A theranostic abscisic acid-based molecular glue

Molecular glues, capable of selectively controlling the interactions between specific pairs or groups of proteins and the associated downstream effects, have become a promising strategy for manipulating cellular functions and developing novel therapies for human diseases. Theranostics with both diagnostic and therapeutic capabilities acting at disease sites has become a powerful tool to achieve both functions simultaneously with high precision. To selectively activate molecular glues at the desired site and monitor the activation signals at the same time, here we report an unprecedented theranostic modular molecular glue platform integrating signal sensing/reporting and chemically induced proximity (CIP) strategies. We have demonstrated for the first time the integration of imaging and activation capacity with a molecular glue on the same platform to create a theranostic molecular glue. A theranostic molecular glue ABA-Fe(ii)-F1 was rationally designed by conjugating a NIR fluorophore dicyanomethylene-4H-pyran (DCM) with a CIP inducer abscisic acid (ABA) using a unique carbamoyl oxime linker. We have also engineered a new version of ABA-CIP with an enhanced ligand-responding sensitivity. We have validated that the theranostic molecular glue can sense Fe2+ and produce turn-on NIR fluorescence for monitoring as well as releasing the active inducer ligand to control cellular functions including gene expression and protein translocation. This novel molecular glue strategy paves the way to building a new class of molecular glues with theranostic capacity for research and biomedical applications.


General information
All reagents were purchased from commercial sources such as Sigma-Aldrich, Ambeed, and Fluka and used without further purification. The solvents were used by dry solvents system. All reactions were monitored by TLC or LC-MS from Shimadzu. Purification was conducted on preparative flash column chromatography and preparative reversed-phase high performance liquid chromatography (RP-HPLC) with solvent systems specified. Nuclear magnetic resonance (NMR) spectra were recorded on automated Bruker AVIII-400 instruments. High-resolution mass spectra (HRMS) were recorded on a Bruker microTOF II instrument in positive ion mode using an Agilent G1969 API-TOF with an electrospray ionization (ESI) source. Ultraperformance liquid chromatography (UPLC) spectra for compounds were acquired using a Shimadzu LabSolutions system.
Selectivity studies of ABA-Fe(II)-F1 toward metal ions, GSH, lysine and cysteine. 10 mL of 10 μM solution of ABA-Fe(II)-F1 was prepared by diluting a 10 mM DMSO stock solution of ABA-Fe(II)-F1 into 50% HEPES/DMSO (10 mM, pH 7.4) in a 1 cm × 1 cm capped quartz cuvette. 500 μL of this solution were added to ten 1 cm × 1 cm capped quartz cuvettes, then the cuvettes were placed in a 37 °C water bath for 5 min. After 5 min, 500 μL of a solution of the metal of interest was added to the cuvette to bring the concentration of transition metals to 100 μM and the concentration of NaCl, KCl, CaCl2ꞏ2H2O, MgCl2ꞏ4H2O to 1 mM, final GSH, Lysine and Cysteine concentration of 5 mM. 500 μL of buffer was added to one cuvette, and this sample served as the blank throughout the experiment. (v/v) GlutaMAX (Life Technologies) and 100 IU/ml penicillin/streptomycin (Life Technologies) in a humidified 37 o C incubator with 5% CO2 atmosphere. Cells were tested for mycoplasma and confirmed mycoplasma-free using MycoAlert TM mycoplasma detection kit (Lonza). For all cell culture experiment, stock solution of ABA and theranostic compound were prepared in DMSO. A 500 mM of stock Fe 2+ solution was prepared by mixing ferrous chloride (Sigma) and sodium citrate (Thermofisher) at 1:1 molar ratio. Fresh Fe 2+ solution was made before every experiment.

