Design of Arylsulfonylhydrazones as Potential FabH Inhibitors: Synthesis, Antimicrobial Evaluation and Molecular Docking.

BACKGROUND: Antimicrobial resistance is a persistent problem regarding infection treatment and calls for developing new antimicrobial agents. Inhibition of bacterial ß-ketoacyl acyl carrier protein synthase III (FabH), which catalyzes the condensation reaction between a CoAattached acetyl group and an ACP-attached malonyl group in bacteria is an interesting strategy to find new antibacterial agents. OBJECTIVE: The aim of this work was to design and synthesize arylsulfonylhydrazones potentially FabH inhibitors and evaluate their antimicrobial activity. METHODS: MIC50 values of sulfonylhydrazones against E. coli and S. aureus were determined. Antioxidant activity was evaluated by DPPH (1-1'-diphenyl-2-picrylhydrazyl) assay and cytotoxicity against LL24 lung fibroblast cells was verified by MTT method. Principal component analysis (PCA) was performed in order to suggest a structure-activity relationship. Molecular docking allowed to propose sulfonylhydrazones interactions with FabH. RESULTS: The most active compound showed activity against S. aureus and E. coli, with MIC50 = 0.21 and 0.44 µM, respectively. PCA studies correlated better activity to lipophilicity and molecular docking indicated that sulfonylhydrazone moiety is important to hydrogen-bond with FabH while methylcatechol ring performs π-π stacking interaction. The DPPH assay revealed that some sulfonylhydrazones derived from the methylcatechol series had antioxidant activity. None of the evaluated compounds was cytotoxic to human lung fibroblast cells, suggesting that the compounds might be considered safe at the tested concentration. CONCLUSION: Arylsufonylhydrazones is a promising scaffold to be explored for the design of new antimicrobial agents.

Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease.

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan-α-glucosaminide N-acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease-causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice-site mutation c.494-1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372-2G>A, and c.234+1G>A present in cis with c.564-98T>C and c.710C>A rare single-nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease-causing variants, and reveal the evolutionary history of MPSIIIC.

Bioinformatic Identification of Functionally and Structurally Relevant Residues and Motifs in Protein S-Acyltransferases.

DHHC palmitoyltransferases (DHHC-PATs) are very peculiar in that, outside the DHHC domain, they are very divergent even across orthologs from closely related species. This represents a challenge for the bioinformatic analyses of these proteins. Sequence-based analyses and predictions require a valid sequence alignment, which for this family of proteins requires extensive manual curation and this is difficult to attain for the nonspecialist. Here we present a simple method for the in silico analysis of the sequence of a particular PAT, that would allow for the identification of important structural features and functional residues in a PAT or PAT family.

A complete enzymatic capacity for long-chain polyunsaturated fatty acid biosynthesis is present in the Amazonian teleost tambaqui, Colossoma macropomum.

In vertebrates, the essential fatty acids (FA) that satisfy the dietary requirements for a given species depend upon its desaturation and elongation capabilities to convert the C18 polyunsaturated fatty acids (PUFA), namely linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), into the biologically active long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA), including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Recent studies have established that tambaqui (Colossoma macropomum), an important aquaculture-produced species in Brazil, is a herbivorous fish that can fulfil its essential FA requirements with dietary provision C18 PUFA LA and ALA, although the molecular mechanisms underpinning such ability remained unclear. The present study aimed at cloning and functionally characterizing genes encoding key desaturase and elongase enzymes, namely fads2, elovl5 and elovl2, involved in the LC-PUFA biosynthetic pathways in tambaqui. First, a fads2-like desaturase was isolated from tambaqui. When expressed in yeast, the tambaqui Fads2 showed Δ6, Δ5 and Δ8 desaturase capacities within the same enzyme, enabling all desaturation reactions required for ARA, EPA and DHA biosynthesis. Moreover, tambaqui possesses two elongases that are bona fide orthologs of elovl5 and elovl2. Their functional characterization confirmed that they can operate towards a variety of PUFA substrates with chain lengths ranging from 18 to 22 carbons. Overall our results provide compelling evidence that demonstrates that all the desaturase and elongase activities required to convert LA and ALA into ARA, EPA and DHA are present in tambaqui within the three genes studied herein, i.e. fads2, elovl5 and elovl2.

Structural insights into bacterial resistance to cerulenin.

UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

Specificity of transmembrane protein palmitoylation in yeast.

Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.

Induction of alternatively spliced spermidine/spermine N1-acetyltransferase mRNA in the human kidney cells infected by venezuelan equine encephalitis and tick-borne encephalitis viruses.

293 and RH cells derived from human embryo kidney were infected by Venezuelan equine encephalitis and tick-borne encephalitis viruses and cDNA libraries representing cellular mRNAs induced or suppressed due to the infection were prepared using suppressive subtractive hybridization. Among the up-regulated clones the RT-PCR and Northern analyses revealed an unusual transcript of the spermidine/spermine N1-acetyltransferase (SSAT) gene that was shown to be an alternatively spliced form containing an additional 110-bp exon. The alternatively spliced transcript is polyadenylated and can be expected to yield only a truncated 71 amino acid polypeptide. This first evidence of the host gene alternatively spliced mRNA induction by RNA viruses raises the questions of its biological role, regulation mechanisms of alternative splicing, and significance for the virus life cycle.