Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8–Atg3 Protein–Protein Interaction

Abstract

New therapies are needed against malaria, a parasitic infection caused by Plasmodium falciparum, as drug resistance emerges against the current treatment, artemisinin. We previously characterized the Atg8-Atg3 protein-protein interaction (PPI), which is essential for autophagy and parasite survival. Herein we illustrate the use of virtual library screening to selectively block the PPI in the parasite without inhibiting the homologous interaction in humans by targeting the A-loop of PfAtg8. This A-loop is important for Atg3 binding in Plasmodium, but is absent from the human Atg8 homologues. In this proof-of-concept study, we demonstrate a shift in lipidation state of PfAtg8 and inhibition of P. falciparum growth in both blood- and liver-stage cultures upon drug treatment. Our results illustrate how in silico screening and structure-aided drug design against a PPI can be used to identify new hits for drug development. Additionally, as we targeted a region of Atg8 that is conserved within apicomplexans, we predict that our small molecule will have cross-reactivity against other disease-causing apicomplexans, such as Toxoplasma, Cryptosporidium, Theileria, Neospora, Eimeria, and Babesia.

Visualizing protein–ligand binding with chemical energy-wise decomposition (CHEWD): application to ligand binding in the kallikrein-8 S1 Site

“…Calculation of binding energies as a screening tool is highlighted by numerous implementations in medicinal chemistry and drug design [6][7][8]59]. Decomposing the binding energy into individual residue contributions can provide useful insight into the nature of the protein-ligand complex.…”

“…Structure-based drug design (SBDD) has benefited significantly over the years from developments in the field of proteomics, comparative genomics and high-end computing [1][2][3][4][5]. Binding affinity is an important metric in the assessment of new drug targets and their optimization [5][6][7][8]. The ability to calculate binding affinities reliably in silico is advantageous, reducing the need for expensive experiments [9].…”

Characterization of the molecular mechanism of the autophagy-related Atg8–Atg3 protein interaction in Toxoplasma gondii

“…In Plasmodium, PfAtg8 -PfAtg3 interaction has been considered as an antimalarial drug target. Several small molecular inhibitors against Plasmodium in blood and liver stages have also been discovered (38,39). However, a recent study verified that three PfAtg8 -PfAtg3 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box do not block TgAtg8 lipidation, although all three inhibitors hinder Toxoplasma growth in a dose-dependent manner (40).…”

“…Currently, targeting protein-protein interaction interfaces has been increasingly considered because of its importance and practicality (42)(43)(44). Recently, many compounds with antimalarial activity have been identified by inhibiting the recombinant PfAtg8 -PfAtg3 interaction in vitro, suggesting that the Atg8 -Atg3 interaction could be an attractive novel drug target in apicomplexan protozoans (28,38,39). In this study, we have developed a quantitative and straightforward AlphaScreen assay, which has enabled high-throughput screening of small-molecule modulators from compound libraries for multifarious biological targets.…”

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii

“…These differences were significant enough to result in an ϳ30-fold preference for the binding of small-molecule inhibitors for PfAtg8 compared to human LC3 (26). Moreover, high-throughput screens have identified numerous compounds that inhibit the interaction between recombinant P. falciparum Atg3 (PfAtg3) and PfAtg8 in vitro, including a group of compounds from the Medicines for Malaria Venture (MMV) Malaria Box with antimalarial activity in tissue culture (25)(26)(27). These findings illustrate the ability to specifically target the parasite Atg3-Atg8 interaction and suggest that the Atg3-Atg8 interaction may be an attractive new drug target in apicomplexan parasites.…”