Systat Software GmbH, Erkrath, Germany). For confocal microscopy cells were seeded at a density of 0.2*10 5 cells/well into 8 well microscopy chambers (Ibidi GmbH, Martinsried, Germany). The following day compounds were added at 125nM final concentration. After 1h the cells were imaged on an ECLIPSE Ti (Nikon) equipped with UltraVIEW VoX spinning disc (Perkin Elmer, Waltham, US), ORCA-R2 camera (Ham Hamamatsu Photonics, Japan) and Volocity software 6.1.1 (Perkin Elmer, Waltham, US). Compound 20 23.

Streptavidin-Pull-down with Ratjadone-biotin and identification of exportin by LC-MSMS
A chemical pull down was performed to verify binding of ratjadone to Crm1. HeLa cells were treated with ratjadone-biotin, ratjadone or both at 0.1μg/ml final concentration for 5h. Cells were detached by scraping, washed twice in PBS and lysed in MPER-buffer (Thermo Fisher, Waltham, USA) supplemented with complete protease inhibitor (Roche, Mannheim, Germany). After 5min incubation on ice the lysate was centrifuged (20min, 13000rpm, 4°C) and the supernatant was incubated with streptavidin sepharose high performance (GE healthcare, Freiburg, Germany) overnight at 4°C. The next day beads were washed twice with PBS and resuspended in elution buffer (Roth) and heated to 95°C for 10min. 5% were tested on WesternBlot with a biotin-specific antibody.