ABSTRACT
Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.
Subject(s)
Antifungal Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Candida albicans/drug effects , Candida albicans/enzymology , Transferases (Other Substituted Phosphate Groups)/antagonists & inhibitors , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Candida albicans/genetics , Carrier Proteins , Computational Biology , Enzyme Activation , Gene Expression , Molecular Sequence Data , Phenotype , Promoter Regions, Genetic , Sequence Alignment , Sequence Deletion , Substrate Specificity , Transferases (Other Substituted Phosphate Groups)/chemistry , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/metabolismABSTRACT
Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.
Subject(s)
Aspergillus fumigatus/enzymology , Aspergillus fumigatus/genetics , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Amino Acid Sequence , Amino Acids, Branched-Chain/biosynthesis , Animals , Aspergillosis/microbiology , Aspergillus fumigatus/pathogenicity , Biosynthetic Pathways , Enzyme Activation , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Hydro-Lyases/chemistry , Male , Mice , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Mutation , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Virulence/geneticsABSTRACT
MRCKα and MRCKß (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKß influence cell shape and motility. We report further evidence for MRCKα and MRCKß contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKß together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKß kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC(50) values for MRCKα and MRCKß. We also describe the crystal structure of MRCKß in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.
Subject(s)
Neoplasm Invasiveness/pathology , Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/chemistry , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Amides/chemistry , Amides/pharmacology , Catalytic Domain , Cell Line, Tumor , Collagen/metabolism , Crystallography, X-Ray , Drug Combinations , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Humans , Inhibitory Concentration 50 , Laminin/metabolism , Models, Molecular , Myotonin-Protein Kinase , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/pharmacology , Protein Multimerization/drug effects , Proteoglycans/metabolism , Pyridines/chemistry , Pyridines/pharmacology , Thiophenes/chemistry , Thiophenes/pharmacology , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolismABSTRACT
The mitochondrial phosphopantetheinyl transferase gene pptB of the opportunistic pathogen Aspergillus fumigatus has been identified and characterised. Unlike pptA, which is required for lysine biosynthesis, secondary metabolism, and iron assimilation, pptB is essential for viability. PptB is located in the mitochondria. In vitro expression of pptA and pptB has shown that PptB is specific for the mitochondrial acyl carrier protein AcpA.
