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

<div><p>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 <em>Francisella</em>, <em>Brucella</em>, <em>Bacillus</em>, <em>Burkholderia</em>, and <em>Yersinia</em>. The importance of the MEP pathway to <em>Francisella</em> is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from <em>F. tularensis</em> LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from <em>F. tularensis</em> LVS (IC₅₀ = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from <em>Mycobacterium tuberculosis</em> 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 <em>M. tuberculosis</em>. In this study, we have identified the GlpT homologs in <em>F. novicida</em> and tested transposon insertion mutants of <em>glpT</em>. We showed that FR900098 also requires GlpT for full activity against <em>F. novicida</em>. 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 <em>F. novicida</em> intracellular infection of eukaryotic cell lines and the caterpillar <em>Galleria mellonella</em> as an <em>in vivo</em> 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.</p> </div

Treatment of <i>Francisella</i>-infected wax moth caterpillars with selected compounds.

<p><i>G. mellonella</i> were injected with 3×10⁴ CFU of <i>F. novicida</i> and treated with 9 µg of antibiotics (or 6 µg of ciprofloxacin). Surviving larvae were counted daily. <b>A</b>) <b>Treatment of </b><b><i>G. mellonella</i></b><b> with FR900098 and ciprofloxacin.</b> The mean time to death for untreated caterpillars was 59 hours, and for FR900098 (9 µg)- or ciprofloxacin (6 µg)-treated caterpillars it was 84 hours and 103 hours, respectively. <b>B</b>) <b>Treatment of </b><b><i>G. mellonella</i></b><b> with fosmidomycin and compound 1.</b> The mean time to death for caterpillars treated with fosmidomycin or compound 1 (both 9 µg) was 102 hours and 93 hours, respectively.</p

Inhibition of bacterial growth by selected compounds.

<p>MICs and EC₅₀s of selected compounds against wild-type and <i>glpT</i> mutant <i>F. novicida</i> were determined. (nd = not determined).</p

<i>In vitro</i> determination of IC₅₀ of selected compounds against <i>F. tularensis</i> LVS DXR.

<p><i>In vitro</i> determination of IC₅₀ of selected compounds against <i>F. tularensis</i> LVS DXR.</p

Model for screening method to identify lipophilic, fosmidomycin-derived analogs effective against intracellular pathogens.

<p>This is the model for a new screening method for a library of fosmidomycin/FR900098 analogs against host cells infected with intracellular bacteria to identify lipophilic derivatives that can cross both eukaryotic and prokaryotic membranes. In this example, the mammalian cell (orange line) is infected with intracellular bacteria, <i>F. novicida</i> (green GlpT) or <i>F. novicida glpT</i> mutant (red GlpT) separately. If intracellular bacterial growth of <i>F. novicida glpT</i> mutant is inhibited by a compound (inhibition of the DXR enzyme), the fosmidomycin analog is likely able to cross both eukaryotic (orange) and prokaryotic (blue) cell membranes. Such analogs would be good candidates for further testing in other models such as host cells infected with TB. If the analog does not inhibit growth, it may be GlpT-dependent for bacterial cell entry, and thus could not reach the intra-bacterial DXR enzyme in the <i>glpT</i> mutant. This would be verified by further testing against wild-type <i>F. novicida</i> infected host cells. Thus, by screening eukaryotic cells infected with <i>F. novicida</i> glpT mutants, we are able to simultaneously screen for the three critical functional properties of the desired compound (eukaryotic & prokaryotic membrane penetration and GlpT independence).</p

GlpT homologs identified in <i>Francisella spp</i>.

<p>GlpT homologs identified in <i>Francisella spp</i>.</p

Inhibition of intracellular <i>F. novicida</i> replication in two cell lines following treatment with selected compounds.

<p>Cell lines were first infected with <i>F. novicida</i> at an MOI of 500. The cells were treated with the following concentrations of antibiotics. The cells were lysed after 20 hours of treatment and the intracellular bacteria were enumerated. <b>A</b>) <b>Inhibition of intracellular </b><b><i>F. novicida</i></b><b> in A549 cells with fosmidomycin (Fos), FR900098 (FR), or compound 1 (C1) for 20 hours.</b> Each compound was tested at 250 µM and at 2× MIC (2× MIC for fosmidomycin = 250 µM, 2× MIC for FR900098 = 500 µM and 2× MIC for compound 1 = 400 µM). All three compounds significantly inhibited intracellular <i>F. novicida</i> growth. At 250 µM, intracellular growth was inhibited 98.0±0.7% by fosmidomycin, 86±6% by FR900098, and 97.0±0.8% by compound 1. At 2× MIC, FR900098 (500 µM) inhibited 85±10% of intracellular growth, while compound 1 inhibited 99.0±0.7% of intracellular growth. <b>B</b>) <b>Inhibition of </b><b><i>F. novicida</i></b><b> in RAW264.7 cells with fosmidomycin, FR900098, and compound 1 for 20 hours.</b> Similar results were seen for the RAW264.7 cells as were seen for the A549 cells. <b>C</b>) <b>Inhibition of the </b><b><i>F. novicida glpT</i></b><b> mutant intracellular replication in A549 cells.</b> The intracellular-replication inhibition experiment was performed using the <i>glpT</i> mutant as previously described for wild-type <i>F. novicida</i>. Replication of intracellular <i>glpT</i> mutant was not affected by fosmidomycin (250 µM) and FR900098 (500 µM), but was susceptible to compound 1 (400 µM).</p

