Translating in vitro CFTR rescue into small molecule correctors for cystic fibrosis using the Library of Integrated Network-based Cellular Signatures drug discovery platform.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The common ΔF508-CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including low-temperature, small molecules, overexpression of miR-138, or knockdown of SIN3A. The challenge remains to translate such interventions into therapies and to understand their mechanisms. One approach for connecting such interventions to small molecule therapies that has previously succeeded for CF and other diseases is via mRNA expression profiling and iterative searches of small molecules with similar expression signatures. Here, we query the Library of Integrated Network-based Cellular Signatures using transcriptomic signatures from previously generated CF expression data, including RNAi- and low temperature-based rescue signatures. This LINCS in silico screen prioritized 135 small molecules that mimicked our rescue interventions based on their genomewide transcriptional perturbations. Functional screens of these small molecules identified eight compounds that partially restored ΔF508-CFTR function, as assessed by cAMP-activated chloride conductance. Of these, XL147 rescued ΔF508-CFTR function in primary CF airway epithelia, while also showing cooperativity when administered with C18. Improved CF corrector therapies are needed and this integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508-CFTR function and identify gene networks underlying such rescue.

Integrative chemogenomic analysis identifies small molecules that partially rescue ΔF508-CFTR for cystic fibrosis.

Rare diseases affect 10% of the first-world population, yet over 95% lack even a single pharmaceutical treatment. In the present age of information, we need ways to leverage our vast data and knowledge to streamline therapeutic development and lessen this gap. Here, we develop and implement an innovative informatic approach to identify therapeutic molecules, using the Connectivity Map and LINCS L1000 databases and disease-associated transcriptional signatures and pathways. We apply this to cystic fibrosis (CF), the most common genetic disease in people of northern European ancestry leading to chronic lung disease and reduced lifespan. We selected and tested 120 small molecules in a CF cell line, finding 8 with activity, and confirmed 3 in primary CF airway epithelia. Although chemically diverse, the transcriptional profiles of the hits suggest a common mechanism associated with the unfolded protein response and/or TNFα signaling. This study highlights the power of informatics to help identify new therapies and reveal mechanistic insights while moving beyond target-centric drug discovery.

Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti.

Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na(+) dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction.

A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development.

AeNAT5 (NCBI, ABZ81822), an orphan member of the insect-specific Nutrient Amino acid Transporter subfamily of SoLute Carrier family 6 (NAT-SLC6) and the first representative of a novel eukaryotic methionine-selective transport system (M), was cloned from cDNA of the vector mosquito, Aedes aegypti. It has orphan orthologs throughout several mosquito genomes, but not in Drosophila or outside Diptera. It shows the highest apparent affinity to L-Met (K(0.5) = 0.021 mM) and its metabolites Homocysteine and Cysteine (K(0.5) = 0.89 and 2.16 mM), but weakly interact with other substrates. It has a Na(+) - coupled mechanism (K(0.5) Na(+) ∼ 46 mM) with 1AA:1Na(+) stoichiometry that maintains ∼60% activity in Cl(-) - free media. In situ hybridization showed accumof AeNAT5 transcript in the absorptive and secretory epithelia, as well as in specific peripheral neurons and the central ganglia of mosquito larvae. The labeling pattern is distinct from that of the previously characterized AeNAT1. RNAi of AeNAT5 increases larval mortality during ecdysis and dramatically suppresses adult emergence. Our results showed that in addition to previously characterized broad spectra and aromatic amino acid selective transport systems, the mosquito NAT-SLC6 subfamily evolved a unique mechanism for selective absorption of sulfur-containing substrates. We demonstrated specific patterns of alimentary and neuronal transcription of AeNAT5 in mosquito larvae that is collateral with the indispensable function of this transporter in mosquito development.

An SLC6 transporter of the novel B(0,)- system aids in absorption and detection of nutrient amino acids in Caenorhabditis elegans.

Nutrient amino acid transporters (NATs) of solute carrier family 6 (SLC6) mediate uptake of essential amino acids in mammals and insects. Phylogenomic analysis of the Caenorhabditis elegans (Ce) SLC6 family identifies five genes paralogous to an insect-specific NAT subfamily. Here we cloned and characterized the first representative of the identified nematode-specific transporters, SNF-5. SNF-5 mediates broad spectrum cation-coupled transport of neutral amino acids with submillimolar affinities and stoichiometry of 1 AA:1 Na(+), except for 1 l-Pro:2 Na(+). Unexpectedly, it transports acidic l-Glu(-) and l-Asp(-) (1 AA(-):3 Na(+)), revealing it to be the first member of a new B(0,-) system among characterized SLC6 transporters. This activity correlates with a unique positively charged His(+) 377 in the substrate-binding pocket. snf-5 promoter-driven enhanced green fluorescent protein labels intestinal cells INT1-9 and three pairs of amphid sensory neurons: ASI, ADF and ASK. These cells are intimately involved in control of dauer diapause, development, metabolism and longevity. The snf-5 deletion mutants do not show apparent morphological disorders, but increase dauer formation while reducing dauer maintenance upon starvation. Overall, the present study characterized the first nematode-specific NAT and revealed important structural and functional aspects of this transporter. In addition to the predictable role in alimentary amino acid absorption, our results indicate possible neuronal roles of SNF-5 as an amino acid provider to specific neuronal functions, including sensing of amino acid availability.

