Determination of fexofenadine in Hank's balanced salt solution by high‐performance liquid chromatography with ultraviolet detection: application to Caco‐2 cell permeability studies

The drug‐transporting proteins can affect the pharmacokinetics and pharmacodymanics of many drugs, resulting in an erratic and unpredictable pharmacological response. The Caco‐2 monolayer is routinely applied to investigate the carrier‐mediated transport of drugs. Therefore, the selection of a marker compound able to characterize the activity of such transporters is crucial. Fexofenadine (FEX), a P‐gp/OATP substrate, can be considered a suitable probe. However, in order to use be used as a marker compound, it is mandatory to develop an analytical method able to quantify this drug during the in vitro permeability assay. An HPLC method with ultraviolet detection was developed; the mobile phase consisted of phosphate buffer (pH 3.2) containing 10 m m of sodium octanosulphonate and acetonitrile (60:40) and the flow rate was set at 1.2 mL/min. Fexofenadine was eluted at 40°C, the retention time was about 4.6 min. The LOD and LOQ values were 1.9 and 6.2 ng/mL, respectively. Verapamil and ketoconazole, the most common P‐gp inhibitors, were eluted as distinct peaks of that corresponding to fexofenadine The method was successfully applied to quantify the amount of FEX transported across the Caco‐2 monolayer and could be an additional tool for those investigating the role of membrane transporters on drug absorption. Copyright © 2014 John Wiley & Sons, Ltd.     

Classifying substrate specificities of membrane transporters from Arabidopsis thaliana

Membrane transporters catalyze the active transport of molecules across biological barriers such as lipid bilayer membranes. Currently, the experimental annotation of which proteins transport which substrates is far from complete and will likely remain so for much longer. Therefore, it is highly desirable to develop computational methods that may aid in the substrate annotation of putative membrane transport proteins. Here, we measured the similarity of membrane transporters from Arabidopsis thaliana by their amino acid composition, higher sequence order information, amino acid characteristics, or sequence conservation. We considered the substrate classes amino acids, oligopeptides, phosphates, and hexoses. Substrate classification based on the amino acid frequency yielded an accuracy of 75% or higher. Integrating additional information improved the prediction performance to 90% and higher.     

Progress in drug delivery to the central nervous system by the prodrug approach

This review describes specific strategies for targeting to the central nervous system (CNS). Systemically administered drugs can reach the brain by crossing one of two physiological barriers resistant to free diffusion of most molecules from blood to CNS: the endothelial blood-brain barrier or the epithelial blood-cerebrospinal fluid barrier. These tissues constitute both transport and enzymatic barriers. The most common strategy for designing effective prodrugs relies on the increase of parent drug lipophilicity. However, increasing lipophilicity without a concomitant increase in rate and selectivity of prodrug bioconversion in the brain will result in failure. In these regards, consideration of the enzymes present in brain tissue and in the barriers is essential for a successful approach. Nasal administration of lipophilic prodrugs can be a promising alternative non-invasive route to improve brain targeting of the parent drugs due to fast absorption and rapid onset of drug action. The carrier-mediated absorption of drugs and prodrugs across epithelial and endothelial barriers is emerging as another novel trend in biotherapeutics. Several specific transporters have been identified in boundary tissues between blood and CNS compartments. Some of them are involved in the active supply of nutrients and have been used to explore prodrug approaches with improved brain delivery. The feasibility of CNS uptake of appropriately designed prodrugs via these transporters is described in detail.     

Molecular activities and ligand-binding specificities of StAR-related lipid transfer domains: exploring integrated in silico methods and ensemble-docking approaches

In this study, cholesterol biotransformation gene-set of human steroidogenic acute regulatory protein-related lipid transfer (START) domains were evaluated from high-throughput gene screening approaches. It was shown that STARD1, STARD3 and STARD4 proteins are better effective transporters of cholesterol than STARD5 and STARD6 domains. Docking studies show a strong agreement with gene ontology enrichment data. According to both complementary strategies, it was found that only STARD1, STARD3 and STARD4 are potentially involved in cholesterol biotransformation in mitochondria through Ω1-loop of C-terminal α4-helical domain. Ensemble docking assessment for a set of selected chemicals of protein–chemical networks has shown possible binding probabilities with START domains. Among those, reproductive toxicity evoked drugs (mifepristone), insecticides (rotenone), tobacco pulmonary carcinogens (benzo(a)pyrene) and endocrine disruptor chemicals (EDCs) including perfluorooctanesulfonic acid (PFOS) and aflatoxin B₁ (AFB₁) potentially bound with novel hotspot residues of the α4-helical domain. Compound representation space and clustering approaches reveal that the START proteins show more sensitivity with these lead scaffolds, so they could provide probable barrier assets in cholesterol and steroidogenic acute regulatory (StAR) binding and leads adverse consequences in steroidogenesis. These findings indicate potential START domains and their binding levels with toxic chemicals; sorted viewpoints could be useful as a promising way to identify chemicals with related steroidogenisis impacts on human health.     

