Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides

Abstract

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

Fig. 1

The high-throughput anti-Wolbachia screening flow. This extensive 16-step screen per batch across all three stages was completed over 3 weeks followed by overflow plate reading and data analysis in the 4th week when at full capacity of four daily batches of 150 plates per week. Manual processes are indicated by dashed boxes and fully automated steps in solid boxes. Bold rectangular boxes indicate the use of the fully automated systems: Agilent technologies BioCel system and HighRes Biosolutions automated system. The fluorescent images are representatives of the host nuclei (Hoechst) and Wolbachia (wBmPAL) staining taken from a whole well image (growth area of 10 mm²) on the acumen® to demonstrate cells incubated with the vehicle/DMSO and doxycycline controls

Fig. 2

Overview of the HTS. a Results in ChemGPS chemical space. Purple spheres represent examples of current anti-Wolbachia agents; black points represent the ~20,000 actives identified in the primary HTS; orange spheres represent the 360 compounds from the 57 clusters tested in the microfilarial (Mf) assay; green spheres represent the 18 confirmed, potent hits. The position in ChemGPS space indicates a compound’s size, hydrophobicity/hydrophilicity and flexibility (T1, T2 and T3 defined in ref. ). The hits span a large proportion of the chemical space covered by the ~6000 compounds. The 3D plot was produced in R using the rgl library (http://cran.r-project.org). b The full screening campaign and triaging process. Each stage includes the total compounds screened (rectangular box on the right) and hits identified (circular box on the right). The screen used (C6/36 (wAlbB) or Brugia malayi Mf) and screening location (AstraZeneca or A·WOL) are indicated on the left. Rational analysis was utilised at every stage based on cheminformatics and available data

Fig. 3

Eighteen selected hits represented in chemical space. The first three principal components (PC1–3) together account for 31.6% of the overall variance in the data set. The chemotypes show clustering in chemical space. The size of each data point is proportional to the Selection Score. Selection Score is calculated by summation of the component scores using the criteria in Supplementary Table 3. The label for each point is the ligand efficiency-dependent lipophilicity index (LELP) with values <10 considered good. A selection of the best compound structures is shown (large spheres) in a 3D plot produced by Datawarrior. The cluster number is indicated by colour: cluster 1 (dark blue sphere), 2 (red sphere), 3 (green sphere), 4 (dark gold sphere), 5 (light blue sphere), 6 (lilac sphere), 7 (orange sphere), 8 (teal sphere), 9 (pink sphere)

Fig. 4

Anti-Wolbachia potency and drug-like properties of six cluster representatives. Nine hit series were categorised based on the balance between potency in the microfilarial assay and drug metabolism and pharmacokinetic (DMPK) properties. The figure shows the best representatives of the top six clusters. Five axes in the radar plots represent: EC₅₀ values (nM) in the anti-Wolbachia microfilarial assay (MF), rat hepatocytes clearance (µl min⁻¹ 1 × 10⁶ cells⁻¹) (Rat Heps), human microsome clearance (µl min⁻¹ mg⁻¹) (Hum Mics), LogD_7.4 (LogD) and log value of aqueous solubility in pH 7.4 PBS buffer (µM) (LogS). The core of each cluster chemotype is highlighted blue within the chemical structures

Fig. 5

Anti-Wolbachia time-kill analysis. Representatives from the top five clusters (1A–5A), as well as established anti-Wolbachia antibacterials (doxycycline (white), minocycline (grey), rifampicin (black)) were assessed after exposure for 1 day and 2 days before washing and further incubation until the standard 6-day read out. wsp:gst ratio was normalised to the DMSO vehicle control resulting in a read out of mean percentage reduction from control. Doxycycline day 6 treatment represents the result for exposure to doxycycline at 5 µM for a full 6 days (no wash). The box extends from the 25th to the 75th percentile with the median presented by the central line. The whiskers are set at the minimum and maximum therefore indicating the range from five replicates. The cluster number is indicated by colour: cluster 1 (dark blue), 2 (magenta), 3 (teal), 4 (dark gold), and 5 (light blue)