Assembly of F0F1-ATPase into solid state nanoporous membrane

A novel ATPase/nanoporous membrane system was prepared. In this system, the activity of F(0)F(1)-ATPase was preserved. The two sides of F(0)F(1)-ATPase were successfully separated macroscopically, and the chemical environments of the two sides could be manipulated in situ individually and freely. Furthermore, this system was also provided with mobility and reusage.     

Correlation of F0F1-ATPase inhibition and antiproliferative activity of apoptolidin analogues

[structure: see text] Apoptolidin (1) exhibits potent and highly selective apoptosis inducing activity against sensitive cancer cell lines and is hypothesized to act by inhibition of mitochondrial F(0)F(1)-ATP synthase. A series of apoptolidin derivatives, including a new intermolecular Diels-Alder adduct, were analyzed for antiproliferative activity in E1A-transformed rat fibroblasts. Potent F(0)F(1)-ATPase inhibition was not a sufficient determinant of antiproliferative activity for several analogues, suggesting the existence of a secondary biological target or more complex mode of action for apoptolidin.     

Dibutyltin-3-hydroxyflavone bromide: A fluorescent inhibitor of F1F0-ATPase

Dibutyltin-3-hydroxyflavone bromide [Bu₂SnBr-(of)] is a fluorescent inhibitor (excitation max, 395 nm; emission max., 450 nm) of mitochondrial F₁F₀–ATPase which does not inhibit F₁–ATPase. Bu₂SnBr(of) binding to mitochondria and submitochondrial particles results in a 10-fold fluorescence enhancement which correlates with the amount of F₁F₀–ATPase in the inner membrane. Enhancement is not affected by respiratory-chain substrates, ATP, uncoupling agents, ionophores or respiratory-chain inhibitors. It is reversed by tributyltin chloride (Bu₃SnCl), indicating competition for a common triorganotin-binding site on the F₀ segment of F₁F₀–ATPase. Enhancement is not reversed by dialkyltins, monoalkyltins, tributyl-lead acetate, efrapeptin or oligomycin. Bu₂SnBr(of) is thus a new class of fluorescent probe of the F₀ segment of F₁F₀–ATPase which titrates F₀.     

Identification of compounds that bind mitochondrial F1F0 ATPase by screening a triazine library for correction of albinism

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.     

Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase

BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase. SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.     

The F1F0-ATPase binding site of dibutyltin-3-hydroxyflavone: Interactions with venturicidin,oligomycin and DCCD

Dibutyltin-3-hydroxyflavone bromide, Bu₂Sn(of), is a fluorescent probe inhibitor of mitochondrial F₁F₀ATPase which reacts with and titrates a component of F₀ with marked fluorescence enhancement and reacts similarly with chloroplast CF₁CF₀ and V-ATPases. Its use to monitor the interactions of other F₀ inhibitors (venturicidin, oligomycin, DCCD) with F₁F₀ATPase, both membrane-bound and purified by solubilization is described. Trialkyltins (Bu₃SnCl) back-titrate all Bu₂Sn(of) interaction sites; whereas the macrolide inhibitor venturicidin backtitrates 60±5% and oligomycin only 30±3% of Bu₂Sn(of) interaction sites. Bafilomycin, the macrolide inhibitor of V-ATPases, is inactive in this assay. DCCD acts in a different fashion from the other inhibitors. Current and potential applications of this fluorescent probe in mitochondrial bioenergetics and biogenesis are discussed.     

Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc₁ complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc₁ activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.     

