Synthesis of Novel Potent Archazolids: Pharmacology of an Emerging Class of Anticancer Drugs.

Vacuolar type ATPase (V-ATPase) has recently emerged as a promising novel anticancer target based on extensive and studies with archazolids, complex polyketide macrolides, which present the most potent V-ATPase inhibitors known to date. Herein, we report a biomimetic, one-step preparation of archazolid F, the most potent and least abundant archazolid, the design and synthesis of five novel, carefully selected archazolid analogues, and the biological evaluation of these antiproliferative agents, leading to the discovery of a very potent but profoundly simplified archazolid analogue. Furthermore, the first general biological profiling of the archazolids against a broad range of more than 100 therapeutically relevant targets is reported, leading to the discovery of novel and important targets.

Interaction of mammalian and plant H/sucrose transporters with 14-3-3 proteins.

The (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1-A4, which show only 20-30% identity of amino acid sequences among each other. All these members exhibit an identity of ∼20% to plant H/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H/sucrose cotransporters.

Putative role of an SLC45 H/sugar cotransporter in mammalian spermatozoa.

In the present study, we describe the detection and analysis of a novel type of sugar transporter in mammalian spermatozoa. This transporter belongs to the SLC45 family for which two features are remarkable and distinguish it from other known families of sugar transporters. Firstly, SLC45 transporters recognise not only the monosaccharides glucose or fructose but also the disaccharide sucrose as a substrate.

Putative role of the H(+)/sucrose symporter SLC45A3 as an osmolyte transporter in the kidney.

The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown.

EPR Studies of V-ATPase with Spin-Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit c.

Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins.

The biological basis for poly-L-lactic acid-induced augmentation.

Background: Granulomatous reactions to poly-L-lactic acid (PLLA)-based filler have been described previously. Neither the biological background of these partly late-onset reactions or the desired augmenting effect of PLLA has been studied to date. Histological studies have revealed foreign body reactions and foreign body giant cell formation.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters.

Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions.

Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions.

Flexibility within the rotor and stators of the vacuolar H+-ATPase.

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°.

Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood.

The SLC45 gene family of putative sugar transporters.

According to the classic point of view, transport of sugars across animal plasma membranes is performed by two families of transporters. Secondary active transport occurs via Na(+) symporters of the SLC5 gene family, while passive transport occurs via facilitative transporters of the SLC2 family. In recent years a new family appeared in the scenery which was called the SLC45 gene family of putative sugar transporters, mainly because of obvious similarities to plant sucrose transporters.

Protein kinase A-dependent and -independent activation of the V-ATPase in Malpighian tubules of Aedes aegypti.

Transepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes.

Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid.

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.

The binding site of the V-ATPase inhibitor apicularen is in the vicinity of those for bafilomycin and archazolid.

The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex.

Understanding the inhibitory effect of highly potent and selective archazolides binding to the vacuolar ATPase.

Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the subnanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular docking, molecular dynamics simulations, and biochemical experiments, resulting in a sound model for the inhibitory mechanism of this class of putative anticancer agents.

Vacuolar H(+)-ATPases: intra- and intermolecular interactions.

V-ATPases in eukaryotes are heteromultimeric, H(+)-transporting proteins. They are localized in a multitude of different membranes and energize many different transport processes. Unique features of V-ATPases are, on the one hand, their ability to regulate enzymatic and ion transporting activity by the reversible dissociation of the catalytic V(1) complex from the membrane bound proton translocating V(0) complex and, on the other hand, their high sensitivity to specific macrolides such as bafilomycin and concanamycin from streptomycetes or archazolid and apicularen from myxomycetes.

Identification of an animal sucrose transporter.

According to a classic tenet, sugar transport across animal membranes is restricted to monosaccharides. Here, we present the first report of an animal sucrose transporter, SCRT, which we detected in Drosophila melanogaster at each developmental stage. We localized the protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells.

Archazolid A-15-O-β-D-glucopyranoside and iso-archazolid B: potent V-ATPase inhibitory polyketides from the myxobacteria Cystobacter violaceus and Archangium gephyra.

Two structurally novel analogues of the macrolides archazolids A and B, archazolid A-15-O-β-D-glucopyranoside (archazolid E, 5) and iso-archazolid B (archazolid F, 6), were isolated from the myxobacterium Cystobacter violaceus and Archangium gephyra, respectively. Macrolactone 5 represents the first 15-O-glycoside of the archazolids. iso-Archazolid B (6) incorporates a C-3 alkene and presents the first constitutional isomer reported for this natural product class.

Archazolid A binds to the equatorial region of the c-ring of the vacuolar H+-ATPase.

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC(50) value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral V(O) complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V(1)V(O) holoenzyme we confirmed binding of archazolid to the V(O) subunit c.

Vacuolar-type proton pumps in insect epithelia.

Active transepithelial cation transport in insects was initially discovered in Malpighian tubules, and was subsequently also found in other epithelia such as salivary glands, labial glands, midgut and sensory sensilla. Today it appears to be established that the cation pump is a two-component system of a H(+)-transporting V-ATPase and a cation/nH(+) antiporter. After tracing the discovery of the V-ATPase as the energizer of K(+)/nH(+) antiport in the larval midgut of the tobacco hornworm Manduca sexta we show that research on the tobacco hornworm V-ATPase delivered important findings that emerged to be of general significance for our knowledge of V-ATPases, which are ubiquitous and highly conserved proton pumps.

Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven rotary molecular motor that is a transmembrane proton pump in all eukaryotic cells. Although its activity is fundamental to many physiological processes, our understanding of the structure and mechanism of the V-ATPase is poor. Using cryo-electron microscopy of the tobacco hornworm (Manduca sexta) enzyme, we have calculated the first 3D reconstruction of the intact pump in its native state.

Inhibitors of V-ATPases: old and new players.

V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic endomembranes and plasma membranes, they energize many different transport processes. Currently, a handful of specific inhibitors of the V-ATPase are known, which represent valuable tools for the characterization of transport processes on the level of tissues, single cells or even purified proteins.

Influence of ATP and ADP on dissociation of the V-ATPase into its V(1) and V(O) complexes.

Although the reversible dissociation of the V(1)V(O) holoenzyme into its V(1) and V(O) complexes is a general mechanism for the regulation of V-ATPases, important aspects are still not understood. By analyzing the endogenous nucleotide content of the V(1)V(O) holoenzyme and of the V(1) complex, both purified from Manduca sexta larval midgut, we found that the V(1) complex contained 1.7 molec.