S6
The nuclear export plasmid cassette including NES-ABI and EGFP-PYL was described previously. 3 The modified expression vector EGFP-PYR* was made by replacing PYL from the parental EGFP-PYL with PYRF61L/A160C using in-fusion cloning (Takara). PYR* was PCR amplified using the sv-VP*2GA construct as a template with respective forward and reversed primers (5'-TGGACGAGCTGTACAAGGGCGCGCCACCATCAGAGCTTACCCCCGA-3' and 5'-TGATTATGATCTAGAGTCGCGGCCGCTCAAGCGTAATCTGGAACATCGT-3') and inserted into the linearized Actin-EGFP vector with AscI and NotI restriction sites.
The inducible TRAIL plasmid was constructed by replacing EGFP gene with the nucleotide sequence encoding IL2R secretion signal and TRAIL (aa 114-281) under control of a minimal CMV promoter and 5xUAS response element.
All plasmid constructs were amplified using DH5 chemically competent E.coli strain and purified using Endo-free Qiagen Miniprep kit. DNA sequences of the final constructs were confirmed by Sanger sequencing (Genewiz). Plasmid concentration and purity (A 260/280 = 1.88-1.90) were measured by a Nanodrop spectrophotometer (Thermofisher).

Cellular localization of probe ABA-Fe(II)-F1.
The subcellular localization of probe was tested on three different cell lines including HEK-293T, HeLa, and MDA-MB-231. The cells were seeded into a 96-well black wall clear bottom plate 1 day prior to drug treatment. On the day of imaging, cells were treated with 10 μM ABA-Fe(II)-F1 in the presence of mixture of 5 mM FeCl2 and 5 mM sodium citrate for 30 min. Media were removed and cells were stained with NucBlue TM Live ReadyProbes TM Reagent (Hoechst 33342, Invitrogen) and either 6 μM ER-Tracker® Green (Cell Signaling Technology) or 500 nM LysoTracker® Green (Cell Signaling Technology) for 20 min. Stained cells were then washed twice with DPBS, submerged in DPBS and imaged immediately by Leica HyVolution SP8 confocal microscope using 20x-water immersion objective. 8. Luciferase reporter assay. 10 4 CHO cells were seeded in triplicate in a 96-well plate and allowed to adhere for 24 h at 37 o C in a CO2 incubator. Cells were transfected with ABA-responsive split transcriptional activator with PYL1 being replaced with PYR1 mutants including E141L, F61L/A160C and F61L/E141L/A160V mutations, and 5xUAS response elements controlling the expression of firefly luciferase. 24 h post-transfection, cells were incubated with varying concentration of ABA or DMSO alone (as control). Luciferase activity was detected using Luciferase Assay System (Promega #E1500), for luminometer (GLOMAX-Multi Detection System) according to manufacturer's protocol. The triplicate data obtained for each mutant condition were averaged and normalized to DMSO treatment. 9. ABA-induced EGFP expression. 10 4 HEK-293T cells were seeded in duplicate in a 96-well plate and allowed to adhere for 24 h at 37°C in a humidified CO2 incubator. Cells were transfected, using PEI, with the optimized ABA-responsive split transcriptional activator and an inducible EGFP expression. 24 hours after transfection, the cells were incubated with either DMSO, 10 μM ABA or 10 μM ABA-Fe(II)-F1. One hour later, the cell culture media were changed with fresh DMEM, and varying concentrations of Fe 2+ were added to the cells treated with ABA-Fe(II)-F1 to uncage the compound and generate free ABA. Expression of EGFP was monitored intermittently at 6, 12 and 24 h.

