Automated Sorting of Beads from a ™One-Bead-Two-Compounds∫ Combinatorial Library of Metalloproteinase Inhibitors Caspar Christensen a , Thomas Groth b , Christine Bruun Schi˘dt c , Niels TÒkker Foged c , and Morten Meldal a * a Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby (Denmark) b Combio A/S, Gamle Carlsberg Vej 10, DK-2500 Valby (Denmark) c Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev (Denmark) Review Article The development and use of an instrument that automati- cally separates non-fluorescent beads from fluorescent beads are described. The scope of the instrument was tested by on-bead screening of a solid phase combinatorial library synthesized on the aqueous compatible PEGA library resin [1]. The one-bead-two-compounds library [12] consisted of approx. 160.000 putative phosphinic peptide inhibitors, encoded by ladder synthesis [22] for facile analysis by MALDI-TOF mass spectrometry, and each bead also contained a common fluorescence-quenched substrate. The library was screened for activity against MMP-9; several beads with inhibitory activity were selected from the library by the novel fully automated sorting method and analyzed by MALDI-TOF MS for structural elucidation. Selected inhibitors were investigated in an enzyme kinetic assay with MMP-9, MMP-13, and MMP-14. The obtained K i values are in the middle to low nanomolar range. A good correlation between the inhibitor activity on solid phase and in solution was demonstrated. Hence, the results validated the on-bead screening and the efficiency of the fully automated sorting approach of the one-bead-two- compounds library format. Introduction The design, preparation and screening of chemical libraries, both in solution and on solid phase, are important for rapid development of proteinase inhibitor lead compounds [2 ± 8]. Application of solid phase combinatorial chemistry offers the advantage of synthesizing huge numbers of compounds, which can be screened simultaneously to yield the most potent inhibitors. In the on-bead assays the selection process in which hits are separated from non-hits has been an exasperating task rarely performed in a quantitative and reproducible manner [9]. Previously, it has been demonstrated, that the hydrophilic high-swelling PEGA resin is suitable for on-bead screenings of FRET substrate libraries for the characterization of proteases [10, 11]. The one-bead-two-compounds library [12] is an extension of the one-bead-one-compound format, where each bead contains two different compounds. One of the compounds is a reaction indicator and the other is the target of molecular interaction. Here, the reaction indicator QSAR Comb. Sci. 22 (2003) DOI: 10.1002/qsar.200320005 ¹ 2003 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim 737 * To receive all correspondence: Morten Meldal, Carlsberg Laboratory, Dept. of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark, Fax:  45 33274708, E-mail: mpm@crc.dk Abbreviations: Aa: amino acid; Abz: 2-amino benzoic acid; Acc: 1-amino-1-cyclohexane carboxylic acid; Ac: acetyl; Azc: L-2- azetidine carboxylic acid; Ad: adamantyl; Boc: tert-butoxycarbon- yl; Bu t : tert-butyl; ClF: p-chlorophenylalanine; CCD: Charged Coupled Device; Cha: b-cyclohexylalanine; Cit: L-citrulline; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DTT: dithiothreitol; EtOAc: ethylacetate; EXT: light scatter; FLU: fluorescence intensity; Fmoc: 9-fluorenaylmethyloxycarbonyl; FRET: fluorescence reso- nance energy transfer; MALDI-TOF MS: matrix assisted laser desorption/ionization time-of-flight mass spectrometry; MeOH: methanol; MMP: matrix metalloproteinase; [Mis]: mass/ionization spacer; NEM: N-ethylmorpholine; PEGA: polyethylene glycol- polydimethyl acrylamide resin; Pfp: pentafluorophenyl; [Pll]: photo labile linker; Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfon- yl; PMT: Photon-multiplier; RP-HPLC: reverse phase high pressure liquid chromatography; SPPS: solid phase peptide synthesis; TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethy- luronium tetrafluoroborate; TIPS: triisopropylsilane; TFA: tri- fluoroacetic acid; TOF: time of flight; Trt: triphenylmethyl; Y(NO 2 ): 3-nitro-L-tyrosine. Three- and one-letter abbreviations for amino acids are used according to the recommendations from IUPAC, see http:// www.chem.qmw.ac.uk/iupac. Automated Sorting of Beads from a ™One-Bead-Two-Compounds∫ Combinatorial Library of Metalloproteinase ... & Combinatorial Science