Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections

Activity of gold standard drugs after 3-day treatment against intra- and extracellular <i>L. donovani</i> amastigotes.

<p>*Assay was set up with amastigotes isolated from spleens of infected Rag2−/− mice with subsequent incubation at 26°C. All assays were run in triplicate with biological repeats (intracellular amphotericin B, n = 4; intracellular miltefosine, n = 3; extracellular amphotericin B, n = 3; extracellular miltefosine, n = 2).</p><p>Activity of gold standard drugs after 3-day treatment against intra- and extracellular <i>L. donovani</i> amastigotes.</p

Dose response curves for amphotericin B and miltefosine against intracellular <i>L. donovani</i> amastigotes.

<p>Parasites were released by conditional lysis with saponin from bone marrow derived macrophages of BALB/c mice after 72 h incubation with anti-leishmanial standard drugs. Using this method, the measured activities of amphotericin B (EC₅₀ 29 nM) and miltefosine EC₅₀ (1.45 µM) were reproducible and comparable to reported values against intracellular L. donovani (43–45). Representative plots for amphotericin B (n = 4) and miltefosine (n = 3) are shown. (R²_AmphoB – 0.9905, R²_Mil – 0.9963).</p

Linear correlation of the number of <i>L. donovani</i> amastigotes and the reduction of Alamar blue.

<p><i>L. donovani</i> amastigotes were isolated from the spleen of an infected Rag-2 −/− mouse. Up to 10⁶ (A) or 10⁵ (B) parasites were suspended in 200 µl/well in supplemented RPMI, Alamar blue was added and the plate incubated at 26°C. At the indicated time points, the fluorescence per well was determined and plotted over the initial number of parasites/well. The correlations between Alamar blue conversion and the initial parasite numbers are highly significant: P<0.0001 as shown by linear regression analysis (R²24h– 0.9979, R²48h – 0.9991, R²72h – 0.9984).</p

Release of parasites from infected macrophages in 96-well plates.

<p>Bone marrow derived macrophages were infected overnight with <i>L. donovani</i> amastigotes. Cells were incubated for a further 3 days and then treated with DMEM containing the indicated concentration of saponin. Supplemented RPMI was added to the released amastigotes and plates were further incubated for 4 days at 26°C before addition of Alamar blue and subsequent measurement of fluorescence in a plate reader. The survival of freed amastigotes is dependent on the concentration of saponin used and the time of lysis. A 5-fold greater signal of reduced Alamar blue from freed amastigotes over uninfected macrophages was achieved with 2 mg·ml-1 saponin for 5 min; this concentration was used for all other experiments performed. All samples were run in triplicate; one representative experiment is shown. MΦ, uninfected macrophages; iMΦ, infected macrophages. *** denotes P<0.0001 as determined by a two-way ANOVA.</p

Cellular activity of compounds with good potency in the extracellular but varying potency in the intracellular model.

<p>All three compounds tested showed cellular activity on the intracellular and extracellular <i>L. donovani</i> amastigotes. When tested against intracellular amastigotes, only DDD100887 generated a reproducible dose-response curve. It was not possible to generate dose-response curves for DDD85646 and DDD86211 as both compounds resulted in an reproducible “activity valley” at higher concentrations before resulting in complete killing of the parasites at the highest concentration tested (see text for details). One representative experiment is shown for each compound. The correlation coefficients for the extracellular amastigotes were R²>0.98, with almost all very close to or >0.99. The correlation coefficient for intracellular DDD100887 is R² 0.9212.</p

Chemical structures of NMT inhibitors.

<p>(A) Resynthesised <i>T. brucei</i> inhibitors identified by the Dundee Drug Discovery Unit as also inhibiting <i>L. major</i> NMT <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd.0003363-Frearson1" target="_blank">[31]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd.0003363-WO20100263651" target="_blank">[37]</a>. Results summarized in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd-0003363-t002" target="_blank">Table 2</a>. (B) Selective inhibitors identified by high throughput screening against <i>L. donovani</i> NMT <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd.0003363-Bell1" target="_blank">[35]</a>. Results summarised in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd-0003363-t003" target="_blank">Table 3</a>.</p

Activity values/properties of NMT inhibitors from DDD85646 series.^*

1<p>resynthesised DDD85646 (Ref 31).</p>2<p>resynthesised from WO2010/026365 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd.0003363-WO20100263651" target="_blank">[Ref 37]</a>.</p><p>*structures shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd-0003363-g004" target="_blank">Fig. 4A</a>.</p><p>n.p. – not possible.</p><p>Activity values/properties of NMT inhibitors from DDD85646 series.^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#nt104" target="_blank">*</a>}</p

Activity values/properties of NMT inhibitors identified in Pfizer high-throughput screen.¹^,²

1<p>Ref 35.</p>2<p>structures shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#pntd-0003363-g004" target="_blank">Fig. 4B</a>.</p><p>Activity values/properties of NMT inhibitors identified in Pfizer high-throughput screen.^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#nt106" target="_blank">1</a>}^,^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003363#nt107" target="_blank">2</a>}</p