Conformational Variability in Ground-State CFTR Lipoprotein Particle Cryo-EM Ensembles.

Cystic fibrosis transmembrane regulator (CFTR) is a dynamic membrane protein belonging to the ABC transporter family. It is unusual within this family as it is an ion channel, as opposed to a transporter. Activation of CFTR requires ATP and phosphorylation by PKA, and dysregulation of CFTR mediated salt and water homeostasis can lead to cystic fibrosis. Recent advancements in structural biological methods have led to more than 10 published CFTR structures, and, so far, all of these structures of CFTR, determined by cryo-EM, have been limited to detergent-purified protein preparations. To visualize CFTR in an environment that more closely represents its native membranous environment, we utilized two different lipoprotein particle encapsulation techniques: one in which the ion channel is first purified and then reconstituted using the membrane scaffolding protein Saposin A and another that uses the solubilizing polymer Sokalan CP9 (DIBMA) to extract CFTR directly from membranes. Structures derived from these types of preparations may better correlate to their function, for instance, the single-channel measurements from membrane vesicles.

Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 °C and remain monodisperse at 4 °C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore.

Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.

Rapid Detection of Direct Compound Toxicity and Trailing Detection of Indirect Cell Metabolite Toxicity in a 96-Well Fluidic Culture Device for Cell-Based Screening Environments: Tactics in Six Sigma Quality Control Charts.

Microfluidic screening tools, in vitro, evolve amid varied scientific disciplines. One emergent technique, simultaneously assessing cell toxicity from a primary compound and ensuing cell-generated metabolites (dual-toxicity screening), entails in-line systems having sequentially aligned culture chambers. To explore dual-tox screens, we probe the dissemination of nutrients involving 1-way transport with upstream compound dosing, midstream cascading flows, and downstream cessation. Distribution of flow gives rise to broad concentration ranges of dosing compound (0→ICcompound100) and wide-ranging concentration ranges of generated cell metabolites (0→ICmetabolites100). Innately, single-pass unidirectional flow retains 1st pass informative traits across the network, composed of nine interconnected culture wells, preserving both compound and cell-secreted byproducts as data indicators in each adjacent culture chamber. Thereafter, to assess effective compound hepatotoxicity (0→ECcompound100) and simultaneously classify for cell-metabolite toxicity (0→ECmetabolite100), we reveal utility by analyzing culture viability against ramping exposures of acetaminophen (APAP) and nefazodone (NEF), compounds of hepatic significance. We then discern metabolite generation with an emphasis on amplification across µchannel multiwell sites. Lastly, using conventional cell functions as indicator tools to assess dual toxicity, we investigate a non-drug induced liver injury (non-DILI) compound and DILI compound. The technology is for predictive evaluations of new compound formulations, new chemical entities (NCE), or drugs that have previously failed testing for unresolved reasons.

Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.

Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

Seminaphthofluorones are a family of water-soluble, low molecular weight, NIR-emitting fluorophores.

A readily accessible new class of near infrared (NIR) molecular probes has been synthesized and evaluated. Specific fluorophores in this unique xanthene based regioisomeric seminaphthofluorone dye series exhibit a combination of desirable characteristics including (i) low molecular weight (339 amu), (ii) aqueous solubility, and (iii) dual excitation and emission from their fluorescent neutral and anionic forms. Importantly, systematic changes in the regiochemistry of benzannulation and the ionizable moieties afford (iv) tunable deep-red to NIR emission from anionic species and (v) enhanced Stokes shifts. Anionic SNAFR-6, exhibiting an unusually large Stokes shift of approximately 200 nm (5,014 cm(-1)) in aqueous buffer, embodies an unprecedented fluorophore that emits NIR fluorescence when excited in the blue/green wavelength region. The successful use of SNAFR-6 in cellular imaging studies demonstrates proof-of-concept that this class of dyes possesses photophysical characteristics that allow their use in practical applications. Notably, each of the new fluorophores described is a minimal template structure for evaluation of their basic spectral properties, which may be further functionalized and optimized yielding concomitant improvements in their photophysical properties.

A Unidirectional 96-Well Fluidic Culture Platform for Upstream Cell Dosing with Subsequent Downstream Nonlinear and Ascending Exposure Gradients for Real-Time and Cell-Based Toxicity Screening Environments.

