Helperby Therapeutics (United Kingdom)

Bacterial infections remain a leading killer worldwide, which is worsened by the continuous emergence of antibiotic resistance. In particular, antibiotic-resistant Enterobacteriaceae are prevalent and extremely difficult to treat. Repurposing existing drugs and improving the therapeutic potential of existing antibiotics represent an attractive novel strategy. Azidothymidine (AZT) is an antiretroviral drug which is used in combination with other antivirals to prevent and to treat HIV/AIDS. AZT is also active against Gram-negative bacteria but has not been developed for that purpose. Here, we investigated the in vitro and in vivo efficacy of AZT in combination with colistin against antibiotic-resistant Enterobacteriaceae, including strains producing extended-spectrum beta-lactamases (ESBLs) or New Delhi metallo-beta-lactamase 1 (NDM) or carrying mobilized colistin resistance (mcr-1). The MIC was determined using the broth microdilution method. The combined effect of AZT and colistin was examined using the checkerboard method and time-kill analysis. A murine peritoneal infection model was used to test the therapeutic effect of the combination of AZT and colistin. The fractional inhibitory concentration index from the checkerboard assay demonstrated that AZT synergized with colistin against 61% and 87% of ESBL-producing Escherichia coli and Klebsiella pneumoniae strains, respectively, 100% of NDM-1-producing strains, and 92% of mcr-1-producing E. coli strains. Time-kill analysis demonstrated significant synergistic activities when AZT was combined with colistin. In a murine peritoneal infection model, AZT in combination with colistin showed augmented activities of both drugs in the treatment of NDM-1 K. pneumoniae and mcr-1 E. coli infections. The AZT and colistin combination possesses a potential to be used coherently to treat antibiotic-resistant Enterobacteriaceae infections.

Previously, we described a small quinoline-derived compound that exhibited selective bactericidal activity against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). It depolarizes the bacterial cell membrane. In this study, we investigated if HT61 was able to enhance the potency of other antibiotics, namely neomycin, gentamicin and mupirocin, and an antiseptic, namely chlorhexidine, against clinical isolates of MSSA and MRSA in vitro and in vivo. The MICs were determined by the broth microdilution method. The effect of combinations was examined using the chequerboard method and time-kill curves. A murine skin infection model was used to evaluate the enhancement by HT61 of other antimicrobials. Using the fractional inhibitory concentration index, no interaction was seen in both MSSA and MRSA for the pair HT61 and gentamicin or the pair HT61 and neomycin. Synergism was seen for 65% of both MSSA and MRSA when HT61 was combined with chlorhexidine. There was also no interaction between HT61 and mupirocin. Time-kill analysis demonstrated significant synergistic activities when a low level of HT61 was combined with neomycin, gentamicin or chlorhexidine. The effect was more dramatic against non-multiplying bacteria against which the antimicrobials used were inactive on their own. Significant synergistic effects were also seen on mouse infected skin. We demonstrate that HT61, developed as a topical agent, acts as an enhancer that accelerates the activities of other antimicrobial agents against both MSSA and MRSA.

Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophage like cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophage like cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4-/- mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.