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Yeast in drug development

Yeast in drug development. Properties: unicellular eukaryote Methodology: forward and reverse genetics, two-hybrid system, surrogate host Applications: Model system for cellular eukaryotic functions (animal or plant): Intracellular signal transduction

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Yeast in drug development

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  1. Yeast in drug development Properties: unicellular eukaryote Methodology: forward and reverse genetics, two-hybrid system, surrogate host Applications: Model system for cellular eukaryotic functions (animal or plant): Intracellular signal transduction DNA repair, replication, cell cycle transcription chromosome biology protein transport water transport etc. Protein-protein interactions Global transcriptional regulation Drug target identification Drug screening/selection Disease diagnostics Model for fungal pathogens (plant/animal)

  2. Advantages of yeast in molecular biology • Short life cycle, easy to cultivate • Compact genome, fully sequenced • Many fundamental processes on cellular level conserved • Lots of information available per gene product • Genetically tractable: • haploid • DNA transformation • multiple genetic markers available, both selection and counterselection • possible • genetic crosses possible • gene knockout by homologous recombination very efficient – • complete set of 4 x 6000 knockout mutants available

  3. Yeast for production of proteins – hepatitis B vaccine Hepatitis B surface antigen (HbSAg) • Advantages: • higher yield, faster growth than animal cells • eukaryotic post-translational modifications + =

  4. Yeast as a genetically tractable organism to identify drug targets, establish cellular disease models, diagnosis, and for screening of receptor ligands and lead compounds

  5. The yeast genome is densely packed with genes Saccharomyces cerevisiae is the most information- dense of all experimental organisms

  6. Saccharomyces cerevisiae - Functional Catalogue number of ORFs which match to at least one category: 3936 ORFs(ORFs not counted that are classified as "classification unknown" or "not yet clear-cut") Last update: Thu Jan 10 20:13:11 CET 2002 • Functional Catalogue version from 06.12.2001 • METABOLISM (1066 ORFs) • amino acid metabolism (204 ORFs) • amino acid biosynthesis (118 ORFs) • biosynthesis of the aspartate family (1 ORF) • biosynthesis of lysine (1 ORF) • biosynthesis of the cysteine-aromatic group (2 ORFs) • biosynthesis of serine (1 ORF) • biosynthesis of the pyruvate family (alanine, isoleucine, leucine, valine) and D-alanine (1 ORF) • regulation of amino acid metabolism (33 ORFs) • amino acid transport (23 ORFs) • amino acid degradation (catabolism) (35 ORFs) • degradation of amino acids of the glutamate group (1 ORF) • degradation of glutamate (1 ORF) • degradation of amino acids of the cysteine-aromatic group (1 ORF) • degradation of glycine (1 ORF) • other amino acid metabolism activities (5 ORFs) • nitrogen and sulfur metabolism (67 ORFs) • nitrogen and sulfur utilization (38 ORFs) • regulation of nitrogen and sulphur utilization (29 ORFs) • nucleotide metabolism (148 ORFs) • purine ribonucleotide metabolism (45 ORFs) • pyrimidine ribonucleotide metabolism (29 ORFs) • deoxyribonucleotide metabolism (11 ORFs) • metabolism of cyclic and unusual nucleotides (8 ORFs) • regulation of nucleotide metabolism (13 ORFs) • polynucleotide degradation (27 ORFs) • RNA degradation (4 ORFs) • nucleotide transport (14 ORFs) • other nucleotide-metabolism activities (7 ORFs) • phosphate metabolism (33 ORFs) • phosphate utilization (14 ORFs) • regulation of phosphate utilization (8 ORFs) • phosphate transport (10 ORFs) • other phosphate metabolism activities (1 ORF) • C-compound and carbohydrate metabolism (415 ORFs) • C-compound and carbohydrate utilization (261 ORFs) • C-compound, carbohydrate anabolism (1 ORF) • polysaccharide biosynthesis (1 ORF) • regulation of C-compound and carbohydrate utilization (120 ORFs) • C-compound, carbohydrate transport (42 ORFs) • other C-compound, carbohydrate metabolism activities (2 ORFs) • lipid, fatty-acid and isoprenoid metabolism (213 ORFs) • lipid, fatty-acid and isoprenoid biosynthesis (119 ORFs) • phospholipid biosynthesis (2 ORFs) • glycolipid biosynthesis (1 ORF) • isoprenoid biosynthesis (1 ORF) • tetracyclic and pentacyclic triterpenes (cholesterin, steroids and hopanoids) biosynthesis (1 ORF) • breakdown of lipids, fatty acids and isoprenoids (25 ORFs) • lipid, fatty-acid and isoprenoid utilization (26 ORFs) • regulation of lipid, fatty-acid and isoprenoid metabolism (20 ORFs) • lipid and fatty-acid transport (21 ORFs) • other lipid, fatty-acid and isoprenoid metabolism activities (13 ORFs) • metabolism of vitamins, cofactors, and prosthetic groups (86 ORFs) • biosynthesis of vitamins, cofactors, and prosthetic groups (63 ORFs) • utilization of vitamins, cofactors, and prosthetic groups (7 ORFs) • regulation of vitamins, cofactors, and prosthetic groups metabolism (3 ORFs) • transport of vitamins, cofactors, and prosthetic groups (3 ORFs) • other vitamin, cofactor, and prosthetic group metabolism activities (8 ORFs) • secondary metabolism (5 ORFs) • metabolism of primary metabolic sugars derivatives (1 ORF) • biosynthesis of glycosides (1 ORF) • biosynthesis of secondary products derived from primary amino acids (4 ORFs) • biosynthesis of amines (4 ORFs)