Susceptibility of <i>F. novicida glpT</i> mutants to antibiotics.

<p>Fosmidomycin, FR900098, compound 1, and compound 2 were tested at concentrations of 200 µg/ml. <i>F. novicida glpT</i> mutants were resistant to fosmidomycin and partially resistant to FR900098, but not at all resistant to compound 1. Compound 2 was less effective against the <i>glpT</i> mutant than wild-type <i>F. novicida</i>. Percent inhibition was calculated by comparing OD₆₀₀ between treated and untreated wells. Fosmidomycin inhibited 99.6±0.2% of wild-type <i>F. novicida</i>, but did not inhibit the growth of the <i>glpT</i> mutant at all. FR900098 inhibited 97.1±0.8% of <i>F. novicida</i>, but only 55±5% of the <i>glpT</i> mutant. Compound 1 inhibited 100% of the growth of both wild-type <i>F. novicida</i> and the <i>glpT</i> mutant. Compound 2 inhibited 27±14% of <i>F. novicida</i>, and 11±7% of the <i>glpT</i> mutant.</p

Kinetic Characterization and Allosteric Inhibition of the <i>Yersinia pestis</i> 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

<div><p>The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from <i>Yersinia pestis</i>. Enzyme assays indicate apparent kinetic constants of K_M^DXP = 252 µM and K_M^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the <i>Y. pestis</i> MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.</p></div

The MVA and MEP biosynthetic pathways.

<p>A) The MVA pathway is utilized by humans and other eukaryotes, archaebacteria, and certain eubacteria to produce IPP and DMAPP, the building blocks of isoprenoids. The pathway is initiated by the enzymatic condensation of 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is then reduced to MVA by HMG-CoA reductase (4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-M1" target="_blank">[54]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Miziorko1" target="_blank">[55]</a> Subsequent phosphorylation and decarboxylation yield IPP (7) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Amdur1" target="_blank">[56]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-BLOCH1" target="_blank">[57]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-DhePaganon1" target="_blank">[58]</a> which is converted to DMAPP (8) by an isomerase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Agranoff1" target="_blank">[59]</a>. B) The MEP pathway is used by higher plants, the plastids of algae, apicomplexan protozoa, and many eubacteria, including numerous human pathogens. Pyruvate (9) is condensed with glyceraldehyde 3-phosphate (10) to yield 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Lange1" target="_blank">[60]</a>, a branch point intermediate with a role in <i>E. coli</i> vitamin B1 and B6 biosynthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Julliard1" target="_blank">[61]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Julliard2" target="_blank">[62]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hill1" target="_blank">[63]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hill2" target="_blank">[64]</a> as well as isoprene biosynthesis. In the first committed step of the <i>E. coli</i> MEP pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase, Dxr or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-AndrewTKoppisch1" target="_blank">[28]</a>. CDP-ME synthase then converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich2" target="_blank">[65]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kuzuyama2" target="_blank">[66]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kuzuyama3" target="_blank">[67]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Lttgen1" target="_blank">[68]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Herz1" target="_blank">[69]</a>. A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek2" target="_blank">[70]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Campos1" target="_blank">[71]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Hecht1" target="_blank">[72]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Kollas1" target="_blank">[73]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich3" target="_blank">[74]</a>, which is then reduced to both IPP and DMAPP in a ∼5:1 ratio <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich1" target="_blank">[8]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek3" target="_blank">[75]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Cunningham1" target="_blank">[76]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Altincicek4" target="_blank">[77]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-McAteer1" target="_blank">[78]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Rohdich4" target="_blank">[79]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Adam1" target="_blank">[80]</a>. C) The reaction catalyzed by MEP synthase. The intermediate 2-C-methyl-D-erythrose 4-phosphate (18), produced by isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Arigoni1" target="_blank">[81]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106243#pone.0106243-Putra1" target="_blank">[82]</a>, is subsequently reduced to yield MEP (12).</p