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

The pantothenate requirement of the different parasite lines observed in this study.

<p>(a) Percentage proliferation of parasites grown in different pantothenate concentrations. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate. Error bars represent SEM and are not visible if smaller than the symbols. For clarity, only data from the Parent (white circles), CJ-A (black diamonds), and CJ-A^+WTPfPanK1 (grey diamonds) lines are shown. (b) The pantothenate stimulatory concentration 50 (SC₅₀) values obtained for Parent, PanOH-A, PanOH-B, CJ-A, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 line parasites, which is the concentration of pantothenate required in the medium to support parasite proliferation by 50% (with 100% set to parasites grown in complete medium containing 1 <i>μ</i>M pantothenate). Errors represent SEM (n ≥ 3). An asterisk indicates that the value is significantly different from that obtained for the Parent line (CJ-A SC₅₀ value 95% CI compared to Parent: 2.7 to 30.9). No significant difference was observed between the SC₅₀ value of the Parent and those of PanOH-A, PanOH-B, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 (95% CI compared to Parent: PanOH-A = -6.8 to 7.1, PanOH-B = -3.8 to 9.3, Parent^+WTPfPanK1 = -9.3 to 3.3 & CJ-A^+WTPfPanK1 = -7.8 to 6.6).</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

The fitness of the different mutant lines generated in this study relative to the Parent line as determined from parasite competition assays.

<p>(a) A flow chart illustrating how the competition assay was performed. For each competition culture, an equal number of parasites from the Parent and a mutant line were combined into a single flask. These mixed cultures were maintained for a period of 6 weeks. The fitness cost associated with the <i>Pfpank1</i> mutations was assessed by determining the PanOH sensitivity of the mixed cultures: (b) Parent vs PanOH-A (grey triangles), (c) Parent vs PanOH-B (grey squares) and (d) Parent vs CJ-A (grey diamonds), at Week 0 (W0; dashed lines) and Week 6 (W6; solid lines). It was expected that the greater the fitness cost to the mutant, the greater the shift of its mixed culture PanOH dose-response curve toward the Parent line curve after 6 weeks. Arrows indicate this shift between W0 and W6. The parasite proliferation curves (dotted lines) of the respective mutant clones (black symbols) and Parent line (white circles) are also shown for comparison. Values for the mixed cultures are averaged from 2 independent experiments, each carried out in triplicate. Error bars represent SEM (n ≥ 4) for the individual cultures and range/2 for the mixed cultures, and are not visible if smaller than the symbols.</p

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 2

<p><b>Percentage proliferation of parasites from the Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) lines in the presence of (a) PanOH, (b) CJ-15,801 or (c) chloroquine.</b> Drug-pressured lines were generated by exposing Parent line parasites to 11 − 13 weeks of continuous drug-pressuring with either PanOH (for PanOH-A and PanOH-B) or CJ-15,801 (for CJ-A), followed by limiting dilution cloning. Values are averaged from ≥ 4 independent experiments, each carried out in triplicate. All error bars represent SEM. Error bars are not visible if smaller than the symbols.</p

Mutations in <i>Pfpank1</i> and the affected residues within the <i>Pf</i>PanK1 protein.

<p>(a) The single nucleotide polymorphisms detected in the <i>Pfpank1</i> gene (accession number: PF3D7_1420600) of PanOH-A, PanOH-B and CJ-A, and the corresponding amino acid changes in the <i>Pf</i>PanK1 protein. (b) A three-dimensional homology model of the <i>Pf</i>PanK1 protein (pink) based on the solved structure of human PanK3 (PDB ID: 5KPR), overlaid on the human PanK3 structure in its active conformation (blue), with an ATP analogue (AMPPNP; carbon atoms coloured green) and pantothenate (carbon atoms coloured yellow) bound. <i>Pf</i>PanK1 shares 28% sequence identity with human PanK3 over the parts of the protein that have been modeled. Red spheres indicate the residues (G95 and D507) affected by the mutations in the parasite proteins. Human PanK3 has been shown to exist as a dimer. Here, individual monomers are shown in different shades of pink and blue.</p

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 10

<p><b>Percentage parasite proliferation of the different lines in the presence of the pantothenate analogues (a) N5-trz-C1-Pan and (b) <i>N</i>-PE-αMe-PanAm, and inhibition of [¹⁴C]pantothenate phosphorylation in parasite lysates by various pantothenate analogues: (c) PanOH, (d) CJ-15,801, (e) N5-trz-C1-Pan and (f) <i>N</i>-PE-αMe-PanAm.</b> Symbols represent Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasite lines. The Y-axes of c−f indicate the percentage of total pantothenate phosphorylation. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate for the parasite proliferation assays and duplicate for the phosphorylation assays. Error bars represent SEM and are not visible if smaller than the symbols.</p

The phosphorylation of [¹⁴C]pantothenate (2 <i>μ</i>M) over time (in minutes) by lysates generated from Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasites.

<p>Inset shows [¹⁴C]pantothenate (2 <i>μ</i>M supplemented with non-radioactive pantothenate to a total pantothenate concentration of 200 <i>μ</i>M) phosphorylation by CJ-A parasite lysates measured over 420 min. Values are averaged from 3 independent experiments, each performed with a different batch of lysate and carried out in duplicate. Error bars represent SEM and are not visible if smaller than the symbols.</p

Confocal micrographs showing the subcellular location of GFP-tagged <i>Pf</i>PanK1 in 3D7 strain parasites harbouring the <i>Pfpank1</i>-pGlux-1 episomal plasmid.

<p>From left to right: Brightfield, GFP-fluorescence, DAPI- (fixed cells; top) or Hoechst 33258- (live cells; bottom) fluorescence, and merged images of erythrocytes infected with trophozoite-stage <i>P</i>. <i>falciparum</i> parasites expressing <i>Pf</i>PanK1-GFP. Arrows indicate the plasma membranes of either the erythrocytes (black) or the parasites (white). Scale bar represents 5 <i>μ</i>m.</p

<i>Pf</i>PanK activity profiles derived from the initial rates of pantothenate phosphorylation by lysates generated from Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasites at various pantothenate concentrations.

<p>The grey diamond indicates a data point that was outside the 95% confidence band when it was included in the non-linear regression. This data point was therefore deemed an outlier and was omitted from the data set used to generate the fitted curve shown. Values are averaged from 3 independent experiments, each performed with a different batch of lysate and carried out in duplicate. Error bars represent SEM and are not visible if smaller than the symbols. Relative specificity constant (rsc) values express the specificity constants obtained for each cell line relative to that of the Parent. The <i>V</i>_max (<i>μ</i>mol/(10¹² cells.h)), <i>K</i>_m (<i>μ</i>M) and rsc values determined from the curves are shown within each box. Errors represent SEM (n = 3). An asterisk indicates that the value is significantly different compared to the Parent line (<i>V</i>_max 95% CI compared to Parent: PanOH-A = 173 to 344, PanOH-B = 5.2 to 21.6 & CJ-A = 112 to 133; <i>K</i>_m 95% CI compared to Parent: PanOH-A = 8.8 to 13.3, PanOH-B = 8.3 to 20.7 & CJ-A = 42 to 484; rsc 95% CI compared to Parent: PanOH-B = -1.366 to -0.519 & CJ-A = -1.404 to -0.557). No significant difference was observed between the rsc of the Parent and PanOH-A (95% CI compared to Parent: -0.698 to 0.179).</p