Researchers at Microbiotix are using a three-year Phase I Small Business Innovation Research grant from the National Institutes of Health to develop cell-based screens for compounds that can be used against the bacterium Burkholderia pseudomallei, a company official told CBA News this week.
The company plans to develop reporter screens for four targets from the potentially fatal bacterium, characterize the resulting hits, and develop them into lead compounds, said Donald Moir, CSO of Microbiotix.
A paper describing initial results of the project appears online in the July issue of the Journal of Biomolecular Screening. The project continues to move forward, and the company is currently identifying and optimizing lead compounds.
Moir, who is the lead author on the paper, said that the four targets are gyrA,which encodes DNA gyrase, a topoisomerase that maintains the negative supercoiling of DNA during replication; secA, which encodes a product that is important for the cell secretion pathway; glmU, which encodes a cell wall product; and coaD, which encodes a product used in making coenzyme A.
The goal of the grant is to get optimized leads against at least some of these four targets that work in animal models of infection, said Moir. He declined to discuss specific timelines.
Microbiotix works with Donald Woods, a professor of microbiology and infectious diseases at the University of Calgary, Canada, because he has a biosafety level-3 facility.
An Antimicrobial Surrogate
B. pseudomallei causes melioidosis, an infectious disease mainly found in Southeast Asia and northern Australia that results in abcesses, pneumonia, and at worst, a fatal septicemia in susceptible hosts, according to the Centers for Disease Control and Prevention
Moir said that B. pseudomallei is a bioterrorist threat and as such, the goal of the SBIR grant is aimed at biodefense applications. He said that the authors used gene targets found in Pseudomonas aeruginosa, a fairly common clinical pathogen, as surrogates for the same gene targets found in B. pseudomallei and then developed reporter screens for these targets.
As proof of concept, the authors focused on DNA gyrase, a well-validated fluoroquinolone target. Moir said that the investigators identified transcriptional units in P. aeruginosa that respond by upregulating to the reduced expression of gyrA and the inhibition of gyrase by the antibiotic ciprofloxacin.
“The investigators identified transcriptional units in P. aeruginosa that respond by upregulation to the reduced expression of gyrA and the inhibition of gyrase by ciprofloxacin.”
They then fused these transcriptional units — 451 nt, 353 nt, and 150 nt of predicted non-coding sequence upstream from genes PA0612, PA0614, and PA0617, respectively — to a Photorhabdus luminescens luciferase reporter and integrated them into the P. aeruginosa chromosome.
The resulting strains were grown without ciprofloxacin and with a range of three different concentrations of ciprofloxacin, and the ratio of luminescence to ciprofloxacin concentration was measured.
The investigators found that the response of the PA0614 promoter region was at least ten-fold stronger than those of the PA0612 and PA 0617 regions. For example, at a concentration of 0.03 µg/mL of ciprofloxacin, the increase in bioluminescence compared to untreated cells was 1.9-fold, 11.4-fold, and 0.8- fold for PA0612, PA0614, and PA0617, respectively.
The authors concluded that the PA00614 upstream region behaved as a promoter that is upregulated by ciprofloxacin and decreased GyrA levels, so they used the PA0614 construct as the basis for a reporter screen.
This gain-of-signal assay gives fewer false positives than traditional loss-of-signal assays, which result in a loss of signal upon target inhibition, Moir said.
The screens have been developed and shown to be effective, said Woods. The company has also completed some preliminary work that has proven the compounds are effective in mice. Woods said he hopes that the compounds could proceed to clinical trials within the next year or so.
Both Moir and Woods said that they have no plans at this time to commercialize these screens or offer them as a service to biotech or pharmaceutical companies.