Structural modeling of human organic cation transporters

Human organic cation transporters (hOCTs) belong to solute carriers (SLC) 22 family of membrane proteins that play a central role in transportation of chemotherapeutic drugs for several clinical and pathological conditions, including cancer and diabetes. These transporters mediate drug transport; however, the precise mechanism of drug-binding and transport by them is not fully uncovered yet, partly due to unavailability of any crystal structure record. In this work, we performed a multi-phasic approach to compute the 3D structural models of seven human organic cation transporters (hOCTs) starting from primary protein sequence. Our structure modeling approach included 1) I-TASSER based comparative sequence alignment, threading and ab-initio protein modeling; 2) models comparison with PSIPRED secondary structure prediction; 3) loop modeling for incongruent secondary structure in Chimera 1.10.1; 4) high resolution structure simulation, refinement, energy minimization using ModRefiner, and 5) validation of the structure models using PROCHECK at SAVEs. From structural point, the computed 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of twelve α-transmembrane helix domains arranged in a manner rendering hOCTs a barrel shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structure of all hOCTs closely resemble to human SLC2A3 (GLUT3) transporter (PDB ID: 5c65) and displayed an outward-open confirmation and putative cyclic C1 protein symmetry. In addition, hOCTs has a large (>100 amino acids) unique extracellular loop between TMH1 and TMH2 having potential glycosylation sites (Asn-Xaa-Ser/Thr) and cysteine residues, both features indicative of putative role in drug binding and uptake. There is an intracellular three/four-helix loop between TMH6 and TMH7 containing putative phosphorylation sites for precise regulation of hOCTs function as drug transporters. There are nine loops of 4 to 11 amino acids length that protrude from membrane, both intracellularly and extracellularly, and connect adjacent TMHs. The 2D structure prediction showed N_in-C_in topology of all hOCTs. In the unavailability of the crystal structures of hOCTs, the 3D structural models computed in-silico and presented herein can be used for studying the mechanism of drug binding and transport by hOCTs.     

Membrane domain-disrupting effects of 4-substitued cholesterol derivatives

A wide range of cellular functions are thought to be regulated not only by the activity of membrane proteins, but also by the local membrane organization, including domains of specific lipid composition. Thus, molecules and drugs targeting and disrupting this lipid pattern, particularly of the plasma membrane, will not only help to investigate the role of membrane domains in cell biology, but might also be interesting candidates for therapy. We have identified three 4-substituted cholesterol derivatives that are able to induce a domain-disrupting effect in model membranes. When applied to giant unilamellar vesicles displaying liquid-ordered-liquid-disordered phase coexistence, extensive reorganization of the membrane can be observed, such as the budding of membrane tubules or changes in the geometry of the domains, to the point of complete abolition of phase separation. In this case, the resulting membranes display a fluidity intermediate between those of liquid-disordered and liquid-ordered phases.     

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.     

Exogenous agents that target transmembrane domains of proteins

Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.     

A chemical genetics approach reveals H,K-ATPase-mediated membrane voltage is required for planarian head regeneration

Biophysical signaling is required for both embryonic polarity and regenerative outgrowth. Exploiting endogenous ion transport for regenerative therapies will require direct regulation of membrane voltage. Here, we develop a pharmacological method to target ion transporters, uncovering a role for membrane voltage as a key regulator of anterior polarity in regenerating planaria. Utilizing the highly specific inhibitor, SCH-28080, our data reveal that H(+),K(+)-ATPase-mediated membrane depolarization is essential for anterior gene expression and brain induction. H(+),K(+)-ATPase-independent manipulation of membrane potential with ivermectin confirms that depolarization drives head formation, even at?posterior-facing wounds. Using this chemical genetics approach, we demonstrate that membrane voltage controls head-versus-tail identity during planarian regeneration. Our data suggest well-characterized drugs (already approved for human use) might be exploited to control adult stem cell-driven pattern formation during the regeneration of complex structures.     

