Novel Strategies towards the Inhibition of ATP-Binding Cassette (ABC) Transporters from Pathogenic Bacteria
Multidrug transporters in the ATP-Binding Cassette (ABC) superfamily play critical roles in pathogenic bacteria. These transport systems are particularly important in conferring antibiotic resistance on the cell by mediating the efflux of a wide range of structurally unrelated compounds and the transport of lipids that form antibiotic impervious membrane structures. Identifying novel antibiotic targets and strategies is required to address the ever-growing antibiotic crisis. ABC transporters are an untapped pool of potential targets. Two examples are the essential lipid transporter MsbA, from *Escherichia coli* and other pathogenic Enterobacteria, and the multidrug efflux transporter PatAB, that is upregulated in fluoroquinolone resistant *Streptococcus pneumoniae*. These proteins primarily utilise nucleotide hydrolysis to drive transport against the inwardly-directed concentration gradient across the cell plasma membrane. Current strategies to inhibit these transporters utilise small molecule drugs that often resemble the transported substrates or that target hydrophobic pockets in the transmembrane domain. While some promising steps have been made, studies of inhibition of bacterial transporters still need to catch up to their eukaryotic counterparts. This work aimed to elucidate the characteristics of inhibition of MsbA and PatAB while introducing novel strategies to target the ABC superfamily, summarising the state of drug development against these proteins and the available functional techniques to study inhibition. First, extensive characterisation of the interactions of PatAB with its transport substrates revealed two distinct responses to substrate binding. While many of these substrates did not affect the rate of nucleotide hydrolysis, suggesting an uncoupling of their transport from nucleotide hydrolysis, a subclass of substrates including ethidium, propidium and aminocoumarin antibiotics showed potent non-competitive inhibition of hydrolysis. This was observed both for protein in detergent solution and in lipidic nanodiscs. Three models for substrate-protein interaction are presented, including a novel binding site near the nucleotide-binding domain. This activity is unique for a heterodimeric ABC transporter. Identification of the inhibitory site might provide a novel specific target to re-sensitise antibiotic resistance of *S. pneumoniae* infections. Second, this work introduces a novel class of inhibitors specifically targeting MsbA. As MsbA is one of the best-characterised proteins of the ABC superfamily required for cell growth, this transporter is an ideal candidate for clinically relevant inhibitor development. Towards this goal, a library of rationally designed peptide inhibitors was generated from the primary sequence of the transmembrane domain. These peptides were designed to be α-helical membrane spanning synthetic peptides that disrupt helix-helix interactions required for conformational change. Three initial approaches were employed; (i) fragmentation of helices seen as cytotoxic in a preliminary study, (ii) generation of solubility-tagged membrane spanning sequences, and (iii) rational design of peptides targeting known motifs involved in helical rearrangements. Initial screening of these peptides identified sequences from transmembrane helix 1 and transmembrane helix 5 that were able to inhibit transport activity by MsbA in *Lactococcus lactis* cells. Further development was carried out on a hit fragment of transmembrane helix 1, including modifications that improved solubility and mutations that engineered an unexpected active disulphide-containing peptide. Potent inhibitory activity was observed *in vitro* and *in vivo* on both the lipid transport and small molecule efflux activity of MsbA. Inhibition was also observed in intact *Escherichia coli* cells, with recoverable growth through high expression of MsbA proteins from a plasmid. Finally, having identified pitfalls and bottlenecks during our inhibitor design process, a novel screening platform was developed combining electrical measurements with ABC transporter activity in supported lipid bilayers. Using optical, biochemical and electrical measurements the platform validated the use of PEDOT:PSS electrodes to measure ABC transporter activity which not only provide the option for high throughput screening in native-like environments but also identified an ATP-dependent ion transport pathway in MsbA. Findings from this work point to novel mechanisms by which ABC transporter activity can be modulated for drug development and highlight some of the crucial considerations required when generating inhibitors against this superfamily.
ABC Transporter, Antimicrobial Resistance, Drug Transport, Membrane Proteins
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