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Plasmodium falciparum subtilisin-like protease 1 (SUB1) is a novel target for the development of innovative antimalarials. We recently described the first potent difluorostatone-based inhibitors of the enzyme ((4 S )-( N -(( N -acetyl- l -lysyl)- l -isoleucyl- l -threonyl- l -alanyl)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine ( 1 ) and (4 S )-( N -(( N -acetyl- l -isoleucyl)- l -threonyl- l -alanylamino)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine ( 2 )). As a continuation of our efforts towards the definition of the molecular determinants of enzyme-inhibitor interaction, we herein propose the first comprehensive computational investigation of the SUB1 catalytic core from six different Plasmodium species, using homology modeling and molecular docking approaches. Investigation of the differences in the binding sites as well as the interactions of our inhibitors 1 , 2 with all SUB1 orthologues, allowed us to highlight the structurally relevant regions of the enzyme that could be targeted for developing pan-SUB1 inhibitors. According to our in silico predictions, compounds 1,2 have been demonstrated to be potent inhibitors of SUB1 from all three major clinically relevant Plasmodium species ( P. falciparum , P. vivax , and P. knowlesi ). We next derived multiple structure-based pharmacophore models that were combined in an inclusive pan-SUB1 pharmacophore (SUB1-PHA). This latter was validated by applying in silico methods, showing that it may be useful for the future development of potent antimalarial agents. [ABSTRACT FROM AUTHOR]

Highlights: [•] PfSUB3 possesses serine protease activity in its C-terminal region. [•] Plasmodium falciparum profilin interacts with mature PfSUB3. [•] Mature PfSUB3 proteolyses recombinant PfPRF in in vitro assays. [•] PfPRF induces secretion of TNF-α and IL-12 in mouse bone marrow dendritic cells. [ABSTRACT FROM AUTHOR]

Abstract: PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database “PlasmoDB”. The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn334 to Glu769 (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr516 to Glu769 (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes. [Copyright &y& Elsevier]

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canis λEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2 , were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1 , SUB2 , and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1 , SUB2 , and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes. [ABSTRACT FROM AUTHOR]