Small molecule G-quadruplex ligands are antibacterial candidates for Gram-negative bacteria

Abstract

With the rise in bacterial antimicrobial resistance (AMR) and decline in antibiotic discovery, global healthcare is at a point of jeopardy. There is great need for the development of novel antimicrobials that target bacteria that have become resistant to our existing antibiotics, in particular Gram-negative species such as Escherichia coli which is responsible for opportunistic infections of already compromised patients. Here we demonstrate that a novel pyridinium-functionalised azobenzene scaffold L20, identified as a candidate ligand able target G-quadruplex (G4) structures in bacterial genomes/transcriptomes, shows promising antibacterial activity (MIC values ≤ 4 µg/ml) against multi-drug resistant E. coli. Tandem Mass Tag (TMT) proteomics applied to cultures of the E. coli type strain ATCC 25922 treated with sub-lethal concentrations of L20, identified seven G4-containing sequences as potential targets for L20. FRET (fluorescence resonance energy transfer) stabilisation assays indicate L20 binds these selected sequences with variable and moderate affinity, in contrast to two comparator G4 ligands (stiff-stilbene L5 and pyridostatin (PDS)) that better stabilise G4 structures but exhibit a lower antimicrobial activity. However, proteomic experiments also reveal that, alongside its superior antibacterial activity, L20 treatment influences expression levels of more G4-associated proteins than either L5 or PDS, and upregulates multiple essential proteins involved in translation. These findings identify strategies discovering potential G4 ligands as approaches that can lead to antibacterial candidates active against priority targets such as multi-drug resistant E. coli, and that targeting G4 sequences, and ligands such as L20, warrant further exploration as potential novel therapeutics with G4-mediated modes of action.

The Rise of Bacterial G-Quadruplexes in Current Antimicrobial Discovery

“…coli genes involved in the cellular translational machinery to a higher extent compared with PDS . 127 It is worth noting that well-known and widely used G4 stabilizers such as BRACO-19 and TMPyP4 ( Figure 5 ) have shown no antimicrobial activity against E . coli and have not progressed further.…”

“…This latter option has been deemed with a higher priority, also considering that L20 has been found to affect the expression of G4-associated proteins and to modulate the expression of E . coli genes involved in the cellular translational machinery to a higher extent compared with PDS . It is worth noting that well-known and widely used G4 stabilizers such as BRACO-19 and TMPyP4 (Figure ) have shown no antimicrobial activity against E .…”

“…coli strains and a reference susceptible strain (e.g., UTI808, IR60, and ATCC25922, respectively), identifying the azabenzene L20 ( Figure 5 ) as the most promising hit with a MIC in the range 2–4 μg/mL. 127 Subsequently, target G4 sequences have been identified by proteomic analysis on cell lysates incubated with L20 as well as reference ligands including pyridostatin ( PDS , Figure 5 ) through LC-MS/MS analysis, while the ligands’ stabilizing efficacy has been evaluated by FRET melting on seven G4-putative sequences identified by LC-MS/MS in pivotal genes (i.e., dppA , thrA , yjbD , pdxA , clpB , glnD , and hemL , Table 1 ). PDS is a 2-quinolinyl derivative that has been developed as a specific G4 binder in anticancer research, thanks to its stabilizing activity against telomeric G4s preventing telomeres elongation.…”