Structure-kinetic relationship of carbapenem antibacterials permeating throughE. coliOmpC porin

Abstract

The emergence of Gram-negative "superbugs" exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram-negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure-translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC- E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC-expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k(on) ≈ 2 × 10(4) M(-1) s(-1)). Zwitterionic compounds with highly polar groups attached to the penem-2 ring, including panipenem, imipenem and doripenem exhibit faster k(on) (>10(4) M(-1) s(-1)), while meropenem and biapenem with fewer exposed polar groups exhibit slower k(on) (∼5 × 10(3) M(-1) s(-1)). Tebipenem pivoxil and razupenem exhibit ∼13-fold slower k(on) (∼1.5 × 10(3) M(-1) s(-1)) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure-kinetic relationships of carbapenem translocation are governed by H-bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups).

Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

“…In compensation, favorable enthalpic antibiotic-protein interactions (40, 41) ease the passage of the polar molecule. Furthermore, the release of protein (and antibiotic) hydration waters, upon formation of antibiotic-protein H-bonds, is accompanied by a favorable entropy increase (42,43). A very recent investigation showed how small changes to the "lateral chains" of these two carbapenems can modulate the penetration through specific channels in Pseudomonas aeruginosa (22).…”

Towardin vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia

“…We showed that binding under dynamic conditions is characterized by time-dependent occupancy (rather than static occupancy/potency), which is governed by kon relative to the rate of binding site buildup, concentration/exposure, and koff relative to the rate of binding site decay. Non-trappable hERG blocker binding clearly falls at the extreme end of the non-equilibrium spectrum,given that the open (blocker-accessible) state of hERG normally builds and decays over a 350-400 ms time window [17].3) We hypothesized that hERG binding is energetically driven by de-solvation and resolvation costs (the putative origin of all non-covalent binding free energy barriers [17,[19][20][21][22][23]). Toward that end, we studied the solvation properties of the hERG pore usingWaterMap and a homology model of the protein [24] (prior to the publication of the cryo-EM structure of open state hERG).…”

“…3) We hypothesized that hERG binding is energetically driven by de-solvation and resolvation costs (the putative origin of all non-covalent binding free energy barriers [17,[19][20][21][22][23]). Toward that end, we studied the solvation properties of the hERG pore using…”

Establishment of a dual-wavelength spectrophotometric method for analysing and detecting carbapenemase-producing Enterobacteriaceae

“…In addition, the outer membrane permeability of imipenem may differ among CPE strains and contribute to the discrepancy. Porins are reported to constitute the primary route of periplasmic entry of imipenem 21 , 22 , and varying expression levels or porin mutations can affect the results. A strain with reduced permeability will hydrolyse lesser imipenem in our system, while still exhibiting a high MIC.…”