AaCAT1 of the yellow fever mosquito, Aedes aegypti: a novel histidine-specific amino acid transporter from the SLC7 family.

Insect yolk protein precursor gene expression is regulated by nutritional and endocrine signals. A surge of amino acids in the hemolymph of blood-fed female mosquitoes activates a nutrient signaling system in the fat bodies, which subsequently derepresses yolk protein precursor genes and makes them responsive to activation by steroid hormones. Orphan transporters of the SLC7 family were identified as essential upstream components of the nutrient signaling system in the fat body of fruit flies and the yellow fever mosquito, Aedes aegypti. However, the transport function of these proteins was unknown. We report expression and functional characterization of AaCAT1, cloned from the fat body of A. aegypti. Expression of AaCAT1 transcript and protein undergoes dynamic changes during postembryonic development of the mosquito. Transcript expression was especially high in the third and fourth larval stages; however, the AaCAT1 protein was detected only in pupa and adult stages. Functional expression and analysis of AaCAT1 in Xenopus oocytes revealed that it acts as a sodium-independent cationic amino acid transporter, with unique selectivity to L-histidine at neutral pH (K(0.5)(L-His) = 0.34 ± 0.07 mM, pH 7.2). Acidification to pH 6.2 dramatically increases AaCAT1-specific His(+)-induced current. RNAi-mediated silencing of AaCAT1 reduces egg yield of subsequent ovipositions. Our data show that AaCAT1 has notable differences in its transport mechanism when compared with related mammalian cationic amino acid transporters. It may execute histidine-specific transport and signaling in mosquito tissues.

Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

The invertebrate B(0) system transporter, D. melanogaster NAT1, has unique d-amino acid affinity and mediates gut and brain functions.

The CG3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B(0) system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to B(0)-specific l-substrates, DmNAT1 equally or more effectively transports d-amino acids with sub-millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral l-amino acids as well as corresponding d-isomers. The absorption of d-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by d-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance of l-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic d-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B(0)-like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of d-isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily are evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.

Diversity and evolution of coral fluorescent proteins.

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.

Synergy and specificity of two Na+-aromatic amino acid symporters in the model alimentary canal of mosquito larvae.

The nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8, preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal, including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions, respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae.

We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes (Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned, nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family.

Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B(0) type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Ordered differential display.

Ordered differential display (ODD) is one of the approaches that uses systematic, rather than random, sampling of transcripts for display and thereby provides means to browse through essentially all the transcripts in the compared mRNA pools. It is specifically adapted for small amounts of starting material. The protocol outlined here, in addition to ODD procedure itself, also describes isolation of RNA and synthesis of double-stranded cDNA from small biological samples.

Ancestry and progeny of nutrient amino acid transporters.

The biosynthesis of structural and signaling molecules depends on intracellular concentrations of essential amino acids, which are maintained by a specific system of plasma membrane transporters. We identify a unique population of nutrient amino acid transporters (NATs) within the sodium-neurotransmitter symporter family and have characterized a member of the NAT subfamily from the larval midgut of the Yellow Fever vector mosquito, Aedes aegypti (aeAAT1, AAR08269), which primarily supplies phenylalanine, an essential substrate for the synthesis of neuronal and cuticular catecholamines. Further analysis suggests that NATs constitute a comprehensive transport metabolon for the epithelial uptake and redistribution of essential amino acids including precursors of several neurotransmitters. In contrast to the highly conserved subfamily of orthologous neurotransmitter transporters, lineage-specific, paralogous NATs undergo rapid gene multiplication/substitution that enables a high degree of evolutionary plasticity of nutrient amino acid uptake mechanisms and facilitates environmental and nutrient adaptations of organisms. These findings provide a unique model for understanding the molecular mechanisms, physiology, and evolution of amino acid and neurotransmitter transport systems and imply that monoamine and GABA transporters evolved by selection and conservation of earlier neuronal NATs.