A direct interaction between the H+-F1F0-ATPase and the K+ transport within the membrane of anaerobically grown bacteria

An N,N′-dicyclohexylcarbodiimide (DCCD) sensitive 2H⁺-K⁺ exchange with fixed stoichiometry is found in anaerobically grown bacteria and requires the F₁F₀-ATPase and the Trk or similar system. These are assumed to interact with each other directly within the membrane and to form a supercomplex, functioning as an H⁺-K⁺ pump. The findings of reversal of 2H⁺-K⁺ exchange to drive coupled synthesis of ATP as well as of DCCD-sensitive ATPase activity stimulated by K⁺ have confirmed such a “supercomplex model” for the H⁺-K⁺ pump. The phenomenon is absent in bacterial grown anaerobically in the presence of nitrate or aerobically. Under these conditions these transport systems are assumed to operate separately and the Trk or similar system works as an ionophore. Conditions have been studied under which the F₁F₀-ATPase and the Trk or similar system work cooperatively as a supercomplex with local transduction of energy, or separately via energetic mediation by Δμ_H, the transmembranedifference in transport systems within the membrane without mediation by Δμ_H during low effective energetic processes (glycolysis) may be carried out by dithiol-disulphide interchange. A source of reducing equivalents and/or Δμ_H is required. Direct interaction between transport systems with formation of supercomplexes for regulation of cell metabolism could be a general feature among membrane proteins in bacteria.     

Identification of the Schistosoma mansoni molecular target for the antimalarial drug artemether

Plasmodium falciparum and Schistosoma mansonii are the parasites responsible for most of the malaria and schistosomiasis cases in the world. Notwithstanding their many differences, the two agents have striking similarities in that they both are blood feeders and are targets of an overlapping set of drugs, including the well-known artemether molecule. Here we explore the possibility of using the known information about the mode of action of artemether in Plasmodium to identify the molecular target of the drug in Schistosoma and provide evidence that artemether binds to SmSERCA, a putative Ca2?-ATPase of Schistosoma . We also predict the putative binding mode of the molecule for both its Plasmodium and Schistosoma targets. Our analysis of the mode of binding of artemether to Ca2?-ATPases also provides an explanation for the apparent paradox that, although the molecule has no side effect in humans, it has been shown to possess antitumoral activity.     

Inhibition of the mitochondrial F1-ATPase by rose bengal mediated photooxidation. Interaction of the Fe2+ chelate of bathophenanthroline with the sensitizer

Rose Bengal mediated photooxidation of mitochondrial F1-ATPase and its beta-subunit resulted in inactivation and loss of about 50 and 60% of their histidine residues, respectively. The beta-subunit was not cleaved upon photooxidation. Photooxidation of histidine probably results in changes in the conformational stability of F1-ATPase leading to its inactivation. The participation of singlet molecular oxygen during the photooxidation process is suggested by the selective loss of histidine residues, while other amino acids, also sensitive to singlet oxygen attack, were not affected. Photochemical damage of F1-ATPase was prevented by various phenanthroline compounds, the order of efficiency being bathophenanthroline-Fe chelate greater than bathophenanthroline greater than orthophenanthroline-Fe chelate greater than bathophenanthroline-sulfonate-Fe chelate. The prevention by bathophenanthroline-Fe chelate of photochemical damage is interpreted on the basis of its interaction with the photosensitizer, Rose Bengal, probably implying a chemical reaction which decreases the actual concentration of the sensitizer and, thereby, the extent of photoinactivation.     

Oxygen analogues of the benzodiazepine alkaloids sclerotigenin and circumdatin F

A new type of fused oxazepinones 9a and 9b , which are analogues of sclerotigenin and circumdatin F, were obtained in a two step synthesis from 2‐(2‐amino‐benzoylamino)‐benzoic acid or the corresponding methyl ester. Secondly a new synthesis of circumdatin F arose from this work, where 2‐(2‐propionylamino‐benzoylamino)‐benzoic acid methyl ester was used as an intermediate.     

