A Technique For Rapidly Screening Of Mixture-Based Ligand Libraries Via Magnetic Recovery Of Target-Ligand Complexes

A technique was proposed for rapidly screening of mixture-based ligand libraries via magnetic recovery of target-ligand complexes, whose procedure included (a) mixing each mixture with one exogenous reference ligand to prepare a processed mixture for screening (PMFS); (b) initiating competitive bindings among all the ligands in a PMFS to a protein target immobilized on magnetic nanoparticles; (c) recovering target-ligand complexes via magnetic force to extract bound ligands; (d) quantifying chromatographic peak areas of every detected ligand in both its PMFS and corresponding extract of bound ligands; (e) deriving relative affinities of detected candidate ligands if all the prerequisites were satisfied, or else optimizing compositions of PMFSs and/or competitive binding systems till all the prerequisites were met to derive their relative affinities; (f) locating potential valuable ligand(s) bearing affinity(ies) not less than those of other candidate ligands in each mixture; (g) identifying potential hit(s) bearing affinity(ies) not less than those of other potential valuable ligands in the library; (h) judging specificity via competitive binding(s) and then accepting hit(s) bearing judged specificity and satisfactory affinity(ies), or else judging specificity of other potential valuable ligands till location of hit(s).Using HPLC-MS analyses and bindings of biotin derivatives to streptavidin immobilized on magnetic nanoparticles as a model system, reliability of this new technique to each reaction mixture of one anticipated candidate ligand via parallel liquid combinatorial synthesis (PLCS), each mixture of multiple candidate ligands via pooling individual compounds, each reaction mixture of multiple anticipated candidate ligands via PLCS, each mixture of multiple anticipated candidate ligands via fractionation of crude natural products, were tested, respectively, and feasibility to identify trace ligands of higher affinities in crude natural products was investigated.As to each reaction mixture of one anticipated candidate ligands via PLCS, after the level of an exogenous reference ligand was kept at a constant level to have its binding ratio below 10% without any candidate ligand, increase of contents of the anticipated candidate ligand in its PMFS caused decrease of its binding ratios, increase of peak areas of the candidate ligand gradually to be five times above the absolute values of their intercepts for linear responses, and continuouis decrease of its relative affinities to a flat bottom after the binding ratios of the candidate ligand and the reference ligand were below 10%, respectively. Consequently, the average of relative affinities within this flat bottom was accepted as its relative affinity.As to each mixture of multiple candidate ligands via pooling individual compounds or PLCS, their quantitative affinities were available to locate potential hit(s) via HPLC-MS analyses with universally optimized experimental conditions as long as differences in contents of candidate ligands and number of candidate ligands in each mixture were within reasonable ranges, respectively. To such two types of mixtures of multiple candidate ligands, reliability of this new technique was determined by compositions of mixtures. Based on competitive bindings to an exogenous reference ligand at a much gigher level and bindings to denatered protein target, specificity of multiple candidate ligands was judged concomitantly.As to each mixture of multiple candidate ligands in crude natural products, their affinities and specificity could also be determined under universally optimized experimental conditions as long as all the components were well-resolved via HPLC-MS analyses and both differences in contents and number of candidate ligands were within reasonable ranges, respectively. By iterative run of the core procedure (competitive bindings, magnetic recovery of target-ligand complexes to extract bound ligands) with all the bound ligands recovered in previous runs as the sample for consecutive runs, exponential enrichement of ligands of higher affinities (EELHA) was feasible. With methanol extractions of rhizoma Chuanxiong containing multiple synthetic biotin derivatives of different affinities and different contents as samples, EELHA was proven to effectively enhance discovery of ligands of higher affinities at relative abundance about 2% equivalence of those of lower affinities. With amounts of the target in consecutive runs accounting for no more than 20% equivalence of ligands recovered in corresponding previous runs, EELHA could be realized at cost for large amounts of the protein target. EELHA was potentially valuable to crude natural products of known biological actions.In conclusion, this screening technique was promising to discover hit(s) in mixture-based libraries, and EELHA was potentially valuable to discover trace ligands of higher affinities in mixtures like crude natural products.