Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (4)
Over 100 million urinary tract infections (UTIs) occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular more ..
Over 100 million urinary tract infections (UTIs) occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular pathogenesis of infection. New insights into the roles of K capsules in UPEC virulence during UTI make capsules an attractive target. Uropathogenic Escherichia coli (UPEC) produces 80% of community-acquired urinary tract infections (UTI). UPEC is also a leading cause of nosocomial UTI, the most prevalent hospital acquired infection. Dissemination of UPEC from the lower urinary tract is associated with morbidity and mortality through infection of the kidneys, bloodstream, and central nervous system. In recent years, the treatment of outpatient and inpatient UTI has been severely compromised by the rising incidence of antibiotic-resistant UPEC.
Investigators have found that encapsulation is an important UPEC virulence factor. The K1 capsule type is closely associated with pathogenic isolates; not only is it the leading type in UTI, but it also accounts for much of the extra-urinary tract complications. Animals studies of E. coli K1 sepsis demonstrated that injection of K1 capsule degrading enzyme abrogates infection. However, the enzyme treatment is immunogenic; accordingly, chemical inhibition may prove to be a superior approach.
Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K types. Furthermore, the medically important infectious agents Campylobacter jejuni, Hemophilus influenzae, Neisseria meningitides, and Salmonella typhimurium among others, use these homologues in the biogenesis of their capsules. By exploiting the properties of a K-type specific phage, we performed a small scale high-throughput screen of >2,100 molecules from the NCI that uncovered several promising inhibitors of K1 and K5 encapsulation. This assay will identify a larger number of inhibitors with different mechanisms of action from which we may determine the optimal targets for capsule biogenesis inhibition and develop analogues with pharmacologically optimized properties.
Chemical modulators of K1 encapsulation might represent a new avenue to combat the catastrophic effects K1 diseases. To this end, this team has successfully developed a 1536-well high-throughput primary screen suitable for the discovery of novel capsule biogenesis inhibitors. This 96-well format specificity-screen for dose-response determination of target specificity of the primary screen hits is detailed below.
T7 Lysis Inhibition: Compounds were received in powder form and resuspended in DMSO to create 10 mM storage stocks . One hundred microliters of each compound was taken and diluted two-fold sequentially to 0.039 mM in 96-well PCR plates to create working stocks. Compounds were kept frozen in sealed vials or PCR plates at -20C (protected from light). An overnight culture of EV36 (K1:K-12 hybrid strain) was grown in LB from a colony on a plate (from freezer stock). The culture was then diluted 1:100 into a clean and sterile 96-well flat bottom tray (99 ml per well). Compounds were then added to each well (1 ul per well for final concentrations ranging from 100 uM to 0.39 uM (for indicated wells, DMSO only was diluted to a final concentration of 1%). Each compound concentration was tested in quadruplicate. Each plate contained controls for growth of EV36 with and without phage as well as C7 chemical treatment to remove capsule and sensitize the strain to T7 phage (phage lysis control). The plate was then sealed with tape and placed in a shaker at 37C. After incubating the plate at 37C with shaking for 1.5 hrs, an absorbance reading (OD600)of the plate was taken and indicated wells were infected with 5 ul of T7 phage (from freshly cleared lysate) . The plate was returned to the shaker and a second growth reading was taken 3 hrs post infection to determine lysis. Since T7 phage can only attach if no capsule is present, true inhibitors of capsule will yield bacteria that are susceptible to T7 phage and lyse within 2 hr of the addition of phage. However, compounds inhibiting phage replication will not lyse.
Percent Inhibition of Phage Lysis (Cell viability) was calculated relative to the mean of the cell control. Data are graphed as the mean A600 at 3 hrs p.i. +/- SD of four wells normalized to the average of EV36 + DMSO + T7 phage (3 hrs p.i.). Percent Inhibition of Phage Lysis was determined at each of 9 tested concentrations ranging from 100 uM to 0.39 uM. Compounds that were selective and showed phage lysis inhibition > 30% in the confirmatory assay were considered Active. IC50 values were calculated only for active compounds using XLFit.
Score: In this secondary dose response screen using synthesized compounds, active compounds were scored on a scale of 81-100 based on the relative IC50 values. Compounds that were not confirmed as active in the dose response screen were given the score 0. Primary screen data is scored on a scale of 0-40, confirmatory data is ranked from 41-100.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)