Immunoaffinity Ultrafiltration with Ion Spray HPLC/MS for Screening Small-Molecule Libraries

Abstract

A solution-phase screening method for libraries of pharmaceutically relevant molecules is presented. The technique is applicable to screening combinatorial libraries of 20-30 closely related molecules. In 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 separated from nonspecifically bound library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and analyzed by HPLC coupled with pneumatically assisted electrospray (ion spray) ionization mass spectrometric detection. Competition by the benzodiazepines for limited antibody binding sites is controlled by varying the stoichiometry of the complexation mixture. This procedure selects library components with the greatest affinity for a particular antibody. 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 compounds. Affinity ultrafiltration and electrospray mass spectrometry complement each other to enhance screening and identification of pooled drug candidates and potentially can be extended to other small-molecule combinatorial libraries and macromolecular receptor preparations.

Analysis and screening of combinatorial libraries using mass spectrometry

“…In addition to combinatorial libraries, complex natural product extracts have been screened [63], and neither plant nor fermentation broth matrices were found to interfere with screening [58]. As another example of the flexibility of this screening system, a centrifuge tube equipped with an ultrafiltration membrane [64] has been used instead of an on-line ultrafiltration chamber. Other applications of pulsed ultrafiltration-mass spectrometry include screening drugs and drug candidates for metabolic stability [65], metabolic activation to reactive metabolites [66] and the measurement of affinity constants for ligandreceptor interactions [61,62].…”

Rapid methods for screening low molecular mass compounds non-covalently bound to proteins using size exclusion and mass spectrometry applied to inhibitors of human cytomegalovirus protease

“…53 However, the technique of microconcentration, which combines centrifugation with ultraÐltration, utilizes membranes which retain molecules above a speciÐc cut-o †, and can be used to rapidly and efficiently M r separate low inhibitors from retained high M r M r proteaseÈinhibitor complexes.54h56 An example of the use of microconcentration coupled with mass spectrometry is the isolation of active benzodiazepine analogs in the presence of polyclonal antibodies raised against speciÐc benzodiazapines, as reported by Henion and coworkers. 56 We have been studying the protease of human cytomegalovirus, in an e †ort to identify low inhibitors M r useful for the treatment of viral infection. Previously, we described an inhibitor (compound CL13933, a thiophile) which forms disulÐde bonds with CMVP57,58 and also a number of additional inhibitors which promote intramolecular disulÐde bond formation within CMVP.…”

Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries

“…Although the nomenclature of the techniques may be different, the solution phase screening methods are all generally based on the same principles. The screening process is initiated by forming a protein͞ligand complex, followed by isolation of the complex by using, for example, size exclusion chromatography (9)(10)(11)(12) or a molecular weight cut-off membrane (13,14). Determination of bound ligands requires dissociation of the complex, followed by chromatographic-mass spectrometric detection.…”