Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H(+) or Na(+) across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H(+)-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1-3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

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P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5. multidrug resistance | ABC transporter | membrane protein | X-ray crystallography | macrocyclic peptide M ultidrug transporters of the ATP-binding cassette (ABC) superfamily, such as P-glycoprotein (P-gp; MDR1; ABCB1), MRP1 (ABCC1), and ABCG2 (BCRP), transport a large number of structurally unrelated compounds with molecular weights ranging up to several thousand Daltons (1, 2). These transporters not only play important roles in normal physiology by protecting tissues from various toxic xenobiotics and endogenous metabolites but also contribute to multidrug resistance (MDR) in tumors, a major obstacle to effective chemotherapeutic treatment (1, 3-7). Their functional forms consist of a minimum of four core domains: two transmembrane domains (TMDs) that create the translocation pathway for substrates and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP to power the transport process (8, 9). These four domains can exist either as two separate polypeptides (half-size) or fused together in a single large polypeptide with an internal duplication (full-size). The crystal structures of mouse and nematode P-gps, as well as their bacterial homologs (10-14), have been determined, and they have provided important insights into the relationships between protein structure and the functional and biochemical characteristics of P-gp. However, the detailed architecture of the TMD machinery and the gating mechanism during the transition between the inward-and outward-open states are poorly understood.Here, we report the structures of a eukaryotic P-gp homolog, unlocked (at 2.6-Å resolution) and locked allosterically with a tailor-made peptide at 2.4-Å resolution. Although CmABCB1 is not a full-length ABC transporter but a half-sized ABC transporter adopting a homodimeric architecture, CmABCB1 showed quite similar functional properties to those of human P-gp (hP-gp). Based on these structures, we...

Transforming growth factor (TGF)-β is a secreted multifunctional cytokine that signals via plasma membrane TGF-β type I and type II receptors and intercellular SMAD transcriptional effectors. Aberrant inter- and intracellular TGF-β signaling can contribute to cancer progression. In normal cells and early stages of cancer, TGF-β can stimulate epithelial growth arrest and elicit a tumor suppressor function. However, in late stages of cancer, when the cytostatic effects of TGF-β in cancer cells are blocked, TGF-β signaling can act as tumor promoter by its ability to stimulate epithelial-to-mesenchymal transition of cancer cells, by stimulating angiogenesis, and by promoting evasion of immune responses. In this review, we will discuss the rationale and challenges of targeting TGF-β signaling in cancer and summarize the clinical status of TGF-β signaling inhibitors that interfere with TGF−β bioavailability, TGF-β/receptor interaction, or TGF-β receptor kinase function. Moreover, we will discuss targeting of TGF-β signaling modulators and downstream effectors as well as alternative approaches by using promising technologies that may lead to entirely new classes of drugs.

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M2+)-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 ± 0.026) min−1. A pH rate profile analysis revealed pKa's of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M2+-free conditions at 37°C.