Small-Molecule Intervention At The Dimerization Interface Of Survivin By Novel Rigidized Scaffolds.

INTRODUCTION: Survivin is a nodal protein involved in several cellular pathways. It is a member of the IAP family and an integral component of the chromosomal passenger complex, where it binds to borealin and INCENP through its dimerization interface. By targeting survivin with a small molecule at its dimerization interface, inhibition of the proliferation of cancer cells has been suggested. With Abbott 8, a small-molecule dimerization inhibitor has been recently reported. The structure-activity relationship of this series of inhibitors implied that the middle pyridin-2(1H)-one ring did not tolerate modifications of any kind. METHODS: Based on the synthetic strategy of Abbott 8 using multicomponent reactions, we synthesized a series of small molecules bearing a novel rigidized core scaffold. This rigidization strategy was accomplished by integrating the pyridin-2(1H)-one and its 6-phenyl substituent into a tricyclic structure, linking position 5 of pyridin-2(1H)-one to the phenyl substituent by rings of different sizes. The new scaffolds were designed based on in silico molecular dynamics of survivin. RESULTS: Binding of these rigidized scaffolds to the recombinant L54M mutant of survivin was evaluated, revealing affinities in the low micromolar range. CONCLUSION: This easily accessible, new class of survivin-dimerization modulators is an interesting starting point for further lead optimization.

Targeting the Gatekeeper MET146 of C-Jun N-Terminal Kinase 3 Induces a Bivalent Halogen/Chalcogen Bond.

We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of Cε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%).

Adenylate-forming enzymes of rubradirin biosynthesis: RubC1 is a bifunctional enzyme with aminocoumarin acyl ligase and tyrosine-activating domains.

The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis.

Identification of a napsamycin biosynthesis gene cluster by genome mining.

Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N-methyl diaminobutyric acid, an ureido group, a methionine and two non-proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5'-amino-3'-deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non-ribosomal peptide synthetase (NRPS)-like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N-methyl diaminobutyric acid, to the generation of m-tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC-ESI-MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.

Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements.

IS256 is the founding member of the IS256 family of insertion sequence (IS) elements. These elements encode a poorly characterized transposase, which features a conserved DDE catalytic motif and produces circular IS intermediates. Here, we characterized the IS256 transposase as a DNA-binding protein and obtained insight into the subdomain organization and functional properties of this prototype enzyme of IS256 family transposases. Recombinant forms of the transposase were shown to bind specifically to inverted repeats present in the IS256 noncoding regions. A DNA-binding domain was identified in the N-terminal part of the transposase, and a mutagenesis study targeting conserved amino acid residues in this region revealed a putative helix-turn-helix structure as a key element involved in DNA binding. Furthermore, we obtained evidence to suggest that the terminal nucleotides of IS256 are critically involved in IS circularization. Although small deletions at both ends reduced the formation of IS circles, changes at the left-hand IS256 terminus proved to be significantly more detrimental to circle production. Taken together, the data lead us to suggest that the IS256 transposase-mediated circularization reaction preferentially starts with a sequence-specific first-strand cleavage at the left-hand IS terminus.

Success through diversity - how Staphylococcus epidermidis establishes as a nosocomial pathogen.

Staphylococcus epidermidis normally is a commensal inhabitant of the healthy human skin and mucosa, but also a common nosocomial pathogen in immunocompromised patients. Living at the edge between commensalism and pathogenicity, S. epidermidis has developed interesting strategies to conquer the hospital environment as a novel ecological niche and to transform into a notorious pathogen. Recent progress in genome analysis and molecular epidemiology gave interesting insights into the enormous flexibility by which these bacteria generate continuously novel phenotypic and genotypic variants. Recent multilocus sequence typing studies identified S. epidermidis as a highly diverse species that evolves mainly by recombination and acquires readily mobile genetic elements. With respect to healthcare-associated isolates, a limited number of epidemic clonal lineages were found to have emerged and established in hospital settings worldwide. These isolates are characterised by the carriage of various SCCmec gene cassettes, conferring methicillin resistance, and by a striking ability to form biofilms on medical devices. Moreover, nosocomial S. epidermidis strains typically harbour multiple copies of the insertion sequence element IS256 in their genomes. Nosocomial S. epidermidis strains vary virulence- and resistance-associated gene expression in the course of an infection to a remarkably high degree. Heterogenous gene expression in S. epidermidis is achieved, on the one hand, by complex regulatory pathways. On the other hand, it is associated with genetic mechanisms that were found to be mediated by the action of the IS256 element which obviously represents an important driving force for the flexibility of the S. epidermidis genome. The data accumulated so far suggest that recombination along with the frequent acquisition of mobile genetic elements are crucial factors for the success of S. epidermidis as a nosocomial pathogen.

A transposase-independent mechanism gives rise to precise excision of IS256 from insertion sites in Staphylococcus epidermidis.

