On 2013 Nov 18, Gary Ward commented:

This paper describes a clever high-throughput assay to identify small molecules that disrupt the interaction of Plasmodium falciparum AMA1 and RON2, and can thereby block merozoite invasion of erythrocytes. The data provide promising proof-of-concept for the development of novel antimalarials that disrupt protein-protein interactions critical for invasion. This particular interaction is thought to happen extracellularly, within the blood, which could facilitate access of such drugs to their targets.

The three inhibitors described have IC50 values in the 20-30 uM range. The authors state that "small-molecule inhibitors will result in reduced or no emission signal depending on the strength of the inhibition". Our group was interested to know what the dynamic range of the AlphaScreen assay is and whether it can capture binding at both ends of the affinity spectrum (subnanomolar to millimolar).

A 1000-fold molar excess of inhibitor was required to disrupt RON2L-AMA1 interaction in the screen, perhaps because the compound is added after RON2L-AMA1 complex formation and must essentially displace the RON2L from AMA1. The assay may have been done this way purposely, to recapitulate what happens in the blood, i.e., secreted RON2 is probably not exposed on the surface of the red cell for long before it is bound by AMA1. It would nonetheless be interesting to know what happens to the IC50 if compound is added to AMA1 before the addition of RON2L.

Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelson, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

On 2013 Nov 18, Gary Ward commented:

This paper describes a clever high-throughput assay to identify small molecules that disrupt the interaction of Plasmodium falciparum AMA1 and RON2, and can thereby block merozoite invasion of erythrocytes. The data provide promising proof-of-concept for the development of novel antimalarials that disrupt protein-protein interactions critical for invasion. This particular interaction is thought to happen extracellularly, within the blood, which could facilitate access of such drugs to their targets.

The three inhibitors described have IC50 values in the 20-30 uM range. The authors state that "small-molecule inhibitors will result in reduced or no emission signal depending on the strength of the inhibition". Our group was interested to know what the dynamic range of the AlphaScreen assay is and whether it can capture binding at both ends of the affinity spectrum (subnanomolar to millimolar).

A 1000-fold molar excess of inhibitor was required to disrupt RON2L-AMA1 interaction in the screen, perhaps because the compound is added after RON2L-AMA1 complex formation and must essentially displace the RON2L from AMA1. The assay may have been done this way purposely, to recapitulate what happens in the blood, i.e., secreted RON2 is probably not exposed on the surface of the red cell for long before it is bound by AMA1. It would nonetheless be interesting to know what happens to the IC50 if compound is added to AMA1 before the addition of RON2L.

Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelson, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward