Currently, Tularemia can be treated with antibiotics such as streptomycin and gentamicin. However, the identification of new therapeutics is very significant, since Francisella can easily be genetically modified and therefore its sensitivity to known antibiotics could be compromised. The manipulation of protein-protein interactions, as targets for therapeutics, is a new and expanding field of research. In this regard, the Francisella MglA/SspA complex is a very attractive system that offers at least three usable interactions: i) with each other, ii) with the RNAP, and iii) with the DNA binding transcription factors FevR and PmrA. Each of these Mepiroxol interactions could potentially be modulated by the action of small molecules. In fact, it was reported that the levels of ppGpp Lomitapide Mesylate modulate the activity of PigR and its interactions with MglA/SspA/RNAP complex in vivo. However, no evidence is available to confirm that the effect is due to a direct interaction with the FevR regulator, the MglA/SspA heterodimer, the RNAP, or due to altered levels of an unknown intracellular metabolite. Here we report the identification of a small molecule that specifically modified MglA and SspA interactions in vitro and in vivo. Using structure guided site directed mutagenesis, we were able to determine that quinacrine hydrochloride binds in the “cleft region” formed by the MglA/SspA heterodimer. The biochemical evidence provided herein suggests that a biologically relevant molecule may act to modulate the expression of pathogenicity determinants in F. novicida, and provide the putative binding residues for such interactions. The identification of small molecules that modulate proteinprotein interactions is an expanding field with important therapeutic applications. We used a fluorescence-based thermal screening assay to uncover chemicals that bind the MglA/SspA heterodimer interface. Though small by today’s standards, the Prestwick chemical library was used to identify several compounds that bind Ft-MglA and Ec-SspA. Of the 1152 compounds present in the Prestwick library, 13 small molecules stabilized Ft-MglA in vitro. The number of positive hits obtained is similar to other transcription factors previously screened in our laboratory. A secondary confirmation assay was designed to link the thermal screening results to the biological function of the target protein. Since MglA and SspA form a heterodimer to bind the RNA polymerase, we used a bacterial two hybrid system as a secondary assay. The rationale was to determine which of the 13 small molecules modulate MglA and SspA interactions. Interestingly, we found that only one compound, quinacrine, decreased the 
interaction of Ft-MglA and Ft-SspA by more than 50%. The other 12 compounds examined in the two hybrid system were determined to be biologically insignificant, as no major reduction in MglA/SspA interaction was observed. The thermal stabilization effect observed with these compounds was most likely due to unspecific binding at locations other than the heterodimer interface. Another possibility, however, is a deficiency in the uptake of those chemicals by the E. coli reporter strain. The decrease in MglA/SspA interactions observed in the two hybrid system, in the presence of quinacrine, was correlated with impaired intramacrophage survival of F. novicida and decreased expression of the FPI genes. An interesting observation was that F. novicida could tolerate rather high concentrations of quinacrine when cultured in liquid media, while 25 uM was sufficient to decrease F. novicida macrophage infection and intramacrophage survival.