Fragments vs plasmepsins

Plasmodium, which causes malaria, is a nasty bag of tricks. These include the plasmepsins, aspartic proteases that – among other duties – digest the hemoglobin in red blood cells. In a recent paper in J. Med. Chem., an international group of collaborators led by Kristaps Jaudzems and Aigars Jirgensons at the Latvian Institute of Organic Synthesis describe how they discovered inhibitors.

The team started by performing a fragment screen against plasmepsin II (Plm II), one of ten plasmepsins encoded in Plasmodium falciparum. A library of 976 rule-of-three compliant fragments (from ChemBridge) were screened in pools of six using three different NMR methods: STD, WaterLOGSY, and T1ρ. A total of 49 fragments hit at least two assays and were competitive with a known aspartic protease inhibitor, and ten of these showed functional inhibition in an enzymatic assay. Fragment 1 was the most potent.

Crystallography was unsuccessful, but the researchers were able to use ILOE NMR to show that another aromatic fragment could bind near fragment 1. Based on this information, the researchers appended a phenyl moiety to produce compound 3a and obtained a 10-fold boost in potency.

Crystallography still didn’t work, but modeling based on similar compounds on a different aspartic protease suggested that adding another hydrophobic substituent could fill another pocket, leading to compound 3b. At this stage the researchers were finally able to obtain a crystal structure of compound 3b bound to Plm II, which confirmed the predicted binding mode and also revealed another pocket that could be grown into as in in the case of compound 4b. This and several related compounds inhibited the growth of Plasmodium falciparum at low micromolar concentrations and were minimally cytotoxic to mammalian cells.

There is still much to do. Selectivity for the one human aspartic protease tested was generally modest. Also, as the researchers acknowledge, the most active compounds are seriously lipophilic. Still, this is another example of fragment-based lead discovery in academia. More importantly, it provides more ideas on how to tackle a pernicious parasite.