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

l-Type amino acid transporter 1 (LAT1), expressed abundantly in the brain and placenta and overexpressed in several cancer cell types, has gained a lot of interest in drug research and development, as it can be utilized for brain-targeted drug delivery, as well as inhibiting the essential amino acid supply to cancer cells. The structure of LAT1 is today very well-known and the interactions of ligands at the binding site of LAT1 can be modeled and explained. However, less is known of LAT1′s life cycle within the cells. Moreover, the functionality of LAT1 can be measured by several different methods, which may vary between the laboratories and make the comparison of the results challenging. In the present study, the usefulness of indirect cis-inhibition methods and direct cellular uptake methods and their variations to interpret the interactions of LAT1-ligands were evaluated. Moreover, this study also highlights the importance of understanding the intracellular kinetics of LAT1-ligands, and how they can affect the regular function of LAT1 in critical tissues, such as the brain. Hence, it is discussed herein how the selected methodology influences the outcome and created knowledge of LAT1-utilizing compounds.     

Designer peptide surfactants stabilize diverse functional membrane proteins

Multi-spanning integral membrane proteins, including G-protein coupled receptors (GPCR), ion channels, and ion transporters, comprise a major class of drug targets. However, despite their vital importance, most molecular structures of membrane proteins remain elusive. This is largely due to lack of effective materials and methods to stabilize their functional conformation for sufficient time. Thus finding optimal surfactants and developing new approaches to study fundamental properties of unstable membrane proteins is urgently needed. In this tutorial review we summarize designer peptides with surfactant properties and their usefulness to stabilize membrane proteins. These peptide surfactants present new opportunities for the stabilization and characterization of diverse membrane proteins. Previous studies on the interaction between surfactant peptides and membrane proteins revealed strategies to design new peptides tailor-made for the stabilization of specific proteins. We review examples of solubilization, purification, long-term stabilization of membrane proteins, and the design principles of peptide sequences. We discuss future trends for exploiting spatial features, thermodynamic parameters, and self-assembling properties to create peptide surfactant structures to facilitate the characterization of diverse membrane proteins.     

Cover Picture

The cover picture shows an array of several hundred synthetically produced variants of the 44 amino acids comprising the hyAP-WW protein domain. The array was produced by a stepwise SPOT synthesis on a cellulose membrane. At each synthesis site (spot) a WW domain is bound to the membrane through a C-terminal ester bond. The secondary structure of the WW domain is shown in green as a ribbon. The domains of the single spots differ only by a single amino acid. All the domains were tested simultaneously for their ability to bind to a peptide motif (red) common to many proteins. The binding was evident when visualized by chemoluminescence, with the Spots having various intensities. The systematic analysis undertaken here enabled molecular biology to be carried out that would have otherwise have required great effort, or not been done at all. This chemical technique also allows the construction of many non-genetically coded building blocks. Through the use of modern synthetic techniques for the coupling of peptides this method should also allow the synthesis of proteins. The combination of molecular biological and chemical methods opens up opportunities for the preparation of protein chips for diagnostics and drug discovery. More about this can be found in the article by Schneider-Mergener et al. on p. 897ff.     

Comparative QSAR studies on PAMPA/modified PAMPA for high throughput profiling of drug absorption potential with respect to Caco-2 cells and human intestinal absorption

Despite the dramatic increase in speed of synthesis and biological evaluation of new chemical entities, the number of compounds that survive the rigorous processes associated with drug development is low. Thus, an increased emphasis on thorough ADMET (absorption, distribution, metabolism, excretion and toxicity) studies based on in vitro and in silico approaches allows for early evaluation of new drugs in the development phase. Artificial membrane permeability measurements afford a high throughput, relatively low cost but labor intensive alternative for in vitro determination of drug absorption potential; parallel artificial membrane permeability assays have been extensively utilized to determine drug absorption potentials. The present study provides comparative QSAR analysis on PAMPA/modified PAMPA for high throughput profiling of drugs with respect to Caco-2 cells and human intestinal absorption.     

