The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family. Specifically, these probes would alter DnaK through interactions with its substrate-binding domain (SBD). DnaK serves as a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression. The identified probe ML076 (CID-25105719) binds to DnaK at a novel allosteric site of the substrate binding domain, in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. ML076 and its closely related analogs described in this probe report might be useful as tools for dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. These molecular probes also might serve to validate DnaK as a possible target for novel antibacterial agents.