ABSTRACT
The continuing ability of bacteria to resist current antibiotic treatments highlights the need for alternative strategies for inhibiting their pathogenicity. Bacterial attachment is a major factor in infectivity and virulence. This key binding phase of bacteria to any potential host is mediated by adhesin proteins and so these present an attractive therapeutic target for antiinfective blocking strategies. However, the natural ligands to adhesins are large, typically complex molecules that are difficult to mimic with small molecules. We describe here a method that creates precise synthetic mimics of glycoproteins that are designed to bind adhesins. By using protein-degrading enzymes as the basis for these mimics we have created large-molecule protein ligands that inhibit aggregation of pathogenic bacteria at levels greater than a million-fold higher than small-molecule inhibitors of adhesins.
Subject(s)
Actinomyces/growth & development , Anti-Bacterial Agents/chemistry , Glycoproteins/chemical synthesis , Glycoproteins/pharmacology , Polymers/chemical synthesis , Actinomyces/metabolism , Anti-Bacterial Agents/pharmacology , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Carbohydrates/chemistry , Glycoproteins/metabolism , Polymers/metabolism , Polymers/pharmacologySubject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Subtilisin/antagonists & inhibitors , Subtilisin/metabolism , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Animals , Avidin/chemistry , Avidin/metabolism , Bacillus subtilis/enzymology , Biotin/metabolism , Catalysis , Enzyme Inhibitors/chemistry , Horses , Ligands , Liver/enzymology , Models, Molecular , Protein Structure, Tertiary , Substrate Specificity , Subtilisin/chemistry , Subtilisin/geneticsABSTRACT
A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.