  7. Pathway and graphical function information in databases

  8. Genetic engineering of yeast for drug sensitivity assays • Problem: • Many hydrophobic low Mw compounds • do not enter the yeast cell • Yeast has multiple transport proteins involved • In drug transport: 35 in major facilitator super- • family, plus 14 ABC transporters • Each transporter is highly promiscuous • Solutions: • make mutants defective in membrane lipid • biosynthesis, e.g.erg6 • make multiple deletions of genes for • transporter proteins. • Nine-tuple deletant strain 100x more • sensitive to wide range of compounds

  9. The ”compendium” approach: Goal: to identify protein targets of drugs with unknown mechanism Principle: disturbance of a pathway by a drug or a mutation should yield similar phenotypes, including on transcript profiles Method: clustering of transcript profiles of yeast deletion mutants with experimental conditions Hughes et al., Cell 102:109 (2000)

  10. Transcript profile clustering identifies similarity between dyclonine treatment and disruption of ergosterol metabolism

  11. The two-hybrid system Gal4 AD Y Prey X Bait Gal4 DB Physical interaction between hybrid proteins activates reporter gene under control of Gal4 transcription factor HIS3 Gal4 binding site No growth on -His medium Gal4 AD Y’ X Gal4 DB HIS3 Growth on -His medium Gal4 binding site

  12. Network of physical protein complexes in yeast Gavin et al. Nature 415:141 (2002)

  13. Internet databases combine physical and genetic interaction information

  14. Genetic and physical interactions visualised by dedicated software

  15. Reverse two-hybrid: selection of interaction-disrupting agents and mapping of interaction domains AD AD Y Y X Z GBD LBD HIS3 URA3 Gal4 binding site LexA binding site • a) Mutagenize Y • or • b) Transform with • library encoding random • peptides, or add library of • organic compounds • 2. Select for 5-FOAR and His+ AD Y AD Y X Z GBD LBD HIS3 URA3 Gal4 binding site LexA binding site

  16. Extensions of two-hybrid for drug target identification: aptamers and DB-anchored drugs Selection for aptamers that bind to the bait Selection for aptamers that disrupt an interaction Selection for proteins that bind to a drug which is anchored to a DB through a covalent link to another drug

  17. Systematic analysis of synthetic lethality by SGA – ”synthetic gene arrays”

  18. SGA analysis clusters related genes and functional groups Two-dimensional hierarchical clustering of the synthetic genetic interactions determined by SGA analysis Network of genetically connected gene functions Tong et al., Science 303:808 – 813 (2004)

  19. The ”Seattle Project” Antitumor treatments (drugs, irradiation) are tested on sets of yeast mutants where different DNA repair and DNA damage response pwahtways are inactivated. Hypothesis: synthetic effects if the drug an the mutation in the tumour affect parallell pathways. Goal: individualised tumour therapy Fig. 3. Toxicity profiles of cytotoxic anticancer agents: topoisomerase poisons, X-rays, bleomycin, and actinomycin D. The graphs show the IC50 (log M for compounds, log k rad for X-rays) for each agent against the strain panel. The vertical line is set at the IC50 of the wild-type strain. The strains are