IMMUNOAFFINITY ULTRAFILTRATION WITH ION-SPRAY HPLC MS FOR SCREENING SMALL-MOLECULE LIBRARIES/

A solution-phase screening method for libraries of pharmaceutically re levant molecules is presented. The technique is applicable to screenin g combinatorial libraries of 20-30 closely related molecules. Zn this report, individual benzodiazepines are selected from a multicomponent library mixture by formation in solution of noncovalent immunoaffinity complexes with antibodies raised to therapeutically proven drugs such as nitrazepam, temazepam, or oxazepam. Captured compounds are separat ed from nonspecifically bound library components by centrifugal ultraf iltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and an alyzed by HPLC coupled with pneumatically assisted electrospray (ion s pray) ionization mass spectrometric detection. Competition by the benz odiazepines for limited antibody binding sites is controlled by varyin g the stoichiometry of the complexation mixture. This procedure select s library components with the greatest affinity for a particular antib ody. Specific capture of benzodiazepines is demonstrated by screening both a pool of structurally similar benzodiazepines and a more complex mixture of benzodiazepines with an additional set of unrelated compou nds, Affinity ultrafiltration and electrospray mass spectrometry compl ement each other to enhance screening and identification of pooled dru g candidates and potentially can be extended to other small-molecule c ombinatorial libraries and macromolecular receptor preparations.