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.

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity

“…For example, radiometric assays utilize 3 H- and 13 C-radiolabeled SAM to capture and directly quantify the transfer of the radiolabeled methyl group to the substrates, , and fluorometric assays indirectly measure fluorescent signals generated from coupled chemical reactions. , As reported in more recent studies, NNMT activity is monitored via measurement of absorbance of HPLC-separated reaction species, independently or coupled to mass spectrometry . Similar direct product detection methods (radiometric, radioisotope filter binding, scintillation proximity assay, antimethylation antibody binding fluoroimmuno assays, and aptamer/riboswitch) and series of enzyme-coupled SAH detection colorimetric assays are used to probe the activity of many disease-linked MTs where the catalytic product readouts are single-point, indirect, slow with multiple steps, labor intensive, insensitive, and/or incorporate radioactive materials. − These assays can be challenging and expensive to adapt for high-throughput screening of large chemical libraries to identify MT inhibitors …”

Antibiotic Therapy of Plague: A Review

“…In vitro, measurement of the minimum inhibitory concentration (MIC) in a microdilution assay that complied with the Clinical and Laboratory Standards Institute’s guidelines for the Enterobacteriaceae revealed that tetracyclines, fluoroquinolones, aminoglycosides, sulfonamides, and most of β-lactams are active against Y. pestis ; the MIC 90 ranged from below 0.125 mg/L to 4 mg/L ( Table 1 ) [ 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ]. However, few studies have determined the MICs in more than one strain, and most of the MIC assays were performed at Yersinia optimal growth temperature (28 °C)—a temperature at which the lipooligosaccharide’s structure is not the same as at 37 °C [ 72 ].…”

Profiling susceptibility of NIAID Category A and B priority and emerging pathogens to define strain panels for drug discovery and active drug classes

“…Evaluation of drug candidates begins with testing for potency, which is often performed against a reference laboratory-adapted bacterial strain or a closely related model organism (3)(4)(5). While laboratory or model strains provide a single reference for comparison of drug candidate performance, they do not adequately represent the spectrum of drug susceptibility of clinical strains (6).…”