Primary and Confirmatory Screening for Inhibitors of Bacterial Capsule Biogenesis
Primary Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million 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. more ..
BioActive Compounds: 56
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Patrick Seed, Duke University, Durham, NC
Award: 1 R03 MH090791-01
Primary Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million 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.
During UTI, UPEC lives in intracellular and extracellular locales. UPEC adheres to the apical bladder epithelium and then invades this layer of cells. Within the bladder epithelium, UPEC typically replicates in a biofilm-like state called intracellular bacterial communities (IBC). After maturation of IBCs, UPEC disperses away from the IBC and exits the infected cells. Extracellular UPEC must then re-adhere, initiating the invasion and intracellular replication phases again. Past studies have revealed bacteria encased in the IBC within a complex matrix of fibrous protein assemblies and polysaccharides. Prior studies have also shown that disruption of the IBC pathway aborts experimental UTI, highlighting the importance of this intracellular lifecycle. A detailed study of urine samples from women with acute UTI demonstrated IBC in shed bladder epithelial cell, showing that the pathway is conserved in humans. Investigations have shown that K capsule contributes to multiple aspects of pathogenesis, including IBC formation.
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.
Primary Assay Protocol: In the 1536 well microplate plate format, 1 ml of phage was required per plate. The assay provider sent 300 ml of phage material to complete the entire screen of 338,000 compounds (10% excess). The phage material is stable for over 2 weeks at 4 degrees C and is tolerant to agitation, making it amenable to transportation from Duke University to the testing center. Bacterial cultures of E. coli K1 strain UTI89(cystitis isolate) and isogenic capsule mutant strains were sent to Southern Research (SR) and grown and prepared at the screening center immediately prior to use.
For the assay, preparation of the bacterial cultures for the screen was performed at SR. Overnight starting cultures were grown at 37 degrees C, shaking at 250 RPM in LB. 65 ml of overnight UTI89 culture was inoculated into 6.45 liters of LB (starting OD600 ~0.03) and was sufficient for screening 300 plates at one concentration.
Compounds from the libraries were added to the plates before the addition of bacteria or phage. Each compound was tested as a single point, and ~1200 compounds were tested per plate. Control wells contained UTI89 (wt K1) with 1% DMSO as a simulated positive control; UTI89 with 1% DMSO and tetracycline for a negative control; and media plus vehicle control. The positive control drug C7 in 1% DMSO (2-(4-phenylphenyl)benzo[g]quinoline-4-carboxylic acid) that we previously identified (ID50 12.5 muM),was provided by Dr. Seed to SR for secondary screening. However, it was not used in the primary screen due to aqueous insolubility. The plates were prepared, incubated for 2 hrs, and an initial OD600 reading was measured to identify compounds that were bacteriostatic or bactericidal in the absence of phage. Next, K1F phage was added and plates were incubated at 37 degrees C for 2 hrs. After that time, alamar blue reagent was added to all the plate wells and the plates were incubated for an additional 30 min at 37 degrees C. Following this, measurements of fluorescence at 560EX nm/590EM nm for phage-mediated lysis were taken. A positive hit was defined by the compound producing greater than a 15% reduction in the 590nm fluorescence following the addition of phage.
Dose Response Assay Protocol: This assay was performed using the primary assay methodology with a dose-response, stacked-plate format. The dose response testing confirmed the primary screen hits and provided the IC50 data necessary to determine the lead agents to be advanced in secondary screens and target localization studies. The assay was performed as described for the primary screen except that each compound was tested at 10 concentration points starting from 300 uM and continuing to lower concentrations by serial 2-fold dilutions. The strain UTI89 delta-kpsM, a K1 capsule export mutant, was evaluated with the wt strain as a phage insensitive control (mimicking 100% capsule inhibition). After the addition of the K1F phage, addition of alamar blue reagent and measurements of fluorescence at 560EX nm/590EM nm for phage-mediated lysis were taken at 2 hr. As with the primary screen, inhibitors of capsule biogenesis will inhibit K1F phage lysis, and will generate a higher signal.
Outcome: Inhibition of phage lysis was calculated relative to the mean of the bacterial (positive) control. Inhibition at each of 10 tested concentrations ranging from 300 uM to 0.58 uM. Compounds that showed >30% inhibition of phage lysis were considered Active. IC50 values were calculated only for active compounds using a 4 parameter Levenburg-Marquardt algorithm, with the maximum and minimum locked at 0 and 100 respectively.
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In this confirmatory (dose response) screen, active compounds were scored on a scale of 41-80 based on the
IC50 result compounds that did not show activity were given the score 0.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)