Assay development for identifying inhibitors of the mycobacterial FadD32 activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the gamma-phosphate of ATP to propionate during L-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate approximate to butyrate), nucleotides (ATP approximate to GTP > CTP approximate to TTP; dATP > dGTP > dCTP) and metal ions (Mg2+ approximate to Mn2+ > Co2+). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, alpha-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the gamma-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer. (C) 2013 Elsevier B.V. All rights reserved.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Metabolic efficiency with fast spiking in the squid axon

Fundamentally, action potentials in the squid axon are consequence of the entrance of sodium ions during the depolarization of the rising phase of the spike mediated by the outflow of potassium ions during the hyperpolarization of the falling phase. Perfect metabolic efficiency with a minimum charge needed for the change in voltage during the action potential would confine sodium entry to the rising phase and potassium efflux to the falling phase. However, because sodium channels remain open to a significant extent during the falling phase, a certain overlap of inward and outward currents is observed. In this work we investigate the impact of ion overlap on the number of the adenosine triphosphate (ATP) molecules and energy cost required per action potential as a function of the temperature in a Hodgkin–Huxley model. Based on a recent approach to computing the energy cost of neuronal action potential generation not based on ion counting, we show that increased firing frequencies induced by higher temperatures imply more efficient use of sodium entry, and then a decrease in the metabolic energy cost required to restore the concentration gradients after an action potential. Also, we determine values of sodium conductance at which the hydrolysis efficiency presents a clear minimum.

Energy and information in Hodgkin-Huxley neurons

[EN]The generation of spikes by neurons is energetically a costly process and the evaluation of the metabolic energy required to maintain the signaling activity of neurons a challenge of practical interest. Neuron models are frequently used to represent the dynamics of real neurons but hardly ever to evaluate the electrochemical energy required to maintain that dynamics. This paper discusses the interpretation of a Hodgkin-Huxley circuit as an energy model for real biological neurons and uses it to evaluate the consumption of metabolic energy in the transmission of information between neurons coupled by electrical synapses, i.e., gap junctions. We show that for a single postsynaptic neuron maximum energy efficiency, measured in bits of mutual information per molecule of adenosine triphosphate (ATP) consumed, requires maximum energy consumption. For groups of parallel postsynaptic neurons we determine values of the synaptic conductance at which the energy efficiency of the transmission presents clear maxima at relatively very low values of metabolic energy consumption. Contrary to what could be expected, the best performance occurs at a low energy cost.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

Combining rimonabant and fentanyl in a single entity: preparation and pharmacological results

Based on numerous pharmacological studies that have revealed an interaction between cannabinoid and opioid systems at the molecular, neurochemical, and behavioral levels, a new series of hybrid molecules has been prepared by coupling the molecular features of two well-known drugs, ie, rimonabant and fentanyl. The new compounds have been tested for their affinity and functionality regarding CB1 and CB2 cannabinoid and mu opioid receptors. In [S-35]-GTP.S (guanosine 5'-O-[gamma-thio] triphosphate) binding assays from the post-mortem human frontal cortex, they proved to be CB1 cannabinoid antagonists and mu opioid antagonists. Interestingly, in vivo, the new compounds exhibited a significant dual antagonist action on the endocannabinoid and opioid systems.

Adaptive evolution of energy metabolism genes and the origin of flight in bats

Bat flight poses intriguing questions about how flight independently developed in mammals. Flight is among the most energy-consuming activities. Thus, we deduced that changes in energy metabolism must be a primary factor in the origin of flight in bats. The respiratory chain of the mitochondrial produces 95% of the adenosine triphosphate (ATP) needed for locomotion. Because the respiratory chain has a dual genetic foundation, with genes encoded by both the mitochondrial and nuclear genomes, we examined both genomes to gain insights into the evolution of flight within mammals. Evidence for positive selection was detected in 23.08% of the mitochondrial-encoded and 4.90% of nuclear-encoded oxidative phosphorylation (OXPHOS) genes, but in only 2.25% of the nuclear-encoded nonrespiratory genes that function in mitochondria or 1.005% of other nuclear genes in bats. To address the caveat that the two available bat genomes are of only draft quality, we resequenced 77 OXPHOS genes from four species of bats. The analysis of the resequenced gene data are in agreement with our conclusion that a significantly higher proportion of genes involved in energy metabolism, compared with background genes, show evidence of adaptive evolution specific on the common ancestral bat lineage. Both mitochondrial and nuclear-encoded OXPHOS genes display evidence of adaptive evolution along the common ancestral branch of bats, supporting our hypothesis that genes involved in energy metabolism were targets of natural selection and allowed adaptation to the huge change in energy demand that were required during the origin of flight.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Enzyme Colorimetric Assay Using Unmodified Silver Nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CLAP) and peptide phosphorylation by protein kinase A (PKA) were studied.

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A An electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA).

Au nanoparticles grafted sandwich platform used amplified small molecule electrochemical aptasensor

We report a sensitively amplified electrochemical aptasensor using adenosine triphosphate (ATP) as a model. ATP is a multifunctional nucleotide thatis most important as a "molecular currency" of intracellular energy transfer. In the sensing process, duplexes consisting of partly complementary strand (PCS1), ATP aptamer (ABA) and another partly complementary strand (PCS2) were immobilized onto Au electrode through the 5'-HS on the PCS1. Meanwhile, PCS2 was grafted with the Au nanoparticles (AuNPs) to amplify the detection signals. In the absence of ATP, probe methylene blue (MB) bound to the DNA duplexes and also bound to guanine bases specifically to produce a strong differential pulse voltammetry (DPV) signal. But when ATP exists, the ABA-PCS2 or ABA-PCS1 part duplexes might be destroyed, which decreased the amount of MB on the electrode and led to obviously decreased DPV signal.

Probe beam deflection study on electrochemically controlled release of 5-fluorouracil

In this paper, the polypyrrole (PPy) film modified electrodes are used as an electroreleasing reservoir. The electrochemically controlled release of 5-fluorouracil (5-FU) from a PPy film modified electrode to aqueous electrolytes is studied by the in situ probe beam deflection (PBD) method combined with cyclic voltammetry (CV) and chronoamperometry (CA). The PBD results reveal that the release of 5-FU from PPy film depends on the electrochemical redox process of the PPy film electrode. The released amount is controlled by the reduction potential and is proportional to the thickness of the film. The exchange of 5-FU anions with Cl- on an open circuit is slow on the time scale of minutes, but the release of 5-FU anions can proceed quickly at -0.6 V (vs Ag/AgCl). The amount of released 5-FU decreases with the time that the PPy film is soaked in aqueous solution. (C) 1998 Elsevier Science Ltd. All rights reserved.

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase

We examined the effects of cofactors and DNA on the stability, oligomeric state and conformation of the human mitochondrial DNA helicase. We demonstrate that low salt conditions result in protein aggregation that may cause dissociation of oligomeric structure. The low salt sensitivity of the mitochondrial DNA helicase is mitigated by the presence of magnesium, nucleotide, and increased temperature. Electron microscopic and glutaraldehyde cross-linking analyses provide the first evidence of a heptameric oligomer and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleoside diphosphate. We find that single-stranded DNA binding occurs in the absence of cofactors and renders the mitochondrial DNA helicase more susceptible to proteolytic digestion. Our studies indicate that the human mitochondrial DNA helicase shares basic properties with the SF4 replicative helicases, but also identify common features with helicases outside the superfamily, including dynamic conformations similar to other AAA⁺ ATPases.