Christopher J. Hipolito

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

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.

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.

Significance P-glycoprotein exports various hydrophobic chemicals in an ATP-dependent manner, determines their absorption and distribution in the body, and is involved in multidrug resistance (MDR) in tumors. Understanding the mechanism of the multidrug transport is important for designing drugs of good bioavailability and efficient cancer chemotherapy. We determined the high-resolution crystal structures of a eukaryotic P-glycoprotein homolog and revealed the detailed architecture of its transmembrane domains, which contain an exit gate for substrates that opens to the extracellular side and two entrance gates that open to the intramembranous region and the cytosolic side. We propose a motion of the transmembrane domains powered by the association of two nucleotide-binding domains on ATP binding that is different from other transporters. 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.

Ribosomal production and in vitro selection of natural product-like peptidomimetics: The FIT and RaPID systems

Bioactive natural product peptides have diverse architectures such as non-standard sidechains and a macrocyclic backbone bearing modifications. In vitro translation of peptides bearing these features would provide the research community with a diverse collection of natural product peptide-like molecules with a potential for drug development. The ordinary in vitro translation system, however, is not amenable to the incorporation of non-proteinogenic amino acids or genetic encoding of macrocyclic backbones. To circumvent this problem, flexible tRNA-acylation ribozymes (flexizymes) were combined with a custom-made reconstituted translation system to produce the flexible in vitro translation (FIT) system. The FIT system was integrated with mRNA display to devise an in vitro selection technique, referred to as the random non-standard peptide integrated discovery (RaPID) system. It has recently yielded an N-methylated macrocyclic peptide having high affinity (Kd=0.60 nM) for its target protein, E6AP.

Selection-based discovery of macrocyclic peptides for the next generation therapeutics

Naturally occurring macrocyclic peptides represent a unique class of compounds that exhibit various biological activities ranging from antibiotics to immunosuppressant. Although the discovery of such macrocyclic peptides had relied on their isolation from living organisms, recent advances in ribosomal peptide synthesis and in display techniques made it possible to use artificially generated macrocyclic peptide libraries for selection of ligands for biologically relevant proteins. In this review, we discuss the technologies and their applications for the discovery of peptide ligands.

A macrocyclic peptide that serves as a cocrystallization ligand and inhibits the function of a MATE family transporter.

The random non-standard peptide integrated discovery (RaPID) system has proven to be a powerful approach to discover de novo natural product-like macrocyclic peptides that inhibit protein functions. We have recently reported three macrocyclic peptides that bind to Pyrococcus furiosus multidrug and toxic compound extrusion (PfMATE) transporter and inhibit the transport function. Moreover, these macrocyclic peptides were successfully employed as cocrystallization ligands of selenomethionine-labeled PfMATE. In this report, we disclose the details of the RaPID selection strategy that led to the identification of these three macrocyclic peptides as well as a fourth macrocyclic peptide, MaD8, which is exclusively discussed in this article. MaD8 was found to bind within the cleft of PfMATE’s extracellular side and blocked the path of organic small molecules being extruded. The results of an ethidium bromide efflux assay confirmed the efflux inhibitory activity of MaD8, whose behavior was similar to that of previously reported MaD5.

Protein cocrystallization molecules originating from in vitro selected macrocyclic peptides.

Transmembrane proteins are intractable crystallization targets due to their low solubility and their substantial hydrophobic outer surfaces must be enclosed within a partial micelle composed of detergents to avoid aggregation. Unfortunately, encapsulation within a partial micelle diminishes specific protein-to-protein contacts needed for crystal lattice formation. In addition, the high conformational flexibility of certain transmembrane proteins reduces sample homogeneity causing difficulty in crystallization. Cocrystallization ligands, based on either antibody scaffolds or other proteinaceous non-antibody scaffolds, have greatly facilitated the crystallization of transmembrane proteins. Recently, in vitro selected macrocyclic peptide ligands have been shown to facilitate protein crystallization as well. In this review, we discuss selection strategies used for the discovery of macrocyclic peptide ligands and the three-dimensional crystal structure of the transporter PfMATE in complex with in vitro selected macrocyclic peptides.

Model foldamers: applications and structures of stable macrocyclic peptides identified using in vitro selection

Foldamers are synthetic molecules that seek to mimic the structure-forming propensity of biomolecules, such as proteins. However, on a short oligomer scale, peptides often do not fold in the same manner as large proteins, despite being composed of the same amino acid building blocks. Constraints to available peptide conformations can improve these folding characteristics. One important constraint that leads to an increase in folding behaviour is the formation of a macrocycle, while doing this by means other than disulfide bond formation ensures that this structural constraint persists in all biological settings. Additional non-natural features, such as incorporation of amino acids with unusual side chains, D-amino acids, N-alkyl amino acids, and β-hydroxy acids, further mimic the synthetic characteristics of foldamers, giving a class of compounds that is intermediate between natural proteins and synthetic foldamers. In vitro selection methods, such as phage and mRNA display, allow access to de novo peptides based solely on their ability to bind a target, potentially giving access to unique structures and functions. Recently, a series of structures have become available for several such partially synthetic macrocyclic peptides derived from in vitro selection. Here we present an overview of the structural features of these stable macrocyclic peptides and their binding to protein targets, as well as some initial indications of their folding behaviour free in solution, and discuss implications for the future design and functions of foldamers.

Combined Use of Oligopeptides, Fragment Libraries, and Natural Compounds: A Comprehensive Approach To Sample the Druggability of Vascular Endothelial Growth Factor

The modulation of protein–protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide‐mediated PPIs efficiently, the inhibition of domain–domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF–VEGFR interaction is a typical domain–domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR‐spectroscopy‐based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.

LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively.

Crystallographic Analysis of MATE-Type Multidrug Exporter with Its Inhibitors

Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247-251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.