Project Name

Development of a HTS assay for Inhibitors of bacterial DnaK

Principal Investigator/Assay Provider:

Michael A. Sturgess, Ph.D, Chaperone Technologies, Inc.

 

Project Goal

Advance the knowledge and utilization of DnaK inhibitors as the next generation of antibacterial agents.

Assay Type:

Luminescence

 

Project Description

Antibiotic resistance is a major concern that has influenced both the clinical uses of established antimicrobials and the development of new agents. L. Escherichia coli DnaK, a homolog of Heat shock protein 70, has been shown to protect denatured proteins from aggregation and promote their refolding by ATP hydrolysis. DnaK, along with its two cohort proteins DnaJ and GrpE, forms a microbial chaperone system that shelters microorganisms from environmental stresses such as temperature, osmotic, and pH changes, carbon and/or nitrogen starvation. Seeking inhibitors against bacterial DnaK chaperone systems will lead to a new direction for the development of antimicrobial agents, as current antimicrobial compounds have shown fewer efficacies due to the rapid emerg

Assay Description:

An in vitro refolding assay was developed by Chaperone Technologies to detect and quantify the activity of DnaK. Briefly, firefly luciferase is chemically denatured and used as a substrate for the chaperone system. Promega’s Stady-Glo Luciferase Assay System is then used to detect luminescence as a measure of the refolding of luciferase. In order to rule out compound-induced refolding, a separate set of plates with only denatured luciferase and compounds were read in a parallel assay. The EMLSC has optimized and adapted the assay to a 384-well format for HTS and has been able to consistently achieve a signal to noise ratio of above 8 and a Z’ factor of above 0.5. HTS results are analyzed using CambridgeSoft. Compounds yielding a luminescence signal more than 3SD below the mean luminescence are identified are positives.