Rotation mechanism of F1-ATPase: crucial importance of the water entropy effect

We propose a novel picture of the rotation mechanism of F(1)-ATPase, a rotary-motor protein complex. Entropy, which originates from the translational displacement of water molecules, is treated as the key factor in the proposal. We calculate the water entropy gains upon formation of the α-β, α-γ, and β-γ subunit pairs. The gain is given as the difference between the hydration entropy of a subunit pair and the sum of the hydration entropies of the separate subunits forming the pair. The calculation is made using a hybrid of a statistical-mechanical theory for molecular liquids and morphometric approach. The water entropy gain is considered as a measure of tightness of the packing at each subunit interface. The results are highly correlated with the numbers of stable contacts at the subunit interfaces estimated by a molecular dynamics simulation. We also calculate the hydration entropies of three different subcomplexes comprising the γ subunit, one of the β subunits, and two α subunits adjacent to them. The major finding is that the packing in F(1)-ATPase is highly asymmetrical, and this asymmetry is ascribed to the water entropy effect. We discuss how the rotation of the γ subunit is induced by such chemical processes as ATP binding, ATP hydrolysis, and release of the products. In our picture, the asymmetrical packing plays crucially important roles, and the rotation is driven by the water entropy effect.     

Total syntheses of the benzodiazepine alkaloids circumdatin F and circumdatin C

Total syntheses of circumdatin F and circumdatin C, which both possess a 3H-quinazolin-4-one as well as a 1,4-benzodiazepin-5-one moiety, are described. A tripeptide derivative was synthesized as a key intermediate and dehydrated to a benzoxazine by reaction with triphenylphosphine, iodine, and a tertiary amine. The natural products were attained via rearrangements to an amidine intermediate, deprotection with 45% HBr in acetic acid, and cyclization on silica gel.     

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.     

Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors

We report the construction of a novel biosensing nanodevice to detect single, sequence-specific target DNA molecules. Nanodevice assembly occurs through the association of an immobilized F1-ATPase molecular motor and a functionalized gold nanorod via a single 3',5'-dibiotinylated DNA molecule. Target-dependent 3',5'-dibiotinylated DNA bridges form by combining ligation and exonucleation reactions (LXR), with a specificity capable of selecting against a single nucleotide polymorphism (SNP). Using dark field microscopy to detect gold nanorods, quantitation of assembled nanodevices is sufficient to distinguish the presence of as few as 1800 DNA bridges from nonspecifically bound nanorods. The rotary mechanism of F1-ATPase can drive gold nanorod rotation when the nanorod is attached via the DNA bridge. Therefore, rotation discriminates fully assembled devices from nonspecifically bound nanorods, resulting in a sensitivity limit of one zeptomole (600 molecules).     

Thermally responsive supramolecular nanomeshes for on/off switching of the rotary motion of F1-ATPase at the single-molecule level

The artificial regulation of protein functions is essential for the realization of protein-based soft devices, because of their unique functions conducted within a nano-sized molecular space. We report that self-assembled nanomeshes comprising heat-responsive supramolecular hydrogel fibers can control the rotary motion of an enzyme-based biomotor (F(1)-ATPase) in an on/off manner at the single-molecule level. Direct observation of the interaction of the supramolecular fibers with a microbead unit tethered to the F(1)-ATPase and the clear threshold in the size of the bead required to stop ATPase rotation indicates that the bead was physically blocked so as to stop the rotary motion of ATPase. The temperature-induced formation and collapse of the supramolecular nanomesh can produce or destroy, respectively, the physical obstacle for ATPase so as to control the ATPase motion in an off/on manner. Furthermore, this switching of the F(1)-ATPase motion could be spatially restricted by using a microheating device. The integration of biomolecules and hard materials, interfaced with intelligent soft materials such as supramolecular hydrogels, is promising for the development of novel semi-synthetic nano-biodevices.     

Reactions at the interface of ultrathin Fe films deposited on ZnO(0 0 0 1) and ZnO(0 0 0−1) single crystal substrates

In this work we focus on the chemical interactions at the interface formed during the room temperature deposition of thin Fe films on polar ZnO surfaces via electron beam evaporation. The Fe is found to grow in layers and to form polycrystalline, disordered films. At the interface a considerable reaction takes place. By X-ray photoelectron spectroscopy, the formation of Fe²⁺ and metallic zinc species is detected.     

Identification of a NBD1-binding pharmacological chaperone that corrects the trafficking defect of F508del-CFTR

Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.