Designed Parasite-Selective Rhomboid Inhibitors Block Invasion and Clear Blood-Stage Malaria

Cell Chem Biol. 2020 Nov 19;27(11):1410-1424.e6. doi: 10.1016/j.chembiol.2020.08.011. Epub 2020 Sep 3.


Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

Keywords: Plasmodium; Ras-converting enzyme; Toxoplasma; apicomplexan parasites; malaria; presenilin; regulated intramembrane proteolysis; rhomboid protease; serine protease; site-2 protease.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amides / chemical synthesis
  • Amides / chemistry
  • Amides / pharmacology*
  • Antimalarials / chemical synthesis
  • Antimalarials / chemistry
  • Antimalarials / pharmacology*
  • Boronic Acids / chemical synthesis
  • Boronic Acids / chemistry
  • Boronic Acids / pharmacology
  • Drug Design*
  • HEK293 Cells
  • Humans
  • Malaria / blood
  • Malaria / drug therapy*
  • Malaria / metabolism
  • Molecular Structure
  • Parasitic Sensitivity Tests
  • Peptide Hydrolases / blood
  • Peptide Hydrolases / metabolism
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / pharmacology
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / metabolism
  • Protease Inhibitors / chemical synthesis
  • Protease Inhibitors / chemistry
  • Protease Inhibitors / pharmacology*
  • Proteolysis / drug effects
  • Protozoan Proteins / antagonists & inhibitors*
  • Protozoan Proteins / blood
  • Protozoan Proteins / metabolism


  • Amides
  • Antimalarials
  • Boronic Acids
  • Peptides
  • Protease Inhibitors
  • Protozoan Proteins
  • Peptide Hydrolases
  • ROM4 protein, Plasmodium falciparum