11 research outputs found
Synthesis and Evaluation of Fluoroalkyl Phosphonyl Analogues of 2‑<i>C</i>‑Methylerythritol Phosphate as Substrates and Inhibitors of IspD from Human Pathogens
Targeting essential bacterial processes
beyond cell wall, protein,
nucleotide, and folate syntheses holds promise to reveal new antimicrobial
agents and expand the potential drugs available for combination therapies.
The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP)
and dimethylallyl diphosphate (DMADP), is vital for all organisms;
however, humans use the mevalonate pathway for production of IDP/DMADP
while many pathogens, including <i>Plasmodium falciparum</i> and <i>Mycobacterium tuberculosis</i>, use the orthogonal
methylerythritol phosphate (MEP) pathway. Toward developing novel
antimicrobial agents, we have designed and synthesized a series of
phosphonyl analogues of MEP and evaluated their abilities to interact
with IspD, both as inhibitors of the natural reaction and as antimetabolite
alternative substrates that could be processed enzymatically to form
stable phosphonyl analogues as potential inhibitors of downstream
MEP pathway intermediates. In this compound series, the <i>S</i>-monofluoro MEP analogue displays the most potent inhibitory activity
against <i>Escherichia coli</i> IspD and is the best substrate
for both the <i>E. coli</i> and <i>P. falciparum</i> IspD orthologues with a <i>K</i><sub>m</sub> approaching
that of the natural substrate for the <i>E. coli</i> enzyme.
This work represents a first step toward the development of phosphonyl
MEP antimetabolites to modulate early isoprenoid biosynthesis in human
pathogens
Growth medium-dependent antimicrobial activity of early stage MEP pathway inhibitors
<div><p>The <i>in vivo</i> microenvironment of bacterial pathogens is often characterized by nutrient limitation. Consequently, conventional rich <i>in vitro</i> culture conditions used widely to evaluate antibacterial agents are often poorly predictive of <i>in vivo</i> activity, especially for agents targeting metabolic pathways. In one such pathway, the methylerythritol phosphate (MEP) pathway, which is essential for production of isoprenoids in bacterial pathogens, relatively little is known about the influence of growth environment on antibacterial properties of inhibitors targeting enzymes in this pathway. The early steps of the MEP pathway are catalyzed by 1-deoxy-d-xylulose 5-phosphate (DXP) synthase and reductoisomerase (IspC). The in vitro antibacterial efficacy of the DXP synthase inhibitor butylacetylphosphonate (BAP) was recently reported to be strongly dependent upon growth medium, with high potency observed under nutrient limitation and exceedingly weak activity in nutrient-rich conditions. In contrast, the well-known IspC inhibitor fosmidomycin has potent antibacterial activity in nutrient-rich conditions, but to date, its efficacy had not been explored under more relevant nutrient-limited conditions. The goal of this work was to thoroughly characterize the effects of BAP and fosmidomycin on bacterial cells under varied growth conditions. In this work, we show that activities of both inhibitors, alone and in combination, are strongly dependent upon growth medium, with differences in cellular uptake contributing to variance in potency of both agents. Fosmidomycin is dissimilar to BAP in that it displays relatively weaker activity in nutrient-limited compared to nutrient-rich conditions. Interestingly, while it has been generally accepted that fosmidomycin activity depends upon expression of the GlpT transporter, our results indicate for the first time that fosmidomycin can enter cells by an alternative mechanism under nutrient limitation. Finally, we show that the potency and relationship of the BAP-fosmidomycin combination also depends upon the growth medium, revealing a striking loss of BAP-fosmidomycin synergy under nutrient limitation. This change in BAP-fosmidomycin relationship suggests a shift in the metabolic and/or regulatory networks surrounding DXP accompanying the change in growth medium, the understanding of which could significantly impact targeting strategies against this pathway. More generally, our findings emphasize the importance of considering physiologically relevant growth conditions for predicting the antibacterial potential MEP pathway inhibitors and for studies of their intracellular targets.</p></div
Checkerboard analysis to assess drug interaction between BAP and fosmidomycin.
<p>a) Representative heat plot showing a synergistic relationship between BAP (5400 μM, 1000 μg/mL) and fosmidomycin (64 μM, 12 μg/mL) in CAMHB growth medium (figure reproduced with permission, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197638#pone.0197638.ref023" target="_blank">23</a>]). b) Representative heat plot showing an indifferent relationship between BAP (5 μM, 1 μg/mL) and fosmidomycin (340 μM, 62 μg/mL) in M9-glucose minimal medium with an FIC index range of 1–1.25. The most extreme FIC index value is reported above each heat plot.</p
Fosmidomycin antibacterial activity in GlpT deficient <i>E</i>. <i>coli</i>.
