Browsing by Author "Thangamani, Shankar"

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    Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens
    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa FN N.; Sobreira, Tiago JP P.; Hedrick, Victoria E.; Paul, Lake N.; Seleem, Mohamed N. (Nature Publishing Group, 2016-03-03)
    Traditional methods employed to discover new antibiotics are both a time-consuming and financially-taxing venture. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). Such an effort led to the discovery of auranofin, a drug initially approved as an anti-rheumatic agent, which also possesses potent antibacterial activity in a clinically achievable range. The present study demonstrates auranofin's antibacterial activity is a complex process that involves inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. We also confirmed that the lack of activity of auranofin observed against Gram-negative bacteria is due to the permeability barrier conferred by the outer membrane. Auranofin's ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins. Additionally, auranofin is capable of eradicating intracellular MRSA present inside infected macrophage cells. Furthermore, auranofin is efficacious in a mouse model of MRSA systemic infection and significantly reduces the bacterial load in murine organs including the spleen and liver. Collectively, this study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antibacterial for treatment of invasive bacterial infections.
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    Antibacterial activity and therapeutic efficacy of Fl-PRPRPL-5, a cationic amphiphilic polyproline helix, in a mouse model of staphylococcal skin infection
    Thangamani, Shankar; Nepal, Manish; Chmielewski, Jean; Seleem, Mohamed N. (Dove Medical Press, 2015-01-01)
    The antibacterial activities and therapeutic efficacy of the cationic, unnatural proline-rich peptide Fl-PRPRPL-5 were evaluated against multidrug-resistant Staphylococcus aureus in a mouse model of skin infection. Fl-PRPRPL-5 showed potent activity against all clinical isolates of S. aureus tested, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA, respectively). Fl-PRPRPL-5 was also superior in clearing established in vitro biofilms of S. aureus and Staphylococcus epidermidis, compared with the established antimicrobials mupirocin and vancomycin. Additionally, topical treatment of an MRSA-infected wound with Fl-PRPRPL-5 enhanced wound closure and significantly reduced bacterial load. Finally, 0.5% Fl-PRPRPL-5 significantly reduced the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) in wounds induced by MRSA skin infection. In conclusion, the results of this study suggest the potential application of Fl-PRPRPL-5 in the treatment of staphylococcal skin infections.
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    Exploring simvastatin, an antihyperlipidemic drug, as a potential topical antibacterial agent
    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa FN N.; Hamed, Maha I.; Sobreira, Tiago JP P.; Hedrick, Victoria E.; Paul, Lake N.; Seleem, Mohamed N. (Nature Publishing Group, 2015-11-10)
    The rapid rise of bacterial resistance to traditional antibiotics combined with the decline in discovery of novel antibacterial agents has created a global public health crisis. Repurposing existing drugs presents an alternative strategy to potentially expedite the discovery of new antimicrobial drugs. The present study demonstrates that simvastatin, an antihyperlipidemic drug exhibited broad-spectrum antibacterial activity against important Gram-positive (including methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative pathogens (once the barrier imposed by the outer membrane was permeabilized). Proteomics and macromolecular synthesis analyses revealed that simvastatin inhibits multiple biosynthetic pathways and cellular processes in bacteria, including selective interference of bacterial protein synthesis. This property appears to assist in simvastatin's ability to suppress production of key MRSA toxins (α-hemolysin and Panton-Valentine leucocidin) that impair healing of infected skin wounds. A murine MRSA skin infection experiment confirmed that simvastatin significantly reduces the bacterial burden and inflammatory cytokines in the infected wounds. Additionally, simvastatin exhibits excellent anti-biofilm activity against established staphylococcal biofilms and demonstrates the ability to be combined with topical antimicrobials currently used to treat MRSA skin infections. Collectively the present study lays the foundation for further investigation of repurposing simvastatin as a topical antibacterial agent to treat skin infections.
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    Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens
    Abushahba, Mostafa FN N.; Mohammad, Haroon; Thangamani, Shankar; Hussein, Asmaa AA A.; Seleem, Mohamed N. (Nature Publishing Group, 2016-02-10)
    There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.
