Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC 50 Vacuolar ATPases (V-ATPases)3 are heteromultimeric proteins that use the energy of ATP hydrolysis to translocate protons from the cytoplasm into intracellular compartments or across the plasma membrane of eukaryotic cells. This transport of protons is mediated by the membrane-integral V O complex, whereas the cleavage of ATP occurs at the cytoplasmatic V 1 complex (1). The V O complex is composed of single copies of subunits a, d, and e, and the ring-forming proteolipid subunits c, cЉ, and in fungi subunit cЈ also (2). Based on the crystal structure from the V O ring of K subunits, a homologue of the H ϩ -translocating subunit c in the V-type Na ϩ -ATPase from Enterococcus hirae, and a cryoelectron microscopy structure from the V-ATPase of Manduca sexta, an arrangement of 10 subunits is proposed for the V O ring (3, 4). The subunits c and cЈ are predicted to have four transmembrane helices (TM 1 to 4), whereas subunit cЉ contains an additional fifth transmembrane helix. All proteolipid subunits contain a conserved glutamate residue, subunits c and cЈ in TM4 and subunit cЉ in TM3, which are essential for proton transport across the membrane (2). This glutamate is a target for the covalent binding inhibitor N,NЈ-dicyclohexylcarbodiimide (DCCD) and its derivatives (5-8).By regulating the pH homeostasis and membrane energization of cells, V-ATPases are involved in a variety of fundamental processes like vesicular trafficking or secondary transport. In addition, plasma membrane V-ATPases are responsible for extracellular acidification, e.g. in osteoclasts or metastasing tumor cells, and therefore play an important role in severe diseases such as osteoporosis or cancer (7). For these reasons the V-ATPase is a promising therapeutic target, and inhibitors of this enzyme are the focus of biomedical research. A variety of such compounds has been discovered of which the plecomacrolide inhibitors bafilomycin and concanamycin are the best studied examples (9). With IC 50 values at low nanomolar concentrations these compounds are highly specific inhibitors of the V-ATPase (10). Throughout the past years the binding site and inhibition mechanism of the plecomacrolides has been studied in more detail. In 2002 Bowman et al. (11) identified via mutagenesis studies in Neurospora crassa amino acids in V O subunit c that contribute to the binding of bafilomycin. In the same year photoaffinity labeling studies with the radioactive concanamycin derivative 125 I-concanolid A also resulted in the *

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

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. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded V(O)-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.