High-throughput screening of a diversity collection using biodefense category A and B priority pathogens

Abstract

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute-based high-throughput screening (HTS) 96-well-based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.

Figure 1

Representative real-time PCR genotyping assay melting curves from a fresh culture (A) used to replace a retired culture (B). DNA was extracted from the bacterial pathogen, in this case Bacillus anthracis Ames, and genetically profiled using real-time PCR by examining the following targets for amplification, capB2P (capsule B), anh^C (unique identifying sequence designed in house; putative anhydrolase), LEF^P (lethal factor), PA^P (protective antigen). “C” and “P” indicate chromosomal or plasmid-borne genes. Amplified products were screened for their melting temperature to discern specificity of the amplification reaction for the desired sequence.

Figure 2

(A) Plate format for initial screening at 16 μg/mL. (B) Plate format for minimum inhibitory concentration determination that will accommodate four test compounds. Dilutions are twofold, ranging in concentration from 0.0625 to 16 μg/mL. Appropriate control wells are shown, including ones for color control.