(W0930-03-21) H-89 Exhibits Synergistic Antimicrobial Efficacy With Colistin Against Klebsiella pneumoniae Through Elevated ROS Level

Purpose: Klebsiella pneumoniae is one of the major pathogenic bacteria causing human respiratory diseases like pneumonia. Recently, K. pneumoniae was reported to be highly associated with lung tumors, significantly increasing the mortality rate. While H-89, a small molecular inhibitor of protein kinase A (PKA), has been reported to show activities in tumor suppression, its antimicrobial potential is relatively underexplored. In this study, we investigated its bacterial inhibition effects against K. pneumoniae and explored the synergy with the antibiotics. The antibacterial mechanism of an identified combination, H-89–colistin, was also evaluated.

Methods: The invasion of K. pneumoniae (ATCC 13883) into firefly luciferase-expressing non-small-cell lung cancer (NSCLC) cells was first assessed by co-incubating the bacteria with the cancer cells at a multiple of infection (MOI) of 25:1 for 4 h, followed by gentamicin treatment (300 μg/mL) for 1 h to remove extracellular bacteria and further incubation for 72 h. The cancer cell viability was assessed with a luciferase assay. The number of intracellular bacteria was then determined by plate counting and visualized by transmission electronic microscopy (TEM) and live cell fluorescent microscopy where K. pneumoniae was pre-stained with SYTO 9 and the cancer cell nucleus was stained with Hoechst 33342. The minimal inhibitory concentration (MIC) of H-89 and various commonly used antibiotics against K. pneumoniae was determined with the microdilution method. The antibacterial profile of H-89 was illustrated with the OD₆₀₀ curve and plate count of K. pneumoniae. The synergy of H-89 with various antibiotics against K. pneumoniae was also evaluated with a checkerboard assay. The antibiofilm assay was conducted with crystal violet staining. The membrane potential, outer membrane permeability, and cytoplastic membrane permeability of treated K. pneumoniae were assessed with the dyes DiSC₃(5), N-phenylnaphthalen-1-amine (NPN), and propidium iodide (PI), respectively. The internal reactive oxygen species (ROS) level was determined with a kit containing the DCFH-DA fluorescent probe.

Results: The co-habitation of K. pneumoniae with three lung cancer cell lines, A549-Luc, HCC827-Luc, and NCI-H1299-Luc, confirmed that the bacteria could invade the cancer cells without affecting their viability (Figure 1). H-89 demonstrated moderate antibacterial activity with the MIC against K. pneumoniae of 64 μg/mL. The inhibitory and anti-biofilm effects were both in a dose-dependent manner (Figure 2A–D). H-89 did not show antibacterial synergy with the tested antibiotics in targeting K. pneumoniae, except colistin, which manifested significant synergistic effects with H-89 (Figure 2E–G). The mechanistic investigation revealed that H-89 altered the membrane potential and increased the membrane permeability and internal ROS levels of K. pneumoniae in a dose-dependent manner, in correspondence with its antibacterial activity. The combination of H-89 (16 μg/mL) and colistin (0.125 μg/mL) at the synergistic ratio showed limited influences on the membrane potential and membrane permeability. Still, they dramatically elevated the ROS level of the bacterium, compared with the monotherapies at the same concentrations (Figure 3).

Conclusion: K. pneumoniae can be harbored in NSCLC cells and coexist with them. The PKA inhibitor H-89 exhibits moderate antibacterial efficacy against K. pneumoniae, while its combination usage with colistin shows significant synergistic bacteria suppression. The mechanism study elucidates that the simultaneous administration of the two compounds synergistically elevates the internal ROS to induce the lethal malfunction of the bacterium.

References: (1) Paczosa, M. K.; Mecsas, J. Klebsiella Pneumoniae: Going on the Offense with a Strong Defense. Microbiol. Mol. Biol. Rev. 2016, 80 (3), 629–661. DOI: 10.1128/mmbr.00078-15.
(2) Ding, L.; Yang, Z.; Lu, J.; Ma, L.; Liu, Y.; Wu, X.; Yao, W.; Zhang, X.; Zhu, K. Characterization of Phenotypic and Genotypic Traits of Klebsiella Pneumoniae from Lung Cancer Patients with Respiratory Infection. Infect. Drug Resist. 2020, 13, 237–245. DOI: 10.2147/IDR.S229085.
(3) Marunaka, Y.; Niisato, N. H89, an Inhibitor of Protein Kinase A (PKA), Stimulates Na+ Transport by Translocating an Epithelial Na+ Channel (ENaC) in Fetal Rat Alveolar Type II Epithelium. Biochem. Pharmacol. 2003, 66 (6), 1083–1089. DOI: 10.1016/S0006-2952(03)00456-8.
(4) Böckmann, S.; Nebe, B. The in Vitro Effects of H-89, a Specific Inhibitor of Protein Kinase A, in the Human Colonic Carcinoma Cell Line Caco-2. Eur. J. Cancer Prev. 2003, 12 (6), 469.
(5) Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A–Based Immunotoxins | Molecular Cancer Therapeutics | American Association for Cancer Research.
(6) Yu, M.; Liu, T.; Chen, Y.; Li, Y.; Li, W. Combination Therapy with Protein Kinase Inhibitor H89 and Tetrandrine Elicits Enhanced Synergistic Antitumor Efficacy. J. Exp. Clin. Cancer Res. 2018, 37 (1), 114. DOI: 10.1186/s13046-018-0779-2.
(7) Shan, G.; Bi, G.; Zhao, G.; Liang, J.; Bian, Y.; Zhang, H.; Jin, X.; Hu, Z.; Yao, G.; Fan, H.; Zhan, C. Inhibition of PKA/CREB1 Pathway Confers Sensitivity to Ferroptosis in Non-Small Cell Lung Cancer. Respir. Res. 2023, 24 (1), 277. DOI: 10.1186/s12931-023-02567-3.

Acknowledgements: This work was supported by the University Grants Committee of Hong Kong (ref. 14307823) and Guangdong-Hong Kong-Macao Joint Laboratory for New Drug Screening, School of Pharmacy, The Chinese University of Hong Kong. T. Yi gratefully acknowledges the provision of the Postgraduate Studentship by The Chinese University of Hong Kong.


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