Introduction: Because of the importance to create in vitro screening tools that better mimic in vivo models, for exposure responses to drugs or toxicants, reproducible and adaptable culture platforms must evolve as approaches to replicate functions that are native to human organ systems. The Stairstep Waterfall (SsWaterfall) Fluidic Culture System is a unidirectional, multiwell, gravity-driven, cell culture system with micro-channels connecting 12 wells in each row (8-row replicates). Materials and Methods: The construct allows for the one-way flow of medium, parent and metabolite compounds, and the cellular signaling between connected culture wells while simultaneously operating as a cascading flow and discretized nonlinear dosing device. Initial cell seeding in SsWaterfall mimics traditional static plate protocols but thereafter functions with controlled flow and ramping concentration versus time exposure environments. Results: To investigate the utility of a microfluidic system for predicting drug efficacy and toxicity, we first delineate device design, fabrication, and characterization of a disposable dosing and gradient-exposure platform. We start with detailed characterizations by demarcating various features of the device, including low nonspecific binding, wettability, biocompatibility with multiple cell types, intra-well and inter-well flow, and efficient auto-mixing properties of dose compounds added into the platform. Discussion: We demonstrate the device utility using an example in sequential testing-screening drug toxicity and efficacy across wide-ranging inducible exposures, 0 → IC100, featuring real-time assessments. Conclusion: The integrated auto-gradient technology, gravity flow with stairstep pathways, offers end-users an easy and quick alternative to evaluate broad-ranging toxicity of new compound entities (e.g., pharmaceutical, environmental, agricultural, cosmetic) as opposed to traditional/arduous manual drug dilutions and/or expensive robotic technology.

Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.

Influence of the number and distribution of NLS peptides on the photosensitizing activity of multimeric porphyrin-NLS.

Porphyrin-peptide conjugates bearing multiple nuclear localization sequences (NLS) could show increased tumor cell uptake and affinity for nuclear receptors, and consequently increased photodynamic activity. Previous studies suggest that an increase number of NLS might enhance the nuclear uptake of proteins and other macromolecules. We report the syntheses and investigation of a series of multimeric porphyrin-NLS conjugates bearing two, three or four peptides with the minimum sequence PKKKRKV, linked via PEG or 5-carbon linkers, and with different distributions at the porphyrin periphery. Our results show that the tumor cell uptake and phototoxicity of these conjugates is mainly determined by their amphiphilic character, and not the number of NLS residues per molecule, contrary to previous studies. The mono- and di-substituted photosensitizers bearing one or two PEG linkers and up to three peptide sequences were found to be the most phototoxic toward human carcinoma HEp2 cells, while the tetra-NLS conjugates symmetrically substituted around the porphyrin ring accumulated the least within cells and were non-phototoxic. All conjugates localized intracellularly within endosomal vesicles and lysosomes, probably as a result of an endocytic mechanism of uptake; as a consequence no nuclear uptake was detected by fluorescence microscopy.

Synthesis, characterization, and metabolic stability of porphyrin-peptide conjugates bearing bifunctional signaling sequences.

A series of four porphyrin-peptide conjugates bearing one linear bifunctional sequence containing a cell penetrating peptide (CPP) and a nuclear localization signal (NLS) were synthesized and their in vitro biological and stability properties investigated. All conjugates accumulated within human HEp2 cells to a significantly higher extent than their porphyrin-PEG precursor, and the extent of their uptake and cytotoxicity depends on the nature and sequence of the amino acids. Conjugates 2 and 5 bearing a NLS-CPP accumulated the most within cells and were the most phototoxic (IC50 approximately 7 microM at 1 J/cm2). All conjugates localized preferentially within the cell lysosomes, and in addition, conjugate 2 was also found in the ER. All conjugates were highly stable under nonenzymatic conditions, but their peptide sequences were cleaved to some extent (ca. 50% after 24 h) by proteolytic enzymes, such as cathepsin B, cathepsin D, prolidase, and plasmin.

Syntheses and properties of a series of cationic water-soluble phthalocyanines.

A series of symmetrical cationic phthalocyanines (Pcs) with either Zn(II) or Si(IV) metal ions and two bulky axial ligands on the silicon complexes was synthesized in high yields. The photophysical (absorption, emission, fluorescence, and singlet oxygen quantum yields) and cellular (uptake, toxicity, and subcellular localization) properties of this series of Pcs were investigated. The Si(IV)-Pcs exist mainly as monomers in aqueous media and have higher fluorescent quantum yields in protic solvents (methanol and water) than the Zn(II)-Pcs. The presence of eight short PEG groups at the periphery of a Zn(II)-Pc significantly increases its solubility in protic solvents, but a centrally chelated silicon ion and associated bulky axial ligands were more efficient in preventing aggregation of the Pc macrocycles. The singlet oxygen quantum yields for this series of Pcs in DMSO are in the range 0.09-0.15. All Pcs were readily taken up by human HEp2 cells, and the extent of their accumulation within cells depends on their hydrophobic character. Intracellularly, all Pcs localized preferentially within the cell lysosomes. The Zn(II)-Pc 11 and Si(IV)-Pcs 12 and 14 were found to be the most phototoxic (IC50 = 2.2 microM at a 1 J cm(-2) light dose) of this series of compounds.

