Exploring ND-011992, a quinazoline-type inhibitor targeting quinone reductases and quinol oxidases.

Bacterial energy metabolism has become a promising target for next-generation tuberculosis chemotherapy. One strategy to hamper ATP production is to inhibit the respiratory oxidases. The respiratory chain of Mycobacterium tuberculosis comprises a cytochrome bcc:aa3 and a cytochrome bd ubiquinol oxidase that require a combined approach to block their activity. A quinazoline-type compound called ND-011992 has previously been reported to ineffectively inhibit bd oxidases, but to act bactericidal in combination with inhibitors of cytochrome bcc:aa3 oxidase. Due to the structural similarity of ND-011992 to quinazoline-type inhibitors of respiratory complex I, we suspected that this compound is also capable of blocking other respiratory chain complexes. Here, we synthesized ND-011992 and a bromine derivative to study their effect on the respiratory chain complexes of Escherichia coli. And indeed, ND-011992 was found to inhibit respiratory complex I and bo3 oxidase in addition to bd-I and bd-II oxidases. The IC50 values are all in the low micromolar range, with inhibition of complex I providing the lowest value with an IC50 of 0.12 µM. Thus, ND-011992 acts on both, quinone reductases and quinol oxidases and could be very well suited to regulate the activity of the entire respiratory chain.

Identification of novel anti-cancer agents by the synthesis and cellular screening of a noscapine-based library.

Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine.

Concise Synthesis of 1,4-Benzoquinone-Based Natural Products as Mitochondrial Complex I Substrates and Substrate-Based Inhibitors.

A short, efficient one-step synthesis of 2-methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural product from Pyrola media is described. The synthesis is based on a direct late C-H functionalization of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1 i), an inhibitor with an IC50 of 5 µM against MCI. The identified molecule showed high selectivity for MCI when tested against other quinone-converting enzymes, including succinate dehydrogenase, and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified inhibitor was also active in cell-based proliferation assays. Therefore, 1 i can be considered as a novel chemical probe for MCI.

Synthesis and Biological Screening of New Lawson Derivatives as Selective Substrate-Based Inhibitors of Cytochrome bo3 Ubiquinol Oxidase from Escherichia coli.

The respiratory chain of Escherichia coli contains two different types of terminal oxidase that are differentially regulated as a response to changing environmental conditions. These oxidoreductases catalyze the reduction of molecular oxygen to water and contribute to the proton motive force. The cytochrome bo3 oxidase (cyt bo3 ) acts as the primary terminal oxidase under atmospheric oxygen levels, whereas the bd-type oxidase is most abundant under microaerobic conditions. In E. coli, both types of respiratory terminal oxidase (HCO and bd-type) use ubiquinol-8 as electron donor. Here, we assess the inhibitory potential of newly designed and synthesized 3-alkylated Lawson derivatives through L-proline-catalyzed three-component reductive alkylation (TCRA). The inhibitory effects of these Lawson derivatives on the terminal oxidases of E. coli (cyt bo3 and cyt bd-I) were tested potentiometrically. Four compounds were able to reduce the oxidoreductase activity of cyt bo3 by more than 50 % without affecting the cyt bd-I activity. Moreover, two inhibitors for both cyt bo3 and cyt bd-I oxidase could be identified. Based on molecular-docking simulations, we propose binding modes of the new Lawson inhibitors. The molecular fragment benzyl enhances the inhibitory potential and selectivity for cyt bo3 , whereas heterocycles reduce this effect. This work extends the library of 3-alkylated Lawson derivatives as selective inhibitors for respiratory oxidases and provides molecular probes for detailed investigations of the mechanisms of respiratory-chain enzymes of E. coli.

Polymerase assays for lead discovery: An overall review of methodologies and approaches.

Polymerases represent an attractive molecular target for antibacterial drug development, antiviral intervention and cancer therapy. Over the past decade, academic groups and scientists from pharmaceutical industry have developed a large plethora of different functional assays to monitor the enzymatic reaction catalyzed by polymerases. These assays were used to enable high-throughput screening (HTS) for lead discovery purposes, as well as hit-to-lead (H2L) drug profiling activities. In both cases the choice of the assay technology is critical and to the best of our knowledge, there is no review available to help scientists to choose the most suitable assay. This review summarizes the most common functional assays developed to monitor the enzymatic activity of polymerases and discusses the advantages and disadvantages of each assay. Assays are presented and evaluated in term of cost, ease of use, high-throughput screening compatibility and liability towards delivering false positives and false negatives.

Identification of 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine as a novel, highly potent and specific inhibitor of mitochondrial complex I.

By probing the quinone substrate binding site of mitochondrial complex I with a focused set of quinazoline-based compounds, we identified substitution patterns as being critical for the observed inhibition. The structure activity relationship study also resulted in the discovery of the quinazoline 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (EVP4593) as a highly potent inhibitor of the multisubunit membrane protein. EVP4593 specifically and effectively reduces the mitochondrial complex I-dependent respiration with no effect on the respiratory chain complexes II-IV. Similar to established Q-site inhibitors, EVP4593 elicits the release of reactive oxygen species at the flavin site of mitochondrial complex I. Recently, EVP4593 was nominated as a lead compound for the treatment of Huntingtons disease. Our results challenge the postulated primary mode-of-action of EVP4593 as an inhibitor of NF-κB pathway activation and/or store-operated calcium influx.

