Megan weitner's 2016 thesis

Thesis here: https://jscholarship.library.jhu.edu/handle/1774.2/39473

The thesis studied various drugs that are orally available and low toxicity*. (*Ciprofloxacin can have devastating side effects. There are patient support groups for those injured by 'flox' antibiotics.)

Various experiments were performed on Borrelia Burgdorferi bacteria to measure the synergistic effects of different drug combinations.

Mechanisms of action

Cell wall disruptor:

• Cefuroxime: This antibiotic is a second generation cephalosporin that functions through the inhibition of cell wall biosynthesis. Cefuroxime specifically disrupts cell wall repair mechanisms by inhibiting the pathways needed for the transportation and insertion of peptidoglycan into the cell wall.
• Daptomycin: It inserts into the cell membrane in a phosphatidylglycerol-dependent fashion, where it then aggregates. The aggregation of daptomycin alters the curvature of the membrane, which creates holes that leak ions. This causes rapid depolarization, resulting in a loss of membrane potential leading to inhibition of protein, DNA, and RNA synthesis, which results in bacterial cell death.

Membrane permeability or homeostasis mechanisms:

• Hydroxychloroquine: As a base, it alters the intracellular pH by attracting acids. This may disurpt the ion transport gradient, preventing cells from acquiring the necessary nutrients in their dormant state. When combined with drugs that act on DNA transcription, activity from transmembrane proteins may also be affected, further preventing the cells from maintaining their necessary nutrient and ion balance.
• Fluconazole: Fluconazole is an antifungal drug that inhibits cytochrome p-450-dependent 14α-sterol demethylase, resulting in an inhibition of ergosterol. While normally exclusive to fungal membranes, Borrelia utilize ergosterol in their membranes to maintain fluidity and membrane integrity. The membrane dysfunction caused by the addition of fluconazole could allow for increased cellular penetration of the other drugs, but it could also function through disruption of nutrition uptake.

Free radical producing:

• Methylene blue: This photosensitive dye produces free radicals and hydroxides when exposed to light. These free radicals cause lipid peroxidation, resulting in the loss of membrane integrity, and possibly damage to the peptides in the bacterial cell wall. If methylene blue is able to penetrate the cell wall, the drug can bind to and modify guanine residues, though the effect of this on inhibition of DNA replication has not been determined.

• Artemisinin: Once activated, artemisinin’s endoperoxide bridge causes the creation of reactive oxygen species which can be rearranged into carbon-centered radicals. Both species of free radicals accumulate in neutrally charged lipids and thiols resulting in lipid peroxidation. The free radicals can also cause damage to DNA and metabolic enzymes resulting in disruption of DNA transcription and cellular metabolism. Artemisinin has also been suggested to be involved in protein alkylation, resulting in inhibition of cysteine proteases.

Protein synthesis inhibitor:

• Doxycycline: Doxycycline is a tetracycline derivative protein synthesis inhibitor. Doxycycline binds to the bacterial ribosome and inhibits the ribosomal binding of aminoacyl t-RNA, a necessary step in the initiation of protein synthesis.
• Azithromycin: This macrolide antibiotic binds to the large bacterial ribosomal subunit, inhibiting the synthesis of fully formed proteins.
• Nitrofurantoin: Functions through the creation of free radicals and protein dysfunction. Nitrofurantoin must be activated through intracellular reduction, which results in the creation of electrophiles. These reactive species result in the inhibition of components of the citric acid cycle, along with causing damage to DNA, RNA and protein synthesis mechanisms.55 Nitrofurantoin also functions as a diamide and causes the creation of nonnative disulfide bonds in bacterial proteins, resulting in protein dysfunction.

DNA transcription:

• Rifabutin: Rifabutin is a spiro-piperidyl-rifampin derivative that inhibits DNA transcription by binding to the beta-subunit of the DNA-RNA polymerase. The prevention of RNA synthesis can result in an inhibition of protein synthesis as well as prevent the cell from regulating gene expression.
• Rifaximin: Another rifampin derivative but with lower intracellular penetration than Rifabutin.
• Ciprofloxacin (not a safe drug): Inhibits RNA synthesis. Ciprofloxacin binds directly to DNA, preventing binding of DNA gyrase and not allowing the DNA to form a negative superhelix.
• Pyrimethamine : This folic acid antagonist interferes with the regeneration of tetrahydrofolic acid from dihydrofolate by competitively inhibiting the enzyme dihydrofolate reductase. Tetrahydrofolic acid is essential for DNA and RNA synthesis in many species, including protozoa.
• Clofazimine : Clofazimine works by binding to the guanine bases of bacterial DNA, thereby blocking the template function of the DNA and inhibiting bacterial proliferation.