Understanding the Inhibitory Effect of Highly Potent and Selective Archazolides Binding to the Vacuolar ATPase Sandra Dreisigacker, †,‡ Dorota Latek, ‡,§ Svenja Bockelmann, ∥ Markus Huss, ∥ Helmut Wieczorek, ∥ Slawomir Filipek, ⊥ Holger Gohlke, # Dirk Menche, †,∇ and Teresa Carlomagno* ,‡ ‡ Structural and Computational Biology Unit, EMBL, Mayerhofstrasse 1, D-69117 Heidelberg, Germany † Institute of Organic Chemistry, Ruprecht-Karls University Heidelberg, Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany ∥ Department of Animal Physiology, Faculty of Biology and Chemistry, University of Osnabrü ck, Barbarastrasse 11, D-49069 Osnabrü ck, Germany ⊥ Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland # Heinrich-Heine-University Dü sseldorf, Institute of Pharmaceutical and Medicinal Chemistry, Universitä tsstrasse 1, D-40225 Dü sseldorf, Germany * S Supporting Information ABSTRACT: 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 sub- nanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular dock- ing, molecular dynamics simulations, and biochemical experi- ments, resulting in a sound model for the inhibitory mech- anism 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. ■ INTRODUCTION The polyketide family of archazolides − isolated from myxobacteria Archangium gephyra and cystobacter violaceus 1−4 − are well-established natural products. In the past years, we reported on the structure elucidation and the first total synthesis of archazolide A (1) (Figure 1), and, more recently, on the syntheses of analogs and their bioactivity. 5−11 Archazolides are interesting molecules both from the structural point of view and in terms of their potential utility as pharmaceutical drugs. They display impressive biological properties, including potent cytotoxicity against several tumor cell lines in the subnanomolar range. 2−4,7 The cytotoxicity is attributed to the binding of archazolides to the vacuolar ATPase (V-ATPase), which leads to an inhibition of this vital enzyme. V-ATPases use the energy from ATP hydrolysis to translocate protons across membranes, thereby energizing secondary active transport processes or acidifying either the extracellular medium or the lumen of intracellular organells. 12 Cleavage of ATP occurs at the cytosolic V 1 -complex, while proton transport is mediated by the membrane-integral V O -complex of the enzyme. The V O -complex comprises a ring of c subunits. Each subunit contains a conserved glutamate residue that is essential for proton translocation. 13 V-ATPase mediated extracellular acidification contributes for example to bone resorption by osteoclasts or to metastasis of tumor cells. Accordingly, V-ATPases represent a potential target for the treatment of severe diseases such as osteoporosis or cancer. 14,15 The function of V-ATPase is effectively inhibited by archazolides, which bind selectively to the c subunits of the membrane-integral V O -rotor. 7 In contrast, other ion translocating enzymes, e.g., F-type and P-type ATPases, are not affected by these inhibitors at concentrations up to 10 μM, 7 which makes archazolides highly interesting as prospective potent and selec- tive therapeutic agents. Furthermore, archazolide F (3), with inhibitory profiles in the picomolar range, is the most active inhibitor of V-ATPase-controlled cell growth discovered so far (Figure 1) and also displays a 10-fold higher efficiency for human cell lines compared to its derivatives archazolide A (1) and B (2). 4 The development of archazolides into new pharmaceuticals requires both easy accessibility of the substance and highly selective biological activity. Unfortunately, neither isolation nor total synthesis of archazolides accomplishes the amount of natural product that would be needed in a pharmaceutical Received: May 23, 2012 Published: July 2, 2012 Article pubs.acs.org/jcim © 2012 American Chemical Society 2265 dx.doi.org/10.1021/ci300242d | J. Chem. Inf. Model. 2012, 52, 2265−2272