Lipid Tales: Optimizing Arylomycin Membrane Anchors

Abstract

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria.

Figure 1

Equilibria that are important in understanding the impact of modifications to the lipid anchor. The serum albumin structure is PDB entry 4BKE, and the LepB structure is PDB entry 6B88. This figure was created with BioRender.

Figure 2

Impact of lipophilic tail composition on three different MIC values. Data are shown for 31 compounds that are identical except for their tail regions. Seven compounds have linear aliphatic tails (light-blue ●), 12 have all-carbon biaryl tails (orange ●), and 12 have biaryl tails with heteroatoms incorporated in the aromatic ring proximal to the amide carbonyl group (green ●). From left to right, the AlogP of each compound is plotted against −log₁₀ MIC against the E. coli IMP strain, E. coli 25922 in a broth medium, and E. coli 25922 in the presence of 50% mouse serum. On each plot, diagonal lines are plotted to represent trajectories with equivalent lipophilic efficiency, with the plotted integer equal to pMIC – AlogP. G0775 is highlighted in each plot with an arrow.

Figure 3

Box-and-whisker plots showing the E. coli IMP to E. coli 25922 MIC ratio and the MIC ratio for E. coli 25922 with and without 50% mouse serum, with the compounds subdivided by tail composition. The central horizontal line indicates the median value for each group, and the box ends indicate the first and third quartiles in each group. The whiskers correspond to the highest nonoutlier points in each group. Outliers are defined as the points outside the median value ±1.5-fold the difference between the third quartile and first quartile. Outlier points are plotted individually. n = 7, 12, and 12, left to right.

Figure 4

E. coli 25922 MIC/f_u plotted against the E. coli 25922 MIC determined in the presence of 50% mouse serum. MIC/f_u was calculated by dividing the broth MIC value determined for E. coli 25922 by the free fraction determined in the presence of human serum measured at 24 h. Compounds marked with an asterisk have a measured human f_u < 0.01.