Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase: design, conformation and conversion to α-ketoamide inhibitors

• Published in: European journal of medicinal chemistry 2003-04, Vol.38 (4), p.351-356
• Main Authors: Bruton, Gordon, Huxley, Anthony, O'Hanlon, Peter, Orlek, Barry, Eggleston, Drake, Humphries, John, Readshaw, Simon, West, Andrew, Ashman, Stephen, Brown, Murray, Moore, Keith, Pope, Andrew, O'Dwyer, Karen, Wang, Lei
• Format: Article
• Language: English
• Subjects: Amides - chemistry; Amides - pharmacology; Amino Acid Sequence; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Binding Sites; Biological and medical sciences; Dose-Response Relationship, Drug; Enzyme Activation; Escherichia coli - enzymology; Escherichia coli - genetics; Hydrogen-Ion Concentration; Inhibitor; Kinetics; Lipopetide; Lipoproteins - chemical synthesis; Lipoproteins - chemistry; Lipoproteins - metabolism; Magnetic Resonance Spectroscopy; Medical sciences; Membrane Proteins; Oligopeptides - chemical synthesis; Oligopeptides - chemistry; Oligopeptides - metabolism; Pharmacology. Drug treatments; Protein Conformation; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - metabolism; S. aureus; Sequence Homology, Amino Acid; Serine Endopeptidases - drug effects; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - pharmacology; Signal peptidase; SpsB; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Substrate; Substrate Specificity; Time Factors
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/S0223-5234(03)00040-0

Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide, as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the P1 and P1′ alanines with a k cat/ K m of 2.3×10 6 M −1 s −1 at pH 8.5. Insertion of proline at P1′ converted substrates to competitive inhibitors, whilst the incorporation of an α-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB.