Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Gene regulatory network wikipedia , lookup
Cell culture wikipedia , lookup
Cell membrane wikipedia , lookup
Signal transduction wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Magnesium transporter wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Transporters as drug carriers and targets in the GIT Pascale Anderle, [email protected], IOSI Jon Christensen, [email protected], IOSI Overview • Introduction • Relevant transporters in the GIT • Detection of new transporters using genomics and proteomics – Classification of transporters – Microarray studies and transporters • Influence of genotypes on oral drug absorption • Transporters and cancers, especially colon cancers Introduction Venter et al. 01 Important families Peptide transporters Nucleoside transporters Amino acid transporters Monosaccharide transporters Organic cation transporters Organic anion transporters Monocarboxylate transporters ABC transporters Bile acid transporters → Supplementary material Genomics and Proteomics • Genomics – Genomics is the study of an organism's entire genome. • Proteomics – Proteomics is the large-scale study of proteins, particularly their structures and functions. • High-throughput techniques – For instance Microarrays, MPSS, SAGE Introduction into Microarray Technology Spotting: Probes Photolithography Printing Oligomers Physical support: Glass slide, nylon membrane PCR products Sample preparation and hybridization: cRNA or cDNA Single-labeling or dual-labeling Affymetrix: Short oligo chip Single labeling Fluorescence or radioactivity or cDNA chip: Oligos or PCR products Dual-labeling Classification of transporters Why is classification important? What type of classification makes sense? What’s typical for a transporter? Classification of transporters Classification according to transport mechanism TC System Gene Ontology PFAM Pumps, channels and transporters Transporters Secondary transporters Membrane Transporter Proteins: Classification Membrane Transport Proteins Specific Carriers Selective Channels Primary Active Transport ATP-powered pumps ATPases: P-type, F-type and ABC-type ATPases (ABC transporters) Primary active transport Energy derived from hydrolysis of ATP to ADP liberating energy from high energy phosphate bond Secondary Active Transport Facilitated Diffusion Uniporters Glut1-5 Facilitated diffusion Like any diffusion, transport from an area of higher concentration to lower concentration. Passive transport is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy Symporters Antiporters Pept1 NHE Secondary active transport Use of energy from another source-another secondary diffusion gradient set up across the membrane using another ion. Because this secondary diffusion gradient initially established using an ion pump, as in primary active transport, the energy is ultimately derived from the same source-ATP hydrolysis. Channels Transport water or specific types of ions down their concentration or electric potential gradients Energetically favorable reaction Form protein-lined passageway across the membrane through which multiple water molecules or ions move simultaneously at a very rapid rate—up to 108 per second Plasma membrane of all animal cells contains potassium-specific channel proteins that are generally open and are critical to generating the normal, resting electric potential across the plasma membrane Many other types of channel proteins are usually closed, and open only in response to specific signals Uniporters Transport is specific and saturable Facilitated “low resistance” diffusion: – Down the concentration gradient – Accelerates reaction that is already thermodynamically favored Reversible Rate much higher than passive diffusion: - Molecule never in contact with hydrophobic core of the membrane Uniporters: Example GLUT1 Facilitated vs. passive diffusion Mechanism of transport Secondary transporters Couple the movement of one type of ion or molecule against its concentration gradient to the movement of a different ion or molecule down its concentration gradient Ability to transport two different solutes simultaneously also called co-transporters Mediate coupled reactions in which an energetically unfavorable reaction coupled to energetically favorable reaction Catalyze “uphill” movement of certain molecules often referred to as “active transporters”, but unlike pumps, do not hydrolyze ATP (or any other molecule) during transport Symporters and antiporters: Examples Pept1 and NHE NHE Pumps Use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient or electric potential. Overall reaction—ATP hydrolysis and the “uphill” movement of ions or small molecules—is energetically favorable P, F, and V classes transport ions only, whereas the ABC superfamily class transports small molecules as well as ions. Pumps: Example ABCB1 TC System: Function/Phylogenetic Milton Saier et al. (http://www.tcdb.org/) Functional/phylogenetic system Analogous to the Enzyme Commission (EC) system for classification of enzymes, except that it incorporates both functional and phylogenetic information. EC strictly functional. Designed for classification of all transmembrane transport protein found in living organisms on Earth Nearly 400 families Affiliation with a family requires rigorous statistical criteria of homology (Saier 1994). Comparison over 60 residues TC System II: Function/Phylogenetic 1. Channels/Pores 1.A. α-Type channels 1.B. β-Barrel porins 1.C. Pore-forming toxins (proteins and peptides 1.D. Non-ribosomally synthesized channel 1.E. Holins 2. Electrochemical Potentialdriven Transporters 2.A. Porters Uniporters, symporters, antiporters 2.B. Nonribosomally synthesized porters 3. Primary Active Transporters 4. Group Translocators 5. Transport Electron Carriers 8. Accessory Factors Involved in Transport 9. Incompletely Characterized Transport Systems 2.C. Ion-gradient-driven energizers Gene Ontology Consortium GO Output Cellular Component L3 L3 L4 GO:X Molecular Function L3 L3 GO:Y Biological processes L3 GO:Z L3 L4 GO:Y ABCB1 Two pragmatic purposes of ontology: 1. Facilitate communication between people and organizations 2. Improve interoperability between systems Ontologies are structured vocabularies in the form of directed acyclic graphs (DAGs) that represent a network in which each term may be a “child” of one or more than one ”parent”. Human Genome Organization: HUGO The Human Genome Organization (HUGO) Nomenclature Committee Database has as a goal to make sure that each symbol is unique, and ensures that each gene locus is only given one approved gene symbol In HUGO Nomenclature Committee Database: SLC series: Currently 43 families and 298 transporter genes Non-SLC human transport-related genes: ATP-driven transporters Channels Ionotropic receptors Aquaporins Transporter and channel subunits auxiliary/regulatory transport proteins http://www.bioparadigms.org/slc/intro.htm PFAM Database Database of protein domain families Contains curated multiple sequence alignments for each family, as well as profile hidden Markov models (profile HMMs) for finding these domains in new sequences Contains functional annotation, literature references and database links for each family There are two multiple alignments for each Pfam family: 1. Seed alignment that contains a relatively small number of representative members of the family 2. The full alignment that contains all members in the database that can be detected The profile HMM is built from the seed alignment using the HMMER package (see http://hmmer.wustl.edu/ ), which is then used to search the pfamseq sequence database Position specific iterative BLAST (PSI-Blast): Position specific scoring matrix (PSSM) is constructed (automatically) from a multiple alignment of the highest scoring hits in an initial BLAST search. The PSSM is generated by calculating position-specific scores for each position in the alignment. Highly conserved positions receive high scores and weakly conserved positions receive scores near zero. The profile is used to perform a second (etc.) BLAST search and the results of each "iteration" used to refine the profile. This iterative searching strategy results in increased sensitivity. Transporters in the GIT ABCB1 SLC5A1 ABCC3 SLC2A2 SLC28A1 SLC15A1 SLC2A1 SLC29A1 facilitated Various forms of transporters and carriers working in an intestinal epithelial cell. Gluc is glucose, Nuc is nucleotide, DP is dipeptides, ABC is ATP-binding cassette family transporter. SLC5A1 is the sodium-dependent glucose transporter, SLC2A2 is the glucose transporter 2, SLC28A1 is the concentrative nucleotide transporter 1, SLC29A1 is the equilibrative transporter 1 and SLC15A1 is the di/tri-peptide transporter. Transporters as drug carriers: Design of prodrugs O N HN H2 N N N N H2 Intestinal cell O O O Valacyclovir O HO N N H N N O O Acyclovir N O O N O O N O N H O H N N O II. Cyclosp orin e A Basolateral side Apical Side I. V al ac ic lo vi r H N O VACV VACV ACV ACV hPEPT1 P-gp Genomics and transporters I Profiling of the intestinal mucosa Genes encoding proteins functioning in metabolism, transport, and cell–cell communication have the most dynamically regulated expression profiles (Bates et al. Gastroenterolology 2002). In contrast to Bates et al. class of transporters was not significantly regulated compared to other GO classes, however, the subclasses carriers, and in particular its daughter classes “antiporters” and “symporters” (Anderle et al. BMC Genomics 2005). Profiling of in vitro systems, namely Caco-2 cells Caco-2 cell differentiation is accompanied by coordinated down-regulation of genes involved in cell cycle progression and DNA synthesis, which reflected the concomitant reduction in cell proliferation (Mariadason et al. Canc Res 2002). In contrast to Mariadason et al. Fleet et al. observed in a subclone of the Caco-2 parental line, the so-called Caco-2 BBe cells, a significant number of transporter genes being regulated upon differentiation (Fleet et al. Physiol. Genomics 2003). Distinctive switch