Disulfide-Based Molecular Assembly On The Magnetic-Silica Nanostructure And Its Application In Drug Target Identification

To gain insight into the mechanism of action of a biologically active small molecule,identifying its biological targets is an essential step.Among the numerous target identification strategies developed over the past decades,affinity purification remains the most commonly used method because it can directly reveal the physical interaction between small molecules and target proteins.However,affinity purification is also quite challenging.One is the issue of ligand immobilization,which involves the study of the structure-activity relationship（SAR）of bioactive small molecules.Derivatization of bioactive small molecules is a time-consuming process,and it is challenging to perform without loss of biological activity.Second,nonspecific protein adsorption;the nonspecific binding of unrelated proteins to the affinity matrix may obscure the target signal or lead to false positives.For the problem of ligand immobilization,the selection of appropriate ligand immobilization methods is the key to preparing the target identification affinity probe according to the different chemical properties of small molecule ligands.In this paper,bioactive small molecules were first immobilized on magnetic silicon nano-matrix by selective coupling method,and a ligand-immobilized affinity probe was constructed by selective coupling method,which was used to identify the small molecular targets of interest,and the effectiveness of the affinity purification method to identify target proteins was demonstrated.Then,a rapid and universal coupling method was proposed.The ligands of small molecules were fixed to the magnetic silicon nano-matrix in a way independent of functional groups through a site-nonselective photo-cross-linking reaction.The non-selective ligand-immobilized affinity probe was constructed,which successfully verified the existence of small molecules on the affinity matrix and the probe could be used for the identification of bioactive small molecule targets.For the non-specific protein adsorption problem,the introduction of disulfide bonds greatly reduces the interference of protein mass spectrometry identification,because breaking the disulfide bonds only allows the release of ligand-bound proteins,and proteins that are not specifically bound to the matrix are not eluted off.Fe₃O₄ nanoparticles（MNPs）with good morphology and uniform size were synthesized by hydrothermal method and then coated with silicon layer（marked as MSP）on the surface of Fe₃O₄ MNPs by Stober method to reduce the aggregation of Fe₃O₄ magnetic core and improve the stability.Then 3-mercaptopropionic acid（MPA）linkage arms containing both sulfhydryl and carboxyl groups were introduced through two sulfhydryl-disulfide bond exchange reactions to functionalize the magnetic nanoparticles,and carboxyl-modified disulfide bond magnetic nanoparticles（MSP-S-S-MPA）were constructed.The MSP-S-S-MPA was successfully constructed and characterized by transmission electron microscopy,hysteresis curve,Fourier transform infrared spectroscopy,and X-ray photoelectron spectroscopy.Then,using carbonic anhydrase 2（CA2,molecular weight 29 KDa）and its binding inhibitor4-（2-aminoethyl）phenylsulfonamide（AEBSA）as a model,the ability of drug ligand-immobilized magnetic silicon affinity probes in drug target identification was systematically investigated.Based on MSP-S-S-MPA magnetic nanoparticles,the selective coupling method was used to immobilize the ligand.AEBSA was immobilized on the surface of MSP-S-S-MPA magnetic nanoparticles by an amide condensation reaction,and the selective coupling method ligand-fixing affinity probe MSP-S-S-MPA-AEBSA was constructed.The ligands were immobilized by non-selective coupling.First,the photoreactive compounds 4-[3-（trifluoromethyl）-3H-diacridine-3-yl]benzylamine hydrochloride（TFMDBA）and Cystamine-Azidobenzoic acid（AZ）were fixed to the surface of MSP-S-S-MPA by an amide condensation reaction,respectively.Two affinity probes,MSP-S-S-MPA-TFMDBA-AEBSA and MSP-S-S-MPA-CD-AZ-AEBSA,were obtained by immobilization of AEBSA on magnetic nanoparticles.Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy,ultraviolet spectroscopy,hydrogen nuclear magnetic resonance spectroscopy,and liquid mass spectrometry were used to demonstrate that AEBSA was successfully immobilized on magnetic nanoparticles by these two methods.It has also been demonstrated that disulfide bonds can be cleaved by the mild reducing agents tris（2-carboxyethyl）phosphine hydrochloride（TCEP）or dithiothreitol（DTT）.The three probes were incubated with the actual samples（human red blood cell lysate or rat brain lysate）.Disulfide bond cleavage allowed the release of the target protein bound to the small molecule ligand,and SDS-PAGE gel electrophoresis was performed to cut a band at about 29 KDa for mass spectrometry analysis.The target protein CA2 of AEBSA was successfully identified by searching the peptide sequence in the database.Western Blot analysis showed that the specific release of AEBSA was caused by the cleavage of the disulfide bond,and the protein at 29 KDa reacted with the Anti-CA2 antibody,indicating that the protein was CA2.This indicates that affinity purification experiments using photo-crosslinked affinity probes are also an effective strategy for target protein identification.In addition,the combination of the selective coupling method,non-selective coupling method with magnetic silicon matrix and mass spectrometry can be used to build a more powerful and versatile platform for identifying unknown targets of small molecule drugs,which is crucial for drug development and the exploration of its mechanism of action.