qHTS for Inhibitors of AMA1-RON; Towards Development of Antimalarial Drug Lead: Primary Screen
Plasmodium, the causative agent of malaria, is one of the world's deadliest parasitic diseases. Currently there is no vaccine available and there is widespread resistance to common anti-malarial drugs. This warrants novel, alternative approaches for developing improved therapy. During infection, one such essential interaction is between two parasite proteins, the apical membrane antigen1 (AMA1) more ..
BioActive Compounds: 735
Depositor Specified Assays
Plasmodium, the causative agent of malaria, is one of the world's deadliest parasitic diseases. Currently there is no vaccine available and there is widespread resistance to common anti-malarial drugs. This warrants novel, alternative approaches for developing improved therapy. During infection, one such essential interaction is between two parasite proteins, the apical membrane antigen1 (AMA1) that is translocated onto the merozoite surface and the rhoptry neck protein 2 (RON2), which is transferred to the red blood cells (RBC) membrane during invasion (Besteiro et.al., 2009). Recently, the Miller lab has shown that the interaction between AMA1 and RON2 is essential for junction formation of the parasite with the host cell (Srinivasan et.al., 2011). The binding of the RON2 peptide to the hydrophobic pocket in AMA triggers the formation of the moving junction which provides a firm anchor for the parasite to pull itself into the RBC using its actin-myosin motor; an irreversible step that commits the parasite for invasion. Only one AMA1 and RON2 genes exists in Plasmodium and there are no alternate pathways. Moreover, the region corresponding to RON2 peptide is conserved in all P. falciparum isolates for which sequences are available. The key residues in the AMA1 hydrophobic pocket that binds RON2 are also conserved and indicate a functional constraint governing this essential step in invasion since a short RON2 peptide (RON2L) corresponding to the binding region on AMA1 is sufficient to compete with the native RON2 protein and inhibits invasion (Tonkin et.al, 2011). Therefore, developing small molecule inhibitors of this interaction offers a promising target opening a new arena for anti-malarial therapeutics drug design.
In collaboration with the Miller lab, a quantitative High Throughput Screen using the AlphScreen technology was developed using a recombinant his-tagged AMA1 and a biotinylated RON2 peptide. This fluorescent screen was assayed against the NIH Molecular Libraries Small Molecule Repository (MLSMR). Inhibition of this protein-protein interaction is the desired outcome of this assay. However, due the limitations of this AlphaScreen assay, a large number of artifacts were observed. An AlphaScreen counterscreen was run to identify the true inhibitors, triage compound activity, and weed out false positives.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH099752
Assay Submitter (PI): Louis Miller, National Institute of Allergy and Infectious Disease (NIAID)
Two microliters of his-tagged AMA1 3D7 allele protein (final concentration 25 nM) was dispensed into a 1,536-well assay plate. Small molecules and positive control peptides were pin-transferred (23 nL or 46 nL) via Kalypsys pin-tool equipped with a 1,536-pin array, resulting in final compound and peptide concentration ranges of 91.5 nM - 57.2 muM, and 15.6 nM - 2.00 muM, respectively. Unlabeled RON2 peptide or R1 peptide (VFAEFLPLFSKFGSRMHILK) that specifically binds the 3D7 AMA1 was used as a positive control that inhibited the binding of RON2L to AMA1. After 15 minutes incubation, 1 uL of biotinylated RON2 peptide (final concentration 25 nM) or buffer were dispensed and the assay plate was incubated for an additional 30 minutes at room temperature and protected from light. This was followed by an addition of 1 uL mixture of donor and acceptor beads (10 ug/mL final concentrations for each). The plates were incubated for 30 min at room temperature and read using the EnVision(R) plate reader (PerkinElmer). Maximum inhibition response was normalized to the positive control signal while no inhibition response was normalized to the DMSO treated wells.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)