Quantification of nuclear export in HEK-293T cells.
Two days before imaging, cells were seeded on 17-mm glass coverslips in a 24-well plate at 50,000 cells/well in a total volume of 500 µL DMEM media and incubated overnight. Transfection was performed using NES-ABI and EGFP-PYR* plasmids at a ratio of 2:1 in PEI transfection reagent (Polyscience). Fresh media was replaced at 6 hours post transfection. 16 h after transfection, cells were treated with either 10 µM ABA, 10 µM ABA-Fe(II)-F1, or 10 µM ABA-Fe(II)-F1 with added 500 eq. of Fe 2+ (prepared in Sodium citrate stock solution at 500 mM). At indicated time points, media were removed, and cells were stained with NucBlue TM Live ReadyProbes TM Reagent (Hoechst 33342, Invitrogen) for 30 min at 37 o C in the dark. Cells were then washed twice with PBS and fixed in 4% formaldehyde fixation solution (Thermofisher) for 10 min at room temperature. Following fixation, cells were washed twice with PBS (5 min each) and mounted on a glass microscopic slide with Vectashield anti-fade mounting media. Samples were stored at 4 o C until ready to use.
To quantify the nuclear translocation, mean green fluorescence intensity of the nuclear and cytoplasmic regions were calculated using ImageJ software. Background-subtracted images were quantified by defining ROI of the nuclear and cytoplasmic regions of each cell for a population of 20-30 cells. The reported nuclear translocation is the ratio between the mean fluorescence intensities of the nuclear (Fn) and cytoplasmic (Fc) regions.

Confocal fluorescence imaging. Confocal fluorescence imaging was performed with Leica
HyVolution SP8 confocal microscope equipped with 20x-water immersion, 40x-and 63x-oil immersion objective lens. Relevant quantifications were performed by the ImageJ software. Red fluorescence (Probe) was detected using Hybrid photodetector at 514 nm excitation and emission was collected at 685-720 nm excitation window. EGFP-expressed cells were excited with a 488 nm PMT laser and emission was collected between 500-550 nm. Excitation of Hoechst 33342 at 405 nm was carried out with diode laser and emission was collected between 415-485 nm. ER-Tracker and LysoTracker dye were excited at 496 nm with argon laser, and the emission was collected between 510-560 nm. The bright field images of the cells were taken with a transmitted light photomultiplier under a 514-nm laser.

Fluorescence imaging experiments.
Cells were imaged using Lionheart™ FX Automated Microscope (BioTek Instruments, Winooski, VT) configured with DAPI, GFP, and Cy5 filter cubes. Hoechst 33342 was detected with a 337/50 excitation filter and a 447/60 emission filter. The GFP light cube uses a 469/35 excitation filter and a 525/39 emission filter. Red fluorophore was detected using a Cy5 filter cube with a 628/50 excitation filter and a 685/50 emission filter.
13. ABA-induced secreted TRAIL and "turn-on" fluorescence. HEK-293T cells were dualtransfected with sv-VP*2GA and the inducible plasmid encoding secreted TRAIL (sTRAIL) under control of minimal CMV promoter and 5xUAS response element. At 24 h post-transfection, cells were treated with either DMSO or 10 µM ABA-Fe(II)-F1. 1 hour later, media were refreshed and varying concentrations of Fe 2+ were then added to corresponding wells, 10 µM ABA was also added to the control well. At 15-minute post-treatment, cells were imaged with Lionheart™ FX Automated Microscope using 628/50 excitation filter and 685/50 emission filter for the generation of probe. Cells were then transferred back into the incubator and continue to grow overnight. At S8 24 h post-iron treatment, cell culture supernatant was collected and quantified for the secretion of TRAIL using human TRAIL PicoKine TM ELISA kit (Boster Bio, Pleasanton, CA).
14. Flow cytometric analysis. Cells for each experimental condition were harvested by trypsinization, washed with DPPS, resuspended in DPBS, then subjected to analysis by flow cytometry using BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences) equipped with a Blue (488 nm) and a Red (640 nm) lasers. Green fluorescent signals were detected on FITC channel and red fluorescent signals were detected on PerCP channel. Data were analyzed with FlowJo software (v10, TreeStar).

Statistical analysis.
Statistical significance was calculated in GraphPad Prism using two tailed unpaired Student's ttest for two independent groups, and ANOVA for multiple groups comparison. All data are presented as mean±S.E.M.   (5 mM