Synthesis and in vitro properties of trimethylamine- and phosphonate-substituted carboranylporphyrins for application in BNCT.

A series of carboranylporphyrins containing either amine or phosphonic acid functionalities and two to six closo-carborane clusters have been synthesized via a [2+2] condensation of a dimethylamino- or diethylphosphonate-substituted dipyrromethane with a dicarboranylmethyl-benzaldehyde. The X-ray structures of four key reaction intermediates (1, 2, 3, and 4a) and of two target porphyrins, the diphosphonate ester- and the diamino-tetracarboranylporphyrins 5b and 6a, are presented and discussed. In vitro studies using human carcinoma HEp2 and human glioblastoma T98G cells show that these porphyrins are non-toxic in the dark up to 100 microM concentrations, and that a tetracarboranylporphyrin bearing two quaternary ammonium groups is the most efficiently taken up by cells at short times (up to 8 h), followed by a dicarboranylporphyrin bearing three phosphonic acid substituents. All carboranylporphyrins delivered therapeutic amounts of boron to T98G cells and localized mainly within the cell lysosomes.

Synthesis and cellular studies of PEG-functionalized meso-tetraphenylporphyrins.

The total syntheses of four PEG-functionalized porphyrins, containing one to four low molecular weight PEG chains linked via amide bonds to the para-phenyl positions of meso-tetraphenylporphyrin, are reported. The hydrophobic character of the PEG-porphyrins decreases with the number of PEG chains linked to the porphyrin ring, while their tendency for aggregation in buffered aqueous solution increases. The porphyrins containing one or two PEG chains accumulated within human HEp2 cells to a much higher extent than those having three or four PEGs at the macrocycle periphery. All PEG-porphyrins were found to be non-toxic in the dark, and only those containing one or two PEG chains were phototoxic (IC(50)=2 microM at 1J/cm(2) light dose). The preferential sites of subcellular localization of the porphyrins containing one or two PEG chains were found to be the mitochondria and endoplasmic reticulum (ER), while those containing three or four PEG chains localize preferentially in the lysosomes.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the ß2-adrenergic G protein-coupled receptor (ß2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of ß2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Peptide-mediated cell transport of water soluble porphyrin conjugates.

Five new porphyrin-peptide conjugates bearing a nuclear localizing sequence SV40 or a fusogenic peptide (HIV-1Tat 40-60 or octa-arginine) linked by low molecular weight poly(ethylene glycol) have been synthesized. In vitro studies using human HEp2 cells show that the cellular uptake of the conjugates depends significantly on the nature and sequence of amino acids in the peptide and on the nature of the substituents on the porphyrin macrocycle. The fusogenic peptide sequences HIV-1Tat 40-60 and octa-arginine were the most effective in delivering the conjugates to the cells. The subcellular distribution of the conjugates was found to be dependent on the nature of substituents on the porphyrin macrocycle. The conjugates bearing a hydrophobic porphyrin localized preferentially in the endoplasmic reticulum and were significantly more phototoxic to HEp2 cells than the carboxylic acid functionalized porphyrin conjugates, which localized mainly in the lysosomes.

Potential sites of CFTR activation by tyrosine kinases.

The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation.

Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.

Synthesis and cellular studies of nonaggregated water-soluble phthalocyanines.

Water-soluble phthalocyanines are promising photosensitizers for application in cancer therapy and in the photoinactivation of viruses. The water-soluble zinc(II) phthalocyanines 5 and 6 were synthesized by converting the corresponding ester derivative 4 into the sodium carboxylate and carboxylic acid species. Compound 5 can be solubilized in water as a monomeric species, as demonstrated by UV/vis and fluorescence spectroscopy. These compounds were characterized by analytical and spectroscopic methods and, in the case of 4, by X-ray crystallography. The water-soluble phthalocyanines were found to have low dark cytotoxicity toward V79 hamster fibroblasts and human HEp2 cells, to be phototoxic at low light and drug doses, to be taken up by cells in culture, and to localize intracellularly, mainly in the cell lysosomes. Conjugation of the anionic phthalocyanines with positively charged LipoGen liposomes resulted in effective delivery of these compounds into the nuclei of cells. It is concluded that these highly water-soluble phthalocyanines are promising sensitizers for the photodynamic therapy of tumors.