Probing the Open Global Health Chemical Diversity Library for Multistage-Active Starting Points for Next-Generation Antimalarials

Abstract

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 μM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 μM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.

Keywords: chemoinformatic analysis; drug discovery; malaria; multistage antimalarials; whole genome sequencing (WGS).

Figure 1

Global Health Chemical Diversity Library screening workflow. A set of 68,614 small molecules was sequentially screened across the Plasmodium life cycle. Horizontal arrows indicate the removal of unqualified compounds at a given criterion while vertical arrows track the progress of desirable ones. Primary screening data were filtered by inhibition to generate a list of reconfirmation worthy hits. Commercially available compounds for each stage were resupplied and reconfirmed in dose response to round off a series of potent leads. Total PbLuc active compounds includes recombinant luciferase inhibitors.

Figure 2

Delayed-death inhibitors. (a) Screening plate view. Heatmaps from the PfLuc dose response assays were superimposed to show preferential compound efficacy at the 48 h (light yellow) or 96 h (red) incubation times. Compounds were titrated in dose response and in identical patterns across both representative plates shown. Artemisinin controls are positive for parasite death at all time points, while delayed-death controls including clindamycin and doxycycline are only active at 96 h. The highest potency ABS inhibitor, DDD01057375, shows a similar trend to delayed-death controls. (b) Dose response curves for clindamycin for ABS and liver stage. (c) From the 68 delayed-death inhibitors, 22 belonged to scaffold families that were enriched for delayed-death inhibitors, relative to the entire library. Active delayed-death members are shown as dark red diamonds while inactive members are shown in pink. Additional data as well as error measurements are available in Tables S1 and S4. Data can be interactively explored at http://www.ndexbio.org/#/networkset/1ac81391-eebd-11e9-bb65-0ac135e8bacf?accesskey=8f0a93f855278c5ffdb1e8e52c3ca7b1983ac8452eb3b8409f99cc39fd783082.

Figure 3

Liver active compounds. (a) 111 compounds belonging to one of 10 clusters enriched for gametocyte activity are shown. Gray diamonds show an activity of less than 10 μM, and green circles show compounds that were not selected for dose response testing or were inactive. Probability values were calculated using the hypergeometric mean function. (b) Activity tests against P. vivax patient isolates for three independent measurements and two compounds from cluster 756 (highlighted in green in (a)) as well as the controls. Parasite exoerythrocytic forms were stained with an HSP70 antibody and were counted on day 7. Three replicates were performed, and the data shows the mean and standard deviation. The number of events (schizonts and hypnozoites) were counted by reading 200 fields per well. Additional data for all compounds can be found in Table S5. Data can be explored at http://www.ndexbio.org/#/networkset/1ac81391-eebd-11e9-bb65-0ac135e8bacf?accesskey=8f0a93f855278c5ffdb1e8e52c3ca7b1983ac8452eb3b8409f99cc39fd783082.

Figure 4

Stage V gametocyte active network. The diagram shows nine clusters (51 compounds) enriched for gametocyte activity (24 active compounds). Dark purples diamonds show the activity of less than 10 μM, and light purple circles show compounds that were not selected for dose response testing or were inactive. Probability values were calculated using the hypergeometric mean function. The cluster identity of the inactive members is shown in Table S3. Data can be explored at http://www.ndexbio.org/#/networkset/1ac81391-eebd-11e9-bb65-0ac135e8bacf?accesskey=8f0a93f855278c5ffdb1e8e52c3ca7b1983ac8452eb3b8409f99cc39fd783082.

Figure 5

Potent multistage and stage-specific enriched clusters. (a) Classification and number of single stage and multistage confirmed hits (IC₅₀ < 10 μM) belonging to enriched clusters (173 compounds). (b) Clusters that have a higher number of active compounds than that expected by chance. The hypergeometric mean was calculated for each cluster, and only those clusters enriched for bioactivity are shown (p < 0.005, 171 compounds). The central node of each cluster represents the maximum common substructure (MCS). Clusters are anchored to each other on the basis of their affinity for a given life cycle stage, while multistage active compounds are further differentiated by color.