Identification of HuR–RNA Interfering Compounds by Dynamic Combinatorial Chemistry and Fluorescence Polarization

The RNA binding protein HuR regulates the post-transcriptional process of different oncogenes and tumor suppressor genes, and its dysregulation is linked with cancer. Thus, modulating the complex HuR–RNA represents a promising anticancer strategy. To search for novel HuR ligands able to interfere with the HuR–RNA complex, the protein-templated dynamic combinatorial chemistry (pt-DCC) method was utilized. The recombinant RRM1+2 protein construct, which contains essential domains for ligand–HuR binding and exhibits enhanced solubility and stability compared to the native protein, was used for pt-DCC. Seven acylhydrazones with over 80% amplification were identified. The binding of the fragments to HuR extracted from DCC was validated using STD-NMR, and molecular modeling studies revealed the ability of the compounds to bind HuR at the mRNA binding pocket. Notably, three compounds effectively interfered with HuR–RNA binding in fluorescence polarization studies, suggesting their potential as foundational compounds for developing anticancer HuR–RNA interfering agents.


Protein expression and purification
The protein expression and purification were performed as previously reported but using the coding sequences for the RRM1+2 protein (residues 1 to 186) cloned into the pETM-11 expression vector.This vector incorporates an N-terminal polyhistidine-tag (His6-tag), which precedes a tobacco etch virus (TEV) protease cleavage site.The recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells.These cells were spread on lysogeny broth (LB)-agar plates containing kanamycin and left to incubate overnight at 37 °C.Thereafter, a single colony was selected and inoculated in LB medium supplemented with kanamycin and incubated overnight at the same temperature.The preculture was then diluted 1:100 with ZYM-5052 medium for auto-induction, which was further enriched with kanamycin and incubated at 37 °C.Upon the cell density (OD600) reaching 0.8, the temperature was reduced to 20 °C, and the culture was incubated overnight.The cells were then harvested via centrifugation, resuspended in lysis buffer, and lysed by sonication.
After the lysate was clarified through centrifugation, the supernatant was loaded onto a Ni-NTA (nickel-nitrilotriacetic acid) agarose column (Qiagen, Germany).The column was washed to eliminate non-specifically bound products, and the target protein was eluted with an elution buffer supplemented with 250 mM imidazole.The flow-through was combined with TEV protease and dialyzed overnight at 4 °C.Both the TEV protease and cleaved His6-tag were removed by a second Ni-NTA affinity chromatography step.The concentrated eluate was ultimately purified through sizeexclusion chromatography (SEC) on a Superdex 75 Hiload 16/60 column (GE Healthcare) in a 20 mM sodium phosphate buffer with pH 7.0, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA.The purified proteins were concentrated, flash-frozen in liquid nitrogen, and stored at -80 °C until use.The protein purity was assessed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), and the concentration was measured by assessing the absorbance at 280 nm.A thermal shift assay (TSA) was performed in their native buffer (20 mM Phosphate buffer pH 7.0) at six different protein concentrations (0.25-10 μM).

Selection of the proper buffer for pt-DCC by TSA
A 96-well plate, holding duplicate preparations of 10 μM protein solutions in sixteen diverse buffers (Table SI-1), water, and 5% DMSO, was maintained at room temperature for a four-day span, and subjected to daily sampling (from day 0 to day 3) for TSA experimentation.The test solution was arranged as depicted in Table SI-  The reaction mixtures were stirred for 36 hours and sampled at regular intervals.For HPLC-UV-MS analysis, samples were prepared as following: 5 μL of each reaction sample were introduced into an Eppendorf tube containing 44 μL of analytical-grade ACN (to precipitate the protein) and 1 μL of 1 M NaOH (to stop the reaction reversibility).The vials were vortexed for 10 seconds and then centrifuged at 14,000 RPM for 5 minutes at 4 °C.The supernatant was collected and immediately analyzed.Previously collected samples were stored at 4 °C.

HPLC method
HPLC-UV-MS analyses were performed on a ThermoScientific Dionex Ultimate 3000 UHPLC System, connected to a ThermoScientific Q Exactive Focus equipped with an electrospray ion source.
Separation was performed on an Acquity Waters Column (BEH, C8 1.7 μm, 2.1 x 150 mm, Waters, Germany) with an accompanying VanGuard Pre-Column (BEH C8, 5 x 2.1 mm, 1.7 μm, Waters, Germany).The elution was performed at a flow rate of 0.250 mL/min in gradient mode.The mobile phase consisted of solvent A (ACN + 0.1% HCOOH) and solvent B (H2O + 0.1% HCOOH).The initial mobile phase composition was maintained at 10% solvent A for 1 min, changed linearly to 95% of A in 16 min and held for 1.5 min, then followed by a return to the initial conditions within 0.1 min and kept 2 min for the chromatograph column equilibrium.The mass spectrum was recorded in positive mode within a range of 100 -700 m/z.

Determination of Equilibrium and Product Amplification Quantification
The products within the reaction mixture were uniquely identified by high-resolution mass, and the peak area was measured on the HPLC-UV-trace at λ = 272 nm.To assess that equilibrium was reached, the area of each peak in blank sample was plotted against time.Once the area reaches a value stable in time, the equilibrium was considered reached and the amplification was investigated in pt-DCC samples.Amplification was calculated for each peak by attributing an arbitrary value of 100% to the area in the blank, following the proportion: AreaDCC: x = Areablank : 100.Then, x = (AreaDCC * 100) / Area blank.
The amplification % is then calculated as A% = x -100.
There is no established cut-off for the selection of amplified products, as this is contingent on the quantity of protein employed in the assay and on the product's affinity for the protein, low A% values might typically be ascribed to slight integration differences rather than actual amplification.

Phosphate Buffer
For compound 2, the equilibrium was reached after 12h.The amplification was measured at 12, 24 and 30 h, and significant values were found at 10 and 24 h with an amplification of 121% and 328% (24h), respectively.
2. Blank and negative control wells were incorporated into each TSA run.Table SI-1.Buffer used for the assessment of pt-DCC.The dye utilized was SyproOrange (Thermo Fischer Scientific), applied at a final concentration of 4x.The plate was subjected to a thermal cycle (from 21 °C to 95 °C, with an incremental temperature ramp of 0.5°C per minute) within a real-time PCR instrument.Table SI-2.Composition of the well for TSA analysis.Melting curves were analyzed using Protein Thermal Shift 1.3 software, and the Tm recorded for each sample was plotted against time (Figure SI-1).

Figure SI- 3 .
Figure SI-3.HPLC-UV trace for the comparative analysis of the pt-DCC assay performed in acetate buffer for compound 2 (marked with an asterisk).

Figure SI- 5 .
Figure SI-5.HPLC-UV trace for the comparative analysis of the pt-DCC assay performed in phosphate buffer for compound 2 is marked with an asterisk.

Figure SI- 7 .
Figure SI-7. 1 H-NMR and STD NMR spectra for the most amplified fragments in the pt-DCC assay.

Figure SI- 9 .
Figure SI-9.% of FP emission of free mRNA in presence of compounds 5 and EGCG.

TABLE OF CONTENT
Table SI-2.Composition of the well for TSA analysis.…SI-4  Table SI-3.1H-NMR characterization for compounds 1-7.
a. o/l = overlap of peak chemical shift in the 1 H NMR spectrum