Rational design of a sensitivity-enhanced tracer for discovering efficient APC–Asef inhibitors

The adenomatous polyposis coli (APC)–Rho guanine nucleotide exchange factor 4 (Asef) protein–protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity (Kd = 0.078 μM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC–Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 ± 0.004 μM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC–Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC–Asef inhibitors, thereby providing insight into PPI drug development.

Reagents and solvents used for synthesis were obtained from commercial suppliers and used without further purification. Proton NMRs were obtained using 600 MHz instruments. Carbon NMRs were obtained using a Bruker 151 MHz NMR spectrometer. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. Mass spectrometry was performed on high-resolution electron impact (EI) or electrospray ionization (ESI) mass spectrometry instruments available at the site of synthesis.
General procedure A for the synthesis of peptide compounds. The desired peptides p53) were conducted in the solid phase using Fmoc-Amide AM Resin (0.35 mmol/g, 572mg) as the solid support. The resin was first swelled in DMF (5 mL) for 5 min, then treated with 20% piperidine in DMF (5 mL) for 15 min. The procedure was repeated one time to completely remove Fmoc protecting group. The beads were washed with DCM (5 mL×3) and DMF (5 mL×3). By coupling of the 0.4 mmol N-Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids using 0. N,N', or 0.6 mmol Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexaflu (PyBOP) as the coupling reagent and 0.8 mmol N,N-Diisopropylethylamine (DIPEA) as the base. Fmoc-protecting groups were removed using 20% piperidine in dimethylformamide (DMF) after each coupling step. After the peptides were assembled, the N-terminal amines were acylated with benzoyl chloride in DIPEA. The peptides were then cleaved from the resin and deprotected using a mixture of trifluoroacetic acid (TFA), water, and triisopropylsilane (TIS) (95:2.5:2.5, v/v/v) for 2.5−3 h. The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and lyophilized to give a white powder. Purified peptides were analyzed by analytical RP-HPLC, and the integrity of the peptides was checked by negative ion electrospray ionization mass spectrometry (ESI-MS). All peptides were purified to ≥98% purity using RP-HPLC and were verified using H-NMR, C-NMR, ESI-MS and HRMS. The use of HBTU poses a skin irritation risk. The use of DIPEA and TFA pose a risk of inhalation toxicity.
General procedure B for the synthesis of tracer compounds. The general synthesis of tracer 1-14, PS-P53-7G, PS-P53-8H, PS-P53-9I, PS-P53-10J, PS-P53-11K were conducted in the solid phase using Rink Amide-MBHA resin (0.646 mmol/g, 100mg) as the solid support. The resin was first swelled in DMF (5 mL) for 5 min, then treated with 20% piperidine in DMF (5 mL) for 15 min. The procedure was repeated one time to completely remove Fmoc protecting group. The beads were washed with DCM (5 mL×3) and DMF (5 mL×3). The Fmoc protected regular amino acid (0.13 mmol), DIC (0.26 mmol) and HOBt (0.26 mmol) were dissolved in 2 mL DMF for 5 min and the solution was added to the resin. The reaction vessel was shaken for 3 hours until the reaction was complete. The reaction cycles were repeated to assemble the desired peptides. After installation of the last Fmoc protected amino acid, the Fmoc group was removed, and FITC (0.13 mmol) in 5 mL DMF and DIPEA (0.39 mmol.) was added to the resin to react overnight. After washing with DMF (5 mL×3) and DCM (5 mL×3), the resin was cleaved using TFA/DCM/TIS (6 mL, 50:48:2, v/v/v) for 3.5 h. The solution was collected, and the resin was washed with DCM (5 mL×2). The solution was combined and evaporated under air flow, and the crude product was subsequently analyzed (1 mL/min flow rate) and purified (16 mL/min flow rate) by Water HPLC system equipped with both analytic and preparative modules. The gradient eluting method was as follows: 5% of solvent B (0.1% TFA in acetonitrile) in A (0.1% TFA in water) was increased to 100% over 50 min was performed. The desired peptides were collected and lyophilized on a Labconco lyophilizer, with the purity determined to be >95% by analytical HPLC. The use of DIC poses acute inhalation toxicity.

Molecular modeling and molecular dynamics
The particular binding structures of tracers 1, 4, and 5 in Supplementary Fig. 1 are generated by molecular modeling and molecular dynamics (MD) simulations as follows: (1) constructed three initial tracer-APC complexes by mutating residue and adding FITC motif into the "GGGGEQLAINELISDGS" segment from Asef based on the crystal structure (PDB ID: 3NMZ) resolved in our previous work 1 , followed by energy minimization of the systems; (2) created the topology file and coordinate file of the three systems for the simulations, then built the water box with a cube with sides of 10 Å for each system and added counter ions (Na + or Cl -) to keep the systems in electric neutrality; (3) performed the MD simulations based on the workflow in our previous work 2 . (4) analyzed the trajectories from each simulation by Pymol and selected a representative conformation of the best energy cluster as the particular structure shown in Supplementary Fig. 1.
In the later research, we also performed an MD simulation for the MDM2-P53 system. The initial structure of the simulation system is the structure published by previous work of others, with the PDB ID of 1YCR 3 . The preparation of FITC-modified P53 is similar to the previous flow of tracer 1-FITC, we firstly mutated the Asparagine of C-Terminal to Lysine, and added the FITC moiety to the Lysine by the Discovery Studio software. After adding blocking terminals of modified P53, we carried the energy minimization for the P53-FITC -MDM2 complex by the software and generated the forcefield for the P53-FITC using Amber. Then we performed the MD simulation followed the preceding workflow of simulation.

Fluorescence Polarization Competition Assays of p53-MDM2
The binding affinity (Kd) of peptides was obtained by fluorescence polarization (FP). GST-MDM2-3-150 containing human MDM2 were expressed in E. coli as previously described by us. The fluorescence polarization assay was conducted by incubating MDM2 (