The mobile element IS256 causes phase variation of biofilm formation in Staphylococcus epidermidis by insertion and precise excision from the icaADBC operon. Precise excision, i.e., removal of the target site duplications (TSDs) and restoration of the original DNA sequence, occurs rarely but independently of functional transposase. Instead, the integrity of the TSDs is crucial for precise excision. Excision increased significantly when the TSDs were brought into closer spatial proximity, suggesting that excision is a host-driven process that might involve most likely illegitimate recombination.

Improved mutasynthetic approaches for the production of modified aminocoumarin antibiotics.

This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1H-NMR spectroscopy.

Spontaneous switch to PIA-independent biofilm formation in an ica-positive Staphylococcus epidermidis isolate.

The ability to form biofilms on abiotic surfaces is considered a major step in Staphylococcus epidermidis pathogenesis. In the majority of isolates, biofilm formation is mediated by the production of the polysaccharide intercellular adhesin PIA which is synthesized by enzymes encoded by the ica operon. Here, we report on a spontaneous switch to proteinaceous biofilm formation in an S. epidermidis icaC::IS256 insertion mutant. Atomic force microscopy analysis of both PIA-dependent and proteinaceous biofilm revealed remarkable differences in biofilm substructures: the PIA-dependent biofilm was characterized by the presence of fibrous, net-like structures which were absent in proteinaceous biofilm. Transcription of aap, encoding the accumulation-associated protein Aap, was enhanced in a variant producing proteinaceous biofilm, while transcription of the Bap-homologous protein gene bhp was down-regulated. Regulation of PIA-independent biofilm differed from the wild type. Thus, ethanol induced proteinaceous biofilm formation, whereas NaCl abolished PIA-independent biofilm formation completely. The combined data indicate that biofilm formation in S. epidermidis is obviously ensured by more than one mechanism suggesting that this life style represents a crucial factor for this organism.

Nosocomial infections by Staphylococcus epidermidis: how a commensal bacterium turns into a pathogen.

Staphylococcus epidermidis is a commensal bacterium of the human skin. However, S. epidermidis and other coagulase-negative staphylococci (CNS) emerge also as common nosocomial pathogens infecting immunocompromized patients carrying medical devices. Antibiotic resistance and the ability of many nosocomial S. epidermidis isolates to form biofilms on inert surfaces make these infections hard to treat. Epidemiological analyses using multilocus sequence typing (MLST) and genetic studies suggest that S. epidermidis isolates in the hospital environment differ from those obtained outside of medical facilities with respect to biofilm formation, antibiotic resistance, and the presence of mobile DNA elements. Since S. epidermidis isolates exhibit high genome flexibility, they are now regarded as reservoirs for the evolution and spread of resistance traits within nosocomial bacterial communities.

Transcriptional organization and posttranscriptional regulation of the Bacillus subtilis branched-chain amino acid biosynthesis genes.

In Bacillus subtilis, the genes of the branched-chain amino acids biosynthetic pathway are organized in three genetic loci: the ilvBHC-leuABCD (ilv-leu) operon, ilvA, and ilvD. These genes, as well as ybgE, encoding a branched-chain amino acid aminotransferase, were recently demonstrated to represent direct targets of the global transcriptional regulator CodY. In the present study, the transcriptional organization and posttranscriptional regulation of these genes were analyzed. Whereas ybgE and ilvD are transcribed monocistronically, the ilvA gene forms a bicistronic operon with the downstream located ypmP gene, encoding a protein of unknown function. The ypmP gene is also directly preceded by a promoter sharing the regulatory pattern of the ilvA promoter. The ilv-leu operon revealed complex posttranscriptional regulation: three mRNA species of 8.5, 5.8, and 1.2 kb were detected. Among them, the 8.5-kb full-length primary transcript exhibits the shortest half-life (1.2 min). Endoribonucleolytic cleavage of this transcript generates the 5.8-kb mRNA, which lacks the coding sequences of the first two genes of the operon and is predicted to carry a stem-loop structure at its 5' end. This processing product has a significantly longer half-life (3 min) than the full-length precursor. The most stable transcript (half-life, 7.6 min) is the 1.2-kb mRNA generated by the processing event and exonucleolytic degradation of the large transcripts or partial transcriptional termination. This mRNA, which encompasses exclusively the ilvC coding sequence, is predicted to carry a further stable stem-loop structure at its 3' end. The very different steady-state amounts of mRNA resulting from their different stabilities are also reflected at the protein level: proteome studies revealed that the cellular amount of IlvC protein is 10-fold greater than that of the other proteins encoded by the ilv-leu operon. Therefore, differential segmental stability resulting from mRNA processing ensures the fine-tuning of the expression of the individual genes of the operon.

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489.

The biosynthetic gene clusters of the gyrase inhibitors coumermycin A(1) and clorobiocin contain two different resistance genes (gyrB(R) and parY(R)). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and GyrB(R) was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and ParY(R) catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.