Transition State Mimetics of the Plasmodium Export Element Are Potent Inhibitors of Plasmepsin V from P. falciparum and P. vivax

Following erythrocyte invasion, malaria parasites export a catalogue of remodeling proteins into the infected cell that enable parasite development in the human host. Export is dependent on the activity of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins within the Plasmodium export element (PEXEL; RxL↓xE/Q/D) in the parasite’s endoplasmic reticulum. Here, we generated transition state mimetics of the native PEXEL substrate that potently inhibit PMV isolated from Plasmodium falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics was completely dependent on the presence of P₁ Leu and P₃ Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired PEXEL processing and killed the parasites. The striking effect of the compounds provides a clearer understanding of the accessibility of the PMV active site and reaffirms the enzyme as an attractive target for the design of future antimalarials

Mass spectrometry identification of peptides containing the sites of frameshifting.

<p>The charge state (Charge), measured m/z value (m/z) and error of measurement in parts per million (Error; ppm) are given for the strongest detected peptide signal for each of the predicted frameshifting events. The scores for peptide identifications are given for both the SequestHT and Mascot search engines. The Mascot spectrum to peptide sequence assignments marked by an asterisk were not considered high confidence identifications, but also did not match any other sequence in the SwissProt sequence database (546,057 sequence entries) and therefore represent the most likely spectrum to sequence match. The peptide intensity is given as the area under the curve (peak area) for the extracted ion chromatogram of the strongest peptide signal of each frameshifting event and can be used as a rough estimation of peptide abundance. No CID: this low intensity signal was not selected for collision induced dissociation tandem mass spectrometry. The peptide detection is therefore based on high mass accuracy precursor mass measurement with an error of <1 ppm (+/−0.0016 Da).</p><p>Mass spectrometry identification of peptides containing the sites of frameshifting.</p

Frameshift enhancer A1 (A) evaluated in a dual luciferase based assay.

<p>Normalized frameshift efficiency of the HIV-1 element (green) is shown as −1 frameshift ratio relative to no compound (left ordinate). Effects of the compounds on the 0-frame control cell line containing the mutagenized slippery sequence is indicated in red, and is shown as translation ratio relative to no compound (right ordinate). (B) Compound A1 counter screen against a CFTR W1282X UGA nonsense mutant (green) and a UGG sense codon control (red). (C) PTC124 screened against CFTR W1282X (green) and the UGG sense codon control (red). Mean and standard deviation for three independent assays, each with n = 5 at 24 h.</p

Comparison of frameshift activity within HIV-1 and human gene <i>PEG10</i> frameshift elements.

<p>(A) Structures of the HIV-1 and <i>PEG10</i> frameshift sites showing slippery sequence (red), intercodon (green) and secondary structural element (extended stem-loop and pseudoknot). (B) Frameshift efficiency at the elements within dual luciferase reporters (<i>Renilla</i> luciferase (hRLuc) upstream and firefly luciferase (Luc⁺ downstream) in the −1 frame. ‘WT’ refers to the native frameshift sequences shown above. ‘Null’ has the slippery sequence modified with mutations to nullify it as a frameshift element. (C) Frameshift efficiency of element with intercodon substitution ‘UGA’ has the GGG/UCC intercodons substituted with the stop codon UGA. Black bars are constructs with empty vector, and grey bars with ~3 fold overexpression of eRF1. Constructs were as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139036#pone.0139036.ref026" target="_blank">26</a>] for HIV-1, and in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139036#pone.0139036.ref016" target="_blank">16</a>] for <i>PEG10</i>. Assays were carried out as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139036#pone.0139036.ref016" target="_blank">16</a>].</p

HIV-1 frameshift enhancers evaluated at both the HIV-1 and the <i>PEG10</i> frameshift elements using dual luciferase reporters and bifluorophore reporters.

<p>Effects of enhancers A1, A2 and A3 with the bifluorophore reporters (open markers), and dual luciferase reporters (closed markers) on HIV-1 frameshifting (upper panel) and <i>PEG10</i> frameshifting (lower panel) in a transient cell based assay. Results show the mean and standard deviation for quadruplicate replicates of three independent assays for the dual fluorophore assays, with the <i>PEG10</i> element tested with two biological replicates in the dual luciferase assay.</p

Effects on frameshifting at the HIV-1 element with increasing concentrations of enhancer A1 and two analogues A2 and A3 at three translational time points.

<p>The frameshift ratio of the cell line containing the HIV-1 frameshift element is indicated by triangles, as a comparison of that with no compound added. The firefly Luc⁺ activity in the control 0-frame cell-line is indicated by circles, and is expressed as the percentage of the activity with no compound. 0 h is indicated in black, 8 h in purple and 24 h in blue.</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