Synthesis And Activity Study Of Multivalent Protein Inhibitors By Dynamic Combinatorial Chemistry

In this study,we presented a new method for the synthesis of multivalent protein inhibitors.Based on the theory of dynamic combinatorial chemistry（DCC）,we generated polymer-scaffolded dynamic combinatorial libraries（PS-DCLs）through reversible acylhydrazone formation reactions between novel aldehyde-functionalized polymer APG and a variety of hydrazide monomer compounds with potential active binding sites.Multivalent proteins,such as acetylcholinesterase,butyrylcholinesterase,E.coli heat labile toxin,were applied to DCLs as templates.Through multivalent interaction between the polymer side chain and biomoleculecular receptors,certain side chains were amplified by reorder the equilibrium of the dynamic systems.HPLC and NMR were used as the main analytical methods to identify the side chains with the best inhibition effect on the multivalent protein in the DCL.Then,multivalent protein inhibitors containing only the optimal side chains were prepared by condensation of the corresponding acylhydrazide monomers with polymer APG.In the in vitro bioactivity evaluation,the inhibitory activity of the polymer inhibitor containing only the optimal side chain,the corresponding acylhydrazide monomer and other clinical reference materials were tested,respectively.The test results showed that the biological activity of the multivalent drug was much better than that of its monovalent ligand and its clinical reference,fully proving the high efficiency of the multivalent protein inhibitor developed by this method.In the in vitro cytotoxicity study,the cytotoxicity of the polymer inhibitor containing only the optimal side chain,the corresponding hydrazine monomer and the polymer APG were tested respectively.It could be seen from the results of cytotoxicity evaluation that the polymer APG did not show obvious cytotoxicity,and monomer acylhydrazide generally had certain toxicity.Therefore,the low toxicity of the multivalent polymer inhibitor containing only the optimal side chain was highly likely due to the introduction of the monomer side chains.It was worth noting that the IC₅₀ of the polymer inhibitor containing only the optimal side chain was significantly higher than its effective inhibition concentration,which fully demonstrated the relative safety of the multivalent protein inhibitor developed by this method.In this study,three multivalent protein inhibitors were synthesized by the above methods,including multivalent acetylcholinesterase inhibitors,multivalent E.coli heat labile toxin inhibitors,and multivalent butylcholinesterase inhibitors.Those results indicated that our method had a certain universal applicability in the development of multivalent protein inhibitors,and thus provided a new method for the subsequent research and development of new multivalent protein inhibitors.In summary,the implementation of this research had basically confirmed the high efficiency,relative safety and general applicability of the proposed method,and had initially developed into a set of mature new methods for the development of multivalent protein inhibitors.