Magnetic Immobilized Dihydrofolate Reductase For Screening Of High Affinity Inhibitors At Low Abundance In Mixtures By Iterative Exponential Enrichment

Mixture-based screening for the discovery of leads has the significant advanteges over the conventional method upon the preparations of pure compounds one-by-one for screening.A new method for screening high-affinity ligands in mixtures by iterative exponential enrichment was developed but still need the validation for screening medicinal ligands at trace level in the mixture library.MtDHFR was expressed and immobilized on magnetic as a target to develop a system for screening high affinity inhibitors at low abounce in mixtures,and forward the applications in the combinatorial synthesis mixture and the natural mixture.The MtDHFR expression plasmid pET28a:dfrA with N-terminal 6×His tag was constructed and successfully expressed in E.coli BL21（DE3）for characterized of enzymatic properties.6×His-MtDHFR was first immobilized on magnetic submicron particles（MSP）with Ni²⁺-NTA.It was found that the immobilization capacity of MtDHFR was（93±12）mg/g MSP（n=3）,but the immobilized enzyme activity retained no more than 32%;Further studies found that Ni²⁺significantly inhibited the enzyme activity,and EDTA and Ni²⁺showed a synergistic inhibitory effect,while Fe³⁺had no remarkable interference.In addition,the stability of the complex of Ni²⁺-NTA and 6×His tagged enzyme is usually maintained at pH 8.0 or higher;however,free 6×His-MtDHFR is susceptible to pH,and the retained activity at pH 8.0 is very low,which does not meet the requirements of drug screening applications.Therefore,the Ni²⁺-NTA MSP can not be applicable for immobilization of MtDHFR for screening of ligand mixture libraries.Immobilization of MtDHFR on MSP functionalized with carboxyl groups.According to the crystal structure（PDB code:4KNE）,MtDHFR contains only one lysine（Lys-53）and one N-terminal primary amino group,both of which are far from the active site.Therefore,it is feasible to form amides between the amino groups and the surface carboxyl groups on MSP for immobilization.The immobilization capacity of MtDHFR was（8.6±0.6）mg/g MSP（n=3）,and the retained activity was（87±4）%（n=3）relative to free enzyme activity.There was no significant difference between the activities of free and the immobilized MtDHFR when stored in binding buffer at 0°C for 16 h,but the free enzyme activity decreased by nearly 60%,while the MtDHFR activity immobilizated on MSP was decreased by only about 35%after storage for 16 h at 25°C;notably,the immobilized enzyme remained stable within 4 h.Thus the stability of the immobilized enzyme was significantly higher than that of the free enzyme,and there was no significant difference in the IC₅₀ of methotrexate（MTX）between the free and MSP-immobilized MtDHFR（P>0.05）.Therefore,immobilization of MtDHFR with carboxyl MSP is competent for screening of high affinity ligands in the library of ligand mixtures by exponential enrichment.The typical inhibitor MTX was used as the parent to design and synthesize a series of derivatives with different order of magnitude affinity for MtDHFR.Artificial mixtures were prepared by pooling the derivatives at a preset ratios,and a reference ligand was added to form a sample for screening.With the immobilized MtDHFR on carboxyl MSP as the target,the competitive binding of ligands in mixture can be achieved for selective enrichment of the higher affinity inhibitors by reducing the amount of target and keeping the average binding ratios less than 10%;by using the ligand extract of the previous round as the sample for the next round of iterative screening as such,an exponential enrichment was achieved for screening MTX of 1%as the high-affinity ligand in the mixture.Then,MTX and aromatic acids were mixed with alcohols and amines for the combinatorial synthesis of a complex mixture containing trace leftover of MTX.The mixture product was diluted properly and the reference was added for iterative screening,and the exponential enrichment of the residual trace amount of MTX was achived by two rounds of iterative screening.Finally,the alcohol extract of turmeric was appropriately diluted to prepare model mixture of natural medicine mixture containing a trace amount of the candidate ligand.Two rounds of screening selectively enriched the trace MTX in the natural mixture.In summary,MtDHFR was recombinant expressed and immobilized as the target;the iterative screening system was validated for exponential enrichment of nanomole affinity at the level of less than 1%using a simulated mixture for preparing by pooling of MTX derivatives.The iterative screening system was successfully applicable for screening such ligand in the combinational synthesis mixture by exponential enrichment,and natural mixtures by selective enrichment.Thus,it is feasible for exponential enrichment of nanomole affinity ligand of trace level and has the promise to discover new ligands as leads from the herbal medicines.