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Spatial chemical distance based on atomic property fields

Similarity of compound chemical structures often leads to close pharmacological profiles, including binding to the same protein targets. The opposite, however, is not always true, as distinct chemical scaffolds can exhibit similar pharmacology as well. Therefore, relying on chemical similarity to known binders in search for novel chemicals targeting the same protein artificially narrows down the results and makes lead hopping impossible. In this study we attempt to design a compound similarity/distance measure that better captures structural aspects of their pharmacology and molecular interactions. The measure is based on our recently published method for compound spatial alignment with atomic property fields as a generalized 3D pharmacophoric potential. We optimized contributions of different atomic properties for better discrimination of compound pairs with the same pharmacology from those with different pharmacology using Partial Least Squares regression. Our proposed similarity measure was then tested for its ability to discriminate pharmacologically similar pairs from decoys on a large diverse dataset of 115 protein–ligand complexes. Compared to 2D Tanimoto and Shape Tanimoto approaches, our new approach led to improvement in the area under the receiver operating characteristic curve values in 66 and 58% of domains respectively. The improvement was particularly high for the previously problematic cases (weak performance of the 2D Tanimoto and Shape Tanimoto measures) with original AUC values below 0.8. In fact for these cases we obtained improvement in 86% of domains compare to 2D Tanimoto measure and 85% compare to Shape Tanimoto measure. The proposed spatial chemical distance measure can be used in virtual ligand screening.     

Cloning of two genes (LAT1,2) encoding specific L: -arabinose transporters of the L: -arabinose fermenting yeast Ambrosiozyma monospora

We identified and characterized two genes, LAT1 and LAT2, which encode specific l-arabinose transporters. The genes were identified in the l-arabinose fermenting yeast Ambrosiozyma monospora. The yeast Saccharomyces cerevisiae had only very low l-arabinose transport activity; however, when LAT1 or LAT2 was expressed, l-arabinose transport was facilitated. When the LAT1 or LAT2 were expressed in an S. cerevisiae mutant where the main hexose transporters were deleted, the l-arabinose transporters could not restore growth on d-glucose, d-fructose, d-mannose or d-galactose. This indicates that these sugars are not transported and suggests that the transporters are specific for l-arabinose.     

活性导向的化学探针在氨基酸反应活性表征中的应用进展

原创药物的研制得益于蛋白质新靶标的发现,而新靶标的发现依赖于高可信度、高通量的药物-蛋白质相互作用分析方法。蛋白质作为生命功能的执行者,其表达量、空间定位与结构差异直接影响药效的发挥。目前,超过85%的蛋白质尚被认为是无法成药的,主要原因是缺少药物分子靶向的空腔以及相应的反应活性位点。因此,基于蛋白质组学层次实现对氨基酸反应活性位点的表征成为原创共价靶向药物设计的关键,也是克服难以成药靶标蛋白问题的关键。近年来,质谱技术的飞速发展极大地推动了基于蛋白质组学技术的药物-靶蛋白相互作用研究。其中基于活性的蛋白质组分析(ABPP)策略是利用活性位点导向的化学探针分子在复杂样品中实现功能状态酶和药物靶标等蛋白质的检测。基于化学探针的开发和质谱定量技术的发展,ABPP技术在氨基酸反应活性表征研究中展现出重要的应用潜力,将助力于药物新靶标的发现和药物先导化合物的开发。ABPP策略主要基于蛋白质的活性特征进行富集,活性探针作为ABPP策略的核心,近年来取得了飞速进展。该文回顾了ABPP策略的发展历程,重点介绍基于广谱活性探针的ABPP技术在多种氨基酸反应活性筛选领域的研究进展,并对其在药物靶点发现中...     

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.     

Amphipathic helices as mediators of the membrane interaction of amphitropic proteins, and as modulators of bilayer physical properties

The amphipathic helix (AH) motif is used by a subset of amphitropic proteins to accomplish reversible and controlled association with the interfacial zone of membranes. Functioning as more than mere membrane anchoring domains, amphipathic helices can serve as autoinhibitory domains to suppress the protein activity in its soluble form, and as sensors or modulators of membrane curvature. Thus amphipathic helices can both respond to and modulate membrane physical properties. These and other features are illustrated by the behavior of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme in PC synthesis. A comparison of the physico-chemical features of CCT's AH motif and 10 others reveals similarities and several differences. The importance of these parameters to the particulars of the membrane interaction and to functional consequences requires more systematic exploration. The membrane partitioning of amphitropic proteins with AH motifs can be regulated by various strategies including changes in membrane lipid composition, phosphorylation, ligand-induced conformational changes, and membrane curvature. Several amphitropic proteins that control budding or tubule formation in cells have AH motifs. The insertion of the hydrophobic face of these amphipathic helices generates an asymmetry in the lateral pressure of the two leaflets resulting in an induction of positive curvature. Curvature induction or stabilization may be a universal property of AHA proteins, not just those involved in budding, but this possibility requires further demonstration.