A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis. Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme͞inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19-and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening of additional new libraries without adding additional enzyme.D uring the last decade new combinatorial techniques have been developed that allow for synthesis of vast quantities of potential therapeutic compounds (1-4). Although remarkable advances in generating complex libraries of molecules have been made, the analytical process of analyzing and screening the immense numbers of compounds generated is currently lacking. To keep pace with the synthetic process, contemporary analytical techniques must be capable of screening a large number of compounds in a high-throughput manner with precision and accuracy.Notable progress has been made in overcoming the problem of screening extensive libraries that are created by combinatorial methods using MS (5-8). At present, a variety of front-end affinity-selection techniques are used in conjunction with MS to determine potentially active compounds. 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 (9-12) or a molecular weight cut-off membrane (13,14). Determination of bound ligands requires dissociation of the complex, followed by chromatographic-mass spectrometric detection. Some examples include pulsed ultrafiltration MS, developed by van Breemen and coworkers, as an elegant combinatorial library screening methodology (13, 15). There are also a vari...

Tetracyclic guanines have been shown to be potent and selective inhibitors of the cGMP-hydrolyzing enzymes PDE1 and PDE5. In general, these compounds are inactive or only weakly active as inhibitors of PDE3, which is a major isozyme involved in cAMP hydrolysis. Structure-activity relationships are developed at N-1, C-2, N-3, and N-5 on the core nucleus. Compound 31, with an IC50 of 70 pM, is the most potent inhibitor of PDE1, while 50, with an IC50 of 4 nM, is the most potent inhibitor of PDE5. Compounds 20, 22, 30, and 50 are potent dual inhibitors with IC50 values below 30 nM for both PDE1 and PDE5. Compounds 12, 20, and 28 reduced blood pressure by more than 45 mmHg when administered orally at 10 mg/kg to the spontaneously hypertensive rat (SHR).