title: Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.
abstract: Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.
authors.affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.
authors.forename: Chrislaine
authors.initials: C
authors.lastname: Withers-Martinez
authors.forename: Catherine
authors.initials: C
authors.lastname: Suarez
authors.forename: Simone
authors.initials: S
authors.lastname: Fulle
authors.forename: Samir
authors.initials: S
authors.lastname: Kher
authors.forename: Maria
authors.initials: M
authors.lastname: Penzo
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Ebejer
authors.forename: Kostas
authors.initials: K
authors.lastname: Koussis
authors.forename: Fiona
authors.initials: F
authors.lastname: Hackett
authors.forename: Aigars
authors.initials: A
authors.lastname: Jirgensons
authors.forename: Paul
authors.initials: P
authors.lastname: Finn
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
mesh_terms: D000962:Antimalarials / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D010961:Plasmodium / Q000201:enzymology*; D010962:Plasmodium berghei / Q000201:enzymology; D010963:Plasmodium falciparum / Q000201:enzymology; D016790:Plasmodium knowlesi / Q000201:enzymology; D010966:Plasmodium vivax / Q000201:enzymology; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000962:Antimalarials; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D013381:Subtilisins; C403176:subtilisin-like protease 1, Plasmodium falciparum