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

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. [Display omitted] •Malaria rhomboid PfROM4 cleaves only cell-surface adhesins required for invasion•Steric clashes alone prevent rhomboid enzymes from cleaving each other's substrates•Inhibitors with designed steric blocks inhibit PfROM4 but not other rhomboid enzymes•Inhibiting PfROM4 impedes resolution of merozoite invasion and clears parasitemia Gandhi et al. combined substrate mapping, structural biology, and manually directed evolution to design potent peptidic inhibitors of the malaria rhomboid PfROM4 that do not target other rhomboid proteases. Treating malaria cultures revealed an essential role for PfROM4 only in invasion, and led to “tethered” parasites that die out.