The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

The role of Ca²âº in the activity of Mycobacterium tuberculosis DNA gyrase.

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.

An integrated approach for identification and target validation of antifungal compounds active against Erg11p.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives.

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.

RNA interference silencing the transcriptional message: aspects and applications.

RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomenon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.

Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

BACKGROUND: Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A2B2 complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations. FINDINGS: We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity. CONCLUSION: We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).

Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino.

Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability. They have become a general and versatile tool for DNA and RNA recognition. LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes. LNA also has therapeutic and diagnostic applications. PNA is another type of DNA analog with neutral charge. The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application. PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies. It was also used to design transcription factor decoy molecules for target gene induction. Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs. It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection. Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry.