Lipophilic Prodrugs of FR900098 Are Antimicrobial against Francisella novicida In Vivo and In Vitro and Show GlpT Independent Efficacy

Abstract

Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC50 = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed “compound 1,” was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad-spectrum antibiotic, and should be effective against most MEP-dependent organisms.

Resistance to the Antimicrobial Agent Fosmidomycin and an FR900098 Prodrug through Mutations in the Deoxyxylulose Phosphate Reductoisomerase Gene ( dxr )

“…These limitations have led to substantial efforts to develop more cell-permeable analogs of FSM or its highly related analog FR900098 (30)(31)(32)(33)(34)(35). Because many FSM derivatives are prodrugs that require intracellular activation, it is often impossible to test the efficacy of these compounds directly on the Dxr target enzyme in vitro.…”

“…Because of this impermeability, FSM is ineffective against many potential pathogens, such as Toxoplasma gondii (29) and M. tuberculosis (10). Thus, substantial efforts have been made to develop more cell-permeable analogs of FSM or its highly related analog, FR900098 (30)(31)(32)(33)(34)(35).…”

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida

“…[23][24][25][26] Using these techniques to develop a phenotypic screen, we identified compounds from the FDAapproved drug library that exerted antibiofilm (but not antimicrobial) activity against F. novicida. More clearly, we defined antibiofilm activity as inhibition of bacterial biofilm formation to a significantly greater amount than inhibition of bacterial growth (cutoff of 50% for growth inhibition).…”

“…24,26,43 To evaluate the ability of selected drugs to prolong survival of infected G. mellonella, larvae were inoculated with 10 mL of PBS containing a lethal dose of F. novicida and treated with selected drugs. We found that the polycyclic compounds maprotiline and chlorpromazine and the SERM, toremifene, all significantly prolonged survival in the waxworm model (Fig.…”

“…The crystal violet assay was performed as previously described, [23][24][25][26] with modifications. First, fold10-dilutions of drug were made in 100 mL of TSBC in a 96-well plate (BD Falcon 353072).…”