PAIN-less identification and evaluation of small molecule inhibitors against protein tyrosine phosphatase 1B.

A highly miniaturized biochemical assay was set up to test a focused set of natural products against the enzymatic activity of protein tyrosine phosphatase 1B (PTP1B). The screen resulted in the identification of the natural product alkaloids, berberine and palmatine as well as α-tocopheryl succinate (α-TOS) as potential inhibitors of PTP1B. In a second step, several read-out and counter assays were applied to confirm the observed inhibitory activity of the identified hits and to remove false positives which target the enzymatic activity of PTP1B by a non-specific mechanism, also known as PAINS (pan-assay interference compounds). Both, berberine and palmatine were identified as false positives which interfered with the assay read-out. Using NMR spectroscopy, self-association via stacking interactions was detected for berberine in aqueous media, which may also contribute to the non-specific inhibition of PTP1B. α-TOS was confirmed as a novel reversible and competitive inhibitor of PTP1B. A concise structure-activity relationship study identified the carboxyl group and the saturated phytyl-side chain as being critical for PTP1B inhibition.

A fluorescence polarization based assay for the identification and characterization of polymerase inhibitors.

A homogenous fluorescence polarization (FP) assay was developed to monitor the enzymatic activity of polymerases. Under the optimized conditions established in this study, the assay provides highly robust and reproducible data. Miniaturization of the assay for high-throughput screening and compound testing was also performed. The sensitivity of the newly developed assay was confirmed using 2',3'-dideoxyadenosine-5'-triphosphate (ddATP), a chain-elongating inhibitor of the polymerase reaction. Side-by-side comparison of the presented fluorescence polarization assay with already well established PicoGreen® fluorescence intensity assay revealed that the performance of both formats is comparable with good assay sensitivity. However, the direct ratiometric readout of the presented FP assay makes it superior over existing colorimetric and fluorescence intensity based assays in terms of susceptibility to false positives. Moreover, due to its generic nature the presented FP assay can be applied to other polymerases and is compatible with identification of inhibitors and requirements of hit-to-lead programs.

Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases.

Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections.

Thermodynamic profiling of inhibitors of Nrf2:Keap1 interactions.

Keap1 binds to the transcription factor Nrf2 and negatively modulates the expression of genes involved in cellular protection against oxidative stress. Small molecules have been discovered to inhibit the Nrf2:Keap1 interactions and act as antagonists of Keap1. The affinities of these small molecules are not very high and need further improvement in follow up hit-to-lead programs. In addition to the affinity parameters Ki, Kd, and IC50 thermodynamic parameters provide useful information for the selection and optimization of these hit molecules at the early stage of the lead discovery process. In this letter a tracer displacement assay was used to determine the thermodynamic signature of some of the known inhibitors of the Nrf2:Keap1 interaction. An optimized assay protocol is presented, which can be applied to other small molecules in hit-to-lead programs in a medium throughput manner.

Targeting a c-MYC G-quadruplex DNA with a fragment library.

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid.

Tracing the tail of ubiquinone in mitochondrial complex I.

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron-sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Production, characterization and determination of the real catalytic properties of the putative 'succinate dehydrogenase' from Wolinella succinogenes.

Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as 'non-classical' succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86-->His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions.

Interplay of 'induced fit' and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch.

Riboswitches are highly structured elements in the 5'-untranslated regions (5'-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Experimental evidence for proton motive force-dependent catalysis by the diheme-containing succinate:menaquinone oxidoreductase from the Gram-positive bacterium Bacillus licheniformis.

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Deltap, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Deltap dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Deltap-dependent catalysis of succinate oxidation by quinone as well as for Deltap generation upon catalysis of fumarate reduction by quinol.

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex.

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic epsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions.

An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs.

Riboswitches are highly structured RNA elements that control the expression of many bacterial genes by binding directly to small metabolite molecules with high specificity and affinity. In Bacillus subtilis, two classes of riboswitches have been described that discriminate between guanine and adenine despite an extremely high degree of homology both in their primary and secondary structure. We have identified intermolecular base triples between both purine ligands and their respective riboswitch RNAs by NMR spectroscopy. Here, specificity is mediated by the formation of a Watson-Crick base pair between the guanine ligand and a C residue or the adenine ligand and a U residue of the cognate riboswitch RNA, respectively. In addition, a second base-pairing interaction common to both riboswitch purine complexes involves a uridine residue of the RNA and the N3/N9 edge of the purine ligands. This base pairing is mediated by a previously undescribed hydrogen-bonding scheme that contributes to the affinity of the RNA-ligand interaction. The observed intermolecular hydrogen bonds between the purine ligands and the RNA rationalize the previously observed change in specificity upon a C to U mutation in the core of the purine riboswitch RNAs and the differences in the binding affinities for a number of purine analogs.