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    Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway
    Thangamani, Shankar; Maland, Matthew; Mohammad, Haroon; Pascuzzi, Pete E.; Avramova, Larisa; Koehler, Carla M.; Hazbun, Tony R.; Seleem, Mohamed N. (Frontiers, 2017-01-18)
    Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofin's potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofin's antifungal activity-mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections.
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    Repurposing celecoxib as a topical antimicrobial agent
    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N. (Frontiers, 2015-07-28)
    There is an urgent need for new antibiotics and alternative strategies to combat multidrug-resistant bacterial pathogens, which are a growing clinical issue. Repurposing existing approved drugs with known pharmacology and toxicology is an alternative strategy to accelerate antimicrobial research and development. In this study, we show that celecoxib, a marketed inhibitor of cyclooxygenase-2, exhibits broad-spectrum antimicrobial activity against Gram-positive pathogens from a variety of genera, including Staphylococcus, Streptococcus, Listeria, Bacillus, and Mycobacterium, but not against Gram-negative pathogens. However, celecoxib is active against all of the Gram-negative bacteria tested, including strains of, Acinetobacter, and Pseudomonas, when their intrinsic resistance is artificially compromised by outer membrane permeabilizing agents such as colistin. The effect of celecoxib on incorporation of radioactive precursors into macromolecules in Staphylococcus aureus was examined. The primary antimicrobial mechanism of action of celecoxib was the dose-dependent inhibition of RNA, DNA, and protein synthesis. Further, we demonstrate the in vivo efficacy of celecoxib in a methicillin-resistant S. aureus (MRSA) infected Caenorhabditis elegans whole animal model. Topical application of celecoxib (1 and 2%) significantly reduced the mean bacterial count in a mouse model of MRSA skin infection. Further, celecoxib decreased the levels of all inflammatory cytokines tested, including tumor necrosis factor-a, interleukin-6, interleukin-1 beta, and monocyte chemo attractant protein-1 in wounds caused by MRSA infection. Celecoxib also exhibited synergy with many conventional antimicrobials when tested against four clinical isolates of S. aureus. Collectively, these results demonstrate that celecoxib alone, or in combination with traditional antimicrobials, has a potential to use as a topical drug for the treatment of bacterial skin infections.
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    Repurposing Clinical Molecule Ebselen to Combat Drug Resistant Pathogens
    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N. (PLoS, 2015-07-29)
    Without a doubt, our current antimicrobials are losing the battle in the fight against newly-emerged multidrug-resistant pathogens. There is a pressing, unmet need for novel antimicrobials and novel approaches to develop them; however, it is becoming increasingly difficult and costly to develop new antimicrobials. One strategy to reduce the time and cost associated with antimicrobial innovation is drug repurposing, which is to find new applications outside the scope of the original medical indication of the drug. Ebselen, an organoselenium clinical molecule, possesses potent antimicrobial activity against clinical multidrug-resistant Gram-positive pathogens, including Staphylococcus, Streptococcus, and Enterococcus, but not against Gram-negative pathogens. Moreover, the activity of ebselen against Gram-positive pathogens exceeded those activities determined for vancomycin and linezolid, drugs of choice for treatment of Enterococcus and Staphylococcus infections. The minimum inhibitory concentrations of ebselen at which 90% of clinical isolates of Enterococcus and Staphylococcus were inhibited (MIC90) were found to be 0.5 and 0.25 mg/L, respectively. Ebselen showed significant clearance of intracellular methicillinresistant S. aureus (MRSA) in comparison to vancomycin and linezolid. We demonstrated that ebselen inhibits the bacterial translation process without affecting mitochondrial biogenesis. Additionally, ebselen was found to exhibit excellent activity in vivo in a Caenorhabditis elegans MRSA-infected whole animal model. Finally, ebselen showed synergistic activities with conventional antimicrobials against MRSA. Taken together, our results demonstrate that ebselen, with its potent antimicrobial activity and safety profiles, can be potentially used to treat multidrug resistant Gram-positive bacterial infections alone or in combination with other antibiotics and should be further clinically evaluated.
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    Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections
    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N. (Nature Publishing Group, 2015-06-26)
    Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections.