Thiol-containing diketopiperazines have been recently identified as novel heterocyclic inhibitors of matrix metalloproteinase (MMPs). The compounds described had similar activities against the MMPs collagenase-1 and gelatinase-B. An inhibitor that showed greater than 10-fold selectivity for collagenase-1 over gelatinase-B was desired. Previously published work with peptidyl hydroxamates and thiols indicated that while preparing gelatinase selective inhibitors was straightforward, there was not an obvious route to selective inhibitors of collagenase-1. Combinatorial libraries were prepared and evaluated for their ability to inhibit collagenase-1 and gelatinase-B substrate hydrolysis. A method for estimating the IC50 values of compounds generated by high-throughput parallel synthesis aided in the identification of compounds with the desired properties. We have found that thiol diketopiperazines derived from nitrophenylalanine are both potent and selective inhibitors of collagenase-1. In addition, we have demonstrated that combinatorial chemistry can be utilized to identify molecules with a desired selectivity profile without access to the traditional tools of rational drug design.
Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.
A high-volume enzyme immunoassay (EIA) system for slaughterhouse screening of sulfamethazine In swine plasma/ serum has been developed. The system Includes a robotic sample processor that performs most of the liquid handling requirements in the assay. The assay gives good correlation with the widely used thin layer chromatographic method for determination of sulfamethazine in serum and plasma (r = 0.826). Inter- and Intra-assay coefficients of variation are less than 10%. Approximately 2,400 serum/ plasma samples can be analyzed In a normal working day (8 h) with this system.
Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788 -10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX 8 GA-NH 2 . Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (؊3, ؊1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme.Bacterial type I signal peptidase is responsible for cleaving the signal peptide from precursor proteins, and its activity is an integral part of the export and maturation of secreted proteins in vivo. The essential function of the enzyme to bacterial cell viability has been demonstrated using genetic approaches with both Gram-positive and Gram-negative organisms (1-3), supporting the notion that the signal peptidase is potentially an antibacterial target (4). Drug discovery efforts with the enzyme, however, may be hampered by the lack of an effective in vitro assay employing a nonprotein substrate such as a peptide (4).Our current understanding is that signal peptides are highly variable in sequence (5). Based on the studies carried out over the past 2 decades, it has been established that the recognition sites for signal peptidases lie between Ϫ6 and ϩ1 in sequences encompassing the site of cleavage (6 -12). Sequence conservation analyses of a large panel of naturally occurring signal peptides in bacteria and eukaryotes reveal that the predominant residue at the P1 site is Ala and that the predominant residues at the P3 site are large aliphatic residues (Leu, Ile, Val) as well as Ala and Ser, a consensus dubbed the (Ϫ3, Ϫ1) rule (9 -11). The (Ϫ3, Ϫ1) rule also holds for the cleavage of engineered preproteins in vivo as well as in vitro (6 -8, 12). The reaction of signal peptidases with synthetic peptides, on the other hand, is not as well explored as with protein substrates. For the signal peptidase LepB from Escherichia coli, the best characterized signal peptidase, Ala was found as the only residue permitted at the P1 site through single amino acid replace...
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