DataSheet_2_Targeting malaria parasites with novel derivatives of azithromycin.pdf

IntroductionThe spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development.MethodsHere, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans).ResultsSeventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.DiscussionThe azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.</p

DataSheet_1_Targeting malaria parasites with novel derivatives of azithromycin.pdf

IntroductionThe spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development.MethodsHere, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans).ResultsSeventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.DiscussionThe azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.</p

Biophotonic imaging of burn wounds.

<p>(<b>A</b>) Timeline of scald injury experiments. (<b>B</b>) Dorsal images of representative BALB/c mice challenged with approx. 1 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29). Mice were subjected to bioluminescent imaging on IVIS Lumina XRMS Series III system at the indicated times. (<b>C</b>), Quantification of photon intensities of bacterial burden showing significant reduction in photon intensities in mupirocin-treated mice. Total flux (means±SEM photons/s; n = 6 mice). *<i>p</i><0.05; **<i>p</i><0.01; multiple <i>t</i>-tests). (<b>D</b>), Total bacterial counts from tissues of control, infected but not treated, and infected + mupirocin-treated mice at the conclusion of the experiment, showing strong correlation with total photon intensities obtained from each treatment group.----denotes limit of detection; ^_____ denotes geometric mean counts.</p

Luminescence signal comparison between groups of CD1 mice challenged IP with <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29).

<p>(<b>A</b>), Quantification of photon intensities on a Xenogen IVIS Lumina XRMS Series III live animal biophotonic imaging system, showing significant reduction in photon intensities in daptomycin-treated mice. Total flux (means±SEM photons/s; n = 6 mice). **<i>p</i><0.01; multiple <i>t</i>-tests). (<b>B</b>), Survival times for CD1 male mice (n = 6) challenged IP with approx. 2.5 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> Xen29 and administered the drug vehicle only or daptomycin (6 mg/kg) IP at 2 and 6 h post-infection. Differences in median survival times (time to moribund) for mice between groups were analyzed by the log-rank (Mantel-Cox) tests. ***, <i>P</i><0.001. (<b>C</b>), Total bacterial counts from blood of control and infected + daptomycin-treated mice at 2, 4 and 6 h post-infection.----denotes limit of detection; ^_____ denotes geometric mean counts.</p

Biophotonic ventral and dorsal images of 2 representative CD1 male mice challenged IP with approx.

<p><b>2.5 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29) and then administered the drug vehicle only or daptomycin IP at 2 and 6 h post-infection</b>. Mice were subjected to bioluminescent imaging on IVIS Lumina XRMS Series III system at the indicated times.</p

Analysis of wound healing in non-infected, infected but not treated, and infected + mupirocin-treated scald burn wounds.

<p>(<b>A</b>), Representative images and graphical analysis of scald wounds over a time-course of 10 days illustrating macroscopic differences in rate of healing between non-infected, infected but not treated, and infected + mupirocin-treated mouse scald wounds. (<b>B</b>), Representative haematoxylin and eosin-stained sections of partial-thickness scald wounds in mice with non-infected, infected but not treated, and infected + mupirocin-treated wounds. Microscopic analysis of scald wounds at day 10 post-wounding suggests that mupirocin treatment of scald wounds is effective in treating <i>S</i>. <i>aureus</i> infected wounds leading to significantly decreased wound length, dermal gape and significantly increased wound re-epithelisation compared to infected wounds. Arrows indicate dermal wound gape distance. Magnification ×4 stitched image. Scale bar = 100 μm. Results represent means and SEM, n = 6 wounds per mice group with a single time-point.</p

Ion-coupled transport in proteoliposomes.

<p>A, B, ³⁶Cl⁻ uptake (100 µM) by LmrA-MD (•), E314A LmrA-MD (⋄), EE LmrA-MD (▪) or empty liposomes (▵) in the presence of a Δψ (interior negative) of −120 mV (A) or -ZΔpH (interior alkaline) of −49 mV (B). In the duplicate experiment for LmrA-MD (□) in (B), the addition of uncoupler (valinomycin plus nigericin, 1 µM each) at the arrow resulted in efflux of accumulated ³⁶Cl⁻, indicating concentrative uptake of the ion. C, ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA (•), EE LmrA (▪) or empty liposomes (▵). D, ΔpH (interior alkaline)-dependent uptake of non-radioactive Cl⁻ (1 mM) by LmrA is observed as a quench in the fluorescence of the SPQ fluorophore trapped in the lumen of the proteoliposomes. Quenching was also observed in empty liposomes (control) in the presence of the Cl⁻/OH⁻ antiporter TBT-Cl (1 µM). E, Kinetic analysis of ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA. F, ΔpH (interior alkaline)-dependent uptake of ²²Na (25 µM) by LmrA-MD. G, Uptake of unlabelled Na⁺ (10 mM) by LmrA was detected as an increase in the fluorescence of the membrane-impermeable sodium green probe trapped in the lumen. H, Na⁺ (100 µM) stimulates the ΔpH-dependent uptake of ³⁶Cl⁻ (100 µM) by LmrA compared to control containing 99 µM NMG⁺ plus 1 µM Na⁺. I, H⁺ efflux in proteoliposomes loaded with pH probe BCECF in the presence of an outwardly directed NaCl gradient. Control, empty liposomes. (<i>n</i> = 5)</p

LmrA activity enhances cell survival.

<p>A, Viability of energized cells (solid bar, LmrA; grey bar, non-expressing control) adapted for 30 min in buffer containing 0.5 M sucrose without or with 100 mM NaCl, 100 mM KCl or 50 mM Na₂SO₄, followed by 100-fold dilution into ultrapure water, or in buffer containing 0.125 M sucrose and 25 mM NaCl (control referred to as 4-fold dilution). B, Effect of mutations in LmrA on the viability after dilution of cells pre-exposed to 100 mM NaCl plus 0.5 M sucrose under conditions as in (A). (<i>n</i> = 10)</p

Ion transport in intact cells.

<p>A, Cl⁻ efflux in cells preloaded with Na³⁶Cl (100 µM) upon the addition of glucose (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; ○, ΔK388 LmrA). B, Effect of the concentration of Na⁺, NMG⁺ or Cl⁻ on the ATPase activity of purified LmrA (open symbols) or LmrCD (•) measured at 2 mM Mg-ATP. C,D,E, H⁺ efflux in energized cells, loaded with pH probe CFDASE to monitor the intracellular pH (pH_in) in the absence (C) or presence of (D) 0.25 M NaCl or (E) 0.672 M sucrose in the external buffer (each equivalent to 521 mOsm) (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; •, E314A LmrA; ▴, ΔK388 LmrA). Metabolic energy was generated in the cells by the addition of 20 mM glucose (at t = 0 min in the figures), 15 min after the addition of the NaCl or sucrose or solvent control. (<i>n</i> = 8)</p

Mass spectra of purified LmrA and LmrA-MD showing predominant homodimer formation for both proteins.

<p>Peaks assigned to binding of one cardiolipin molecule are labelled (blue stars), and measured molecular masses are shown.</p