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Link to original content: https://pubmed.ncbi.nlm.nih.gov/24422534
Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity - PubMed Skip to main page content
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. 2014 Feb 5;136(5):1922-9.
doi: 10.1021/ja410385c. Epub 2014 Jan 24.

Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity

Daniel W Carney  1 , Karl R Schmitz, Jonathan V Truong, Robert T Sauer, Jason K Sello
Affiliations

Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity

Daniel W Carney et al. J Am Chem Soc. .

Abstract

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.

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Figures

Figure 1
Figure 1
Structures of ADEP natural products and optimized synthetic analogs thereof.
Figure 2
Figure 2
Transannular hydrogen bonding in an ADEP. (A) Stereocartoon of an ADEP (gray ball-and-sticks) bound to Escherichia coli ClpP (adjacent subunits in green and orange), generated from crystal structure 3MT6. Two predicted hydrogen bonds are observed within the ADEP (black; distances in Å), and several hydrogen bond networks (cyan) occur either directly between the ADEP and ClpP or via ordered water molecules. (B) Schematic representation of ADEP transannular hydrogen bonds. (C) Overlay of 1H NMR spectra of compound 1a over time in CD3OD. Amides participating in bonds are highlighted in blue and the nonbonding amide is highlighted in red. The half-lives of the hydrogens of the alanine and difluorophenylalanine residues were 26.8 and 3.87 min, respectively (see Supporting Information).
Figure 3
Figure 3
Library of ADEP analogs. The N-methylalanine, pipecolate, 4-methylpipecolate and 4-isopropylpipecolate residues are highlighted in red. Serine and allo-threonine residues are highlighted in blue.
Figure 4
Figure 4
ADEP hydrogen–deuterium exchange in CD3OD. Deuterium exchange rates were measured for 2 mM solutions of each ADEP under pseudo-first order conditions in deuterated methanol at 25 °C. The exchange rates for the hydrogen atoms of the alanine residues within the peptidolactone are shown.
Figure 5
Figure 5
Activation of ClpP and competition with ClpX by ADEPs in vitro. (A) Rigidified ADEPs are more potent activators of ClpP peptide cleavage. Hydrolysis of a fluorogenic decapeptide substrate (15 μM) by E. coli ClpP (25 nM) was assayed in the presence of increasing concentrations of ADEP compounds, and activity was fit to a noncooperative binding model (solid lines). Error bars represent standard deviation among three replicates or standard error of the fit. Tighter apparent affinities correlate with increased ADEP rigidity, with the exception of compound 1d. See also Table 1. (B) ADEPs with greater macrocycle rigidity compete more strongly with ClpX for binding to ClpP. Fold change in ATPase activity of E. coli ClpXΔN (10 nM) in the presence of E. coli ClpP (50 nM) was assayed over increasing concentrations of ADEPs, compared to the activity of ClpXΔN alone, and was fit as above (no fit was obtained for 1d). More rigid ADEPs better compete for binding to ClpP and, thus, more effectively relieve ClpP-mediated repression of ClpXΔN ATPase activity (Table 1).
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