in gene expression patterns upon formation of cell-cell contacts. Proliferating, non-polarized Caco-2 cells more similar to human colon cancer, once differentiated more closely to normal colonocytes. Clustering identifies “normal epithelial cluster” (genes expressed in normal coloncytes and postmitotic, polarizing Caco-2 cells) containing SLC39A5, SLC26A2, SLC17A4 (Saaf et al. Mol Biol Cell 2007) Genomics and transporters II Comparison of intestinal mucosa and Caco-2 cells Comparison of 750 genes encoding transporter and channel proteins in differentiated and undifferentiated Caco-2 cells, human small intestine and colon indicates that Caco-2 cells consist of characteristics of colonocytes, small intestinal enterocytes and tumor cells (Anderle et al. Pharm Res 2003). 26, 38 and 44% of 443 genes, of which 170 are transporters or channels, detectable in 4-day-old Caco-2 cells, 16-day-old Caco-2 cells and human duodenum, respectively. In vivo/in vitro drug permeability measurements correlated well for passively absorbed drug, whereas the correlation coefficient decreased for carrier-mediated drugs. Observed permeability of carrier-mediated drugs higher in human duodenum than in Caco-2 cells.Most of the transporters expressed in the human duodenum also expressed in Caco-2 cells. (Sun et al. Pharm Res 2002) In silico approach exploiting publicly available data sets (results fron 9 different labs, different platforms) in order to assess the Caco-2 model system → Increase of robustness of resulting findings. Principal component analysis showed that Caco-2 cells express a transport protein profile which to some extent represents the absorptive enterocytes, but also colonocytes (Calcagno et al. Mol Pharm 2006). Genomics and transporters III GENOTYPE: The genetic makeup of an individual. The fundamental constitution of an organism in terms of its hereditary factors PHENOTYPE: The physical and physiological traits of an individual resulting from genotype and environment VARIANT: An alteration or change in the genetic sequence. HAPLOTYPE A combination of alleles at multiple linked loci that are transmitted together (Greek haploos = simple). Genomics and transporters III UCSF “Pharmacogenetics of Membrane Transporters Project” (PMT, http://pharmacogenetics.ucsf.edu/index.htm) → Identification of sequence variants in genes encoding selected membrane transport proteins and the functional characterization of these variants. PharmGKB database (http://www.pharmgkb.org/) → Publicly available database that serves as a genotype-phenotype resource focused on pharmacogenetics and pharmacogenomics. NCBI project (http://www.ncbi.nlm.nih.gov/SNP/) Relevant variants cf. Supplementary material Transporters and diseased states of the intestine • Transporters as drug carriers involved in: – – chemo-resistance (i.e. ability of cancer cells to become resistant to the effects of the chemotherapy drugs) and chemo-sensitivity (i.e. susceptibility of tumor cells to the cell-killing effects of chemotherapy drugs) • Transporters as tumor suppressors: SLC5A8, SLC26A3, SLC1A2 (Chapman et al. , Cancer Res 2002, Li et al. PNAS 2003, Schweinfest et al. J Biol Chem 2006, Ueno et al. Tumour Biol 2004) • Transporters in intestinal tumor-initiating cells (stem cells, CD133+ cells): ABCG2 (O'Brien et al. Nature 2007, Monzani et al. Eur J Cancer 2007) • Transporters and EMT: – – • SLC16A3 (MCT4) (Gallagher et al. Cancer Res 2007, Ho et al. 2006 Regulation of SLC15A1, CDH17 and CDX2 (Shimakura et al. , Biochem Pharmacol 2006, Nduati et al. J Biol Chem 2007, Guo et al. Cancer Biol Ther 2004, Suh et al. Mol Cell Biol 1996, Witek et al. Clin Cancer Res 2005, Gross et al. Oncogene 2008) Inflammatory bowel disease: PEPT1-mediated transport of bacteria-derived n-formyl peptides such as formyl-Met-Leu-Phe (fMLP) induces basolateral to apical neutrophil migration in a neutrophil//Caco-2-BBE cell model (Merlin et al. J Clin Invest 1998) Chemo-sensitivity and -resistance: NCI Database Scherf et al. 2000, Nature Genetics Chemo-sensitivity and -resistance: SLCs Sorted correlation coefficients SLC29A1 ABCB1 Effects of expression of transporters on dose-response curves Role in cancer Precarcinogen Metabolic activation Carcinogen Normal cell Metastasis (>1 year) Detoxification Secretion Solute carriers playing a role in EMT, invasion and metastasis formation ABC transporters as efflux pumps of toxic compounds incl. chemotherapeutics Initiation (1-2 days) Invasion, EMT (>1 year) ABC transporters In stem cells Promotion (>10 years) Initiated cell Progression (>1 year) Preneoplastic cell Solute carriers as tumor suppressors Neoplastic cell Metaplastic cell Solute carriers as tumor markers and as drug carriers Summary What’s the relevance of transporters in oral drug absorption? What’s the relevance of transporters as targets in the GIT? What was the big break through with the development of “genomics” technology? What are the limitations of “genomics” methods? Why is it important to know genotypes and phenotypes of variants?