Structural studies of β-hairpin peptidomimetic antibiotics that target LptD in Pseudomonas sp.

Abstract

Abstract:We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the -barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T(1)W(2)L(3)K(4)K(5)R(6)R(7)W(8)K(9)K(10)A(11)K(12)) linked to a DPro-LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys(6) (called LB-01), and another containing the same loop sequence, but linked to an LPro-DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt -hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not -hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa, whereas LB-02 is essentially inactive. Thus the -hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W(2)/W(8)) displayed on opposite faces of the -hairpin represent key groups contributing to antimicrobial activity. conformations. L27-11 and LB-01 both show antimicrobial activity in the nanomolar range against P. aeruginosa, whereas LB-02 is essentially inactive. Thus the ß-hairpin structure of the peptides appears to be very important for their antimicrobial activity.We surmise that a ß-hairpin-shaped peptide might be especially well suited to bind to proteins rich in ß-structure such as LptD in a membrane environment.

Synthesis and antimicrobial activity against Pseudomonas aeruginosa of macrocyclic β-hairpin peptidomimetic antibiotics containing N-methylated amino acids

“…1,14 The twelve N-methylated analogs of LB-01, each containing a single N-methyl residue in the hairpin loop, were prepared in the same way. Some of the necessary protected NMe amino acids were commercially available (Fmoc-NMe-Trp(Boc)-OH, Fmoc-NMeLeu-OH, Fmoc-NMe-Ala-OH) and could be incorporated using Fmoc chemistry.…”

“…1 The backbone adopts a stable folded ß-hairpin conformation according to several NMR criteria, including 3 …”

“…including the important human pathogen P. aeruginosa (PA). 1,2 Their unique mechanism of action involves binding to the outer membrane (OM) protein LptD in PA and inhibition of its essential role in lipopolysaccharide (LPS) transport to the cell surface. 3 Seven essential LPS transport (Lpt) proteins (LptA-G) in Gram-negative bacteria mediate LPS transport from the inner to the OM.…”

“…1 An alanine-scan in the parent compound L27-11 ( Figure-1A) showed that the side chains of Trp2 and Trp8, located on opposite faces of the ß-hairpin, are critical for activity. 1 In this work, we explore the importance of individual peptide bonds for antimicrobial activity, by analyzing analogs of LB-01 containing N-methylated amino acid residues. Crossstrand pairs of amino acids in ß-hairpins occupy alternately internal hydrogenbonding (HB) and non-hydrogen-bonding (NHB) positions.…”

Design, synthesis and evaluation of peptides and peptidomimetics inhibiting the bacterial DsbA-DsbB interaction

“…For further investigation on the binding mechanism to LptD, a third peptidomimetic structure from the same series, L27-11 (Figure 1.14C) was analyzed by NMR spectroscopy and SAR by alanine scanning, proving that the β-hairpin conformation was essential for interaction with LptD (Schmidt et al, 2013). Moreover, only two side chains (the indoles of tryptophan 2 and 8) were essential to retain strong antibacterial activity (Schmidt et al, 2013), hence leaving 10 amino acid sidechains free for further improvements.…”