<p>GlpT transporter-containing (■BW25113) and deficient ( Δ<i>glpT</i> BW25113) <i>E</i>. <i>coli</i> strains treated with 2800 μM (512 μg/mL) fosmidomycin in CAMHB (a), M9-glucose (b), and M9-glycerol (c) growth medium. Cell growth was assessed at 16 h (CAMHB, M9-glucose) or 40 h (M9-glycerol) to ensure culture saturation. The large MIC shift between the parent and GlpT deficient strain in CAMHB and M9-glycerol media indicate that GlpT is a fosmidomycin transporter in these growth conditions. (n = 3, error bars represent standard error).</p
BAP is bacteriostatic.
<p><i>E</i>. <i>coli</i> cultures at an initial inoculum density of 10<sup>6</sup> CFU/mL in M9-glucose were incubated in the presence of BAP () at 4 × MIC (80 μM, or 15 μg/mL), compared to control in the absence of BAP (■) in biological triplicate. Enumeration of bacteria on agar plates over time (0, 2, 4, 6, and 20 h) indicates that BAP is bacteriostatic. (n = 3, error bars represent standard error).</p
Two early stage MEP pathway inhibitors and their targets are shown in the context of the <i>E</i>. <i>coli</i> branchpoint metabolite, DXP.
<p>Butylacetylphosphonate (BAP) is an inhibitor of DXP synthase, and fosmidomycin is an inhibitor of IspC, the first committed step in isoprenoid biosynthesis. (Pi = PO<sub>4</sub><sup>2−</sup>).</p
Fosmidomycin accumulation in <i>E</i>. <i>coli</i>.
<p><i>E</i>. <i>coli</i> was treated with 110 μM (20 μg/mL) or 550 μM (100 μg/mL) fosmidomycin in CAMHB (■) or M9-glucose () medium. Intracellular fosmidomycin accumulation was monitored by LC-MS (SRM method). Fosmidomycin uptake is robust and dose-dependent in CAMHB medium, and poor in M9-glucose medium. (n = 3, error bars are standard error, <i>p</i>-values above charts were calculated using an unpaired, 2-sample t-test).</p
BAP accumulation in <i>E</i>. <i>coli</i>.
<p><i>E</i>. <i>coli</i> treated with 250 μM (47 μg/mL) or 1250 μM (233 μg/mL) BAP in CAMHB (■) or M9-glucose () medium. Intracellular BAP accumulation was monitored by LC-MS (SRM method). BAP uptake is robust and dose-dependent in M9-glucose medium, and poor in CAMHB medium. (n = 3, error bars are standard error, <i>p</i>-values were calculated using an unpaired, 2-sample t-test).</p
Minimum inhibitory concentration (MIC) of fosmidomycin against pathogenic bacteria grown in CAMHB or M9-glucose minimal medium.
<p>Minimum inhibitory concentration (MIC) of fosmidomycin against pathogenic bacteria grown in CAMHB or M9-glucose minimal medium.</p
Identification of an Atg8-Atg3 Protein–Protein Interaction Inhibitor from the Medicines for Malaria Venture Malaria Box Active in Blood and Liver Stage <i>Plasmodium falciparum</i> Parasites
Atg8 is a ubiquitin-like
autophagy protein in eukaryotes that is
covalently attached (lipidated) to the elongating autophagosomal membrane.
Autophagy is increasingly appreciated as a target in diverse diseases
from cancer to eukaryotic parasitic infections. Some of the autophagy
machinery is conserved in the malaria parasite, <i>Plasmodium</i>. Although Atg8’s function in the parasite is not well understood,
it is essential for <i>Plasmodium</i> growth and survival
and partially localizes to the apicoplast, an indispensable organelle
in apicomplexans. Here, we describe the identification of inhibitors
from the Malaria Medicine Venture Malaria Box against the interaction
of <i>Pf</i>Atg8 with its E2-conjugating enzyme, <i>Pf</i>Atg3, by surface plasmon resonance. Inhibition of this
protein–protein interaction prevents <i>Pf</i>Atg8
lipidation with phosphatidylethanolamine. These small molecule inhibitors
share a common scaffold and have activity against both blood and liver
stages of infection by <i>Plasmodium falciparum</i>. We
have derivatized this scaffold into a functional platform for further
optimization