Defining new chemical space for drug penetration into Gram-negative bacteria

Abstract

We live in the era of antibiotic resistance, and this problem will progressively worsen if no new solutions emerge. In particular, Gram-negative pathogens present both biological and chemical challenges that hinder the discovery of new antibacterial drugs. First, these bacteria are protected from a variety of structurally diverse drugs by a low-permeability barrier composed of two membranes with distinct permeability properties, in addition to active drug efflux, making this cell envelope impermeable to most compounds. Second, chemical libraries currently used in drug discovery contain few compounds that can penetrate Gram-negative bacteria. As a result of these challenges, intensive screening campaigns have led to few successes, highlighting the need for new approaches to identify regions of chemical space that are specifically relevant to antibacterial drug discovery. Herein we provide an overview of emerging insights into this problem and outline a general approach to addressing it using prospective analysis of chemical libraries for the ability of compounds to accumulate in Gram-negative bacteria. The overall goal is to develop robust cheminformatic tools to predict Gram-negative permeation and efflux, which can then be used to guide medicinal chemistry campaigns and the design of antibacterial discovery libraries.

Fig. 1 ∣. The Gram-negative cell envelope and pathways of drug fluxes across it.

The envelope includes a network of lipopolysaccharides that limits penetration of large and hydrophobic compounds (green C) (1), porin channels that permit passage of small, hydrophilic molecules (red C) (2), and trans-envelope efflux pumps that capture their substrates from the periplasm (3) or cytosol (4) and pump them out of the cell. The kinetic scheme describes four compartments: outside the cell (O), within the outer membrane (M), in the periplasm (P), and in the cytoplasm (I). Active efflux is approximated as a Michaelis–Menten process, where V is the maximum achievable velocity, P is the concentration of drug in the periplasm, and K_M is the Michaelis constant. The binding to the membrane is postulated to be saturable, with the maximal flux F. The degree of saturation is denoted as ϕ, while k₁ through k₄ are microscopic rate constants. Additional transporters can be readily integrated into this model.

Fig. 2 ∣. Comprehensive approach to developing cheminformatic tools to predict Gram-negative bacterial compound accumulation.

Chemical libraries are designed to probe regions of chemical space and to assess impacts of scaffolds, regiochemistry, and substituents. For each bacterial species, four isogenic strains are used – wild-type, hyperporinated, efflux-deficient, and doubly-compromised – to dissect the contributions of the OM, IM, and efflux to net accumulation (blue = native porins, green = engineered large-bore porins, purple = efflux pumps). Compounds are screened in high-throughput assays and analyzed by high-throughput detection methods, such as solid-phase extraction–mass spectrometry. Steady-state analyses are performed for large collections of compounds while detailed kinetic analyses are carried out for smaller subsets of compounds to enable calculation of kinetic parameters. Regression and classification approaches are used to develop cheminformatic models that correlate physiochemical properties with accumulation, which are then tested further in additional analogues.