Grant: Research Tools and Instruments

Low membrane permeability and extensive efflux are bacterial defence mechanisms which synergistically reduce compound accumulation in Gram-negative bacteria (GNB) resulting in intrinsic resistance that prevent antibacterial agents from achieving their (intended) biological effect(s). Polybasic amphiphiles (PAs) are an emerging class of “potentiator molecules” termed adjuvants that rescue antibacterials from resistance in GNB by enhancing outer membrane permeability and reducing efflux. PAs have been widely neglected by the pharmaceutical industry as a source of therapeutics because of perceived toxicity and selectivity concerns in humans. However, there are multiple examples of PA-based approved drugs for human use including the polymyxin- and aminoglycoside-based classes of antibiotics as well as the macrolide azithromycin. In addition, PA-based antimicrobial peptides (cathelicidins) are released at the site of human infection as a first line of defense indicating that the liabilities of PAs can be minimized/suppressed in humans. ******In contrast to non-basic amphiphiles, PAs are challenging molecules to purify. PAs are highly polar molecules that (depending on pH) coexist in neutral, mixed or polyionized (polycationic) forms. Currently my group purifies PAs by reverse-phase using outdated low-pressure reverse phase flash chromatography. This technique results in (i) low purity levels and (ii) accumulation of small quantities (typically 10 active research collaborations) to spend up to 30 % of their research time on purification. To resolve these challenges, to help students to become more competitive and to train students in modern purification techniques using various solid phase reverse phase extraction techniques, an automated high-pressure purification system for PAs is required to enable rapid access to PAs with high purity ( 95%) on semi-preparative scale (5-50 mg). ******The proposed purification system for polybasic amphiphiles is expected to make significant contributions, including potential breakthroughs in a wide variety of areas associated with biological chemistry of bacterial systems, bioorganic chemistry and microbiology. The information gathered by these studies will have immediate, direct strong impacts on the development of chemical strategies to disarm bacterial defence mechanisms and counter antimicrobial resistance in GNB with multiple applications including food and water safety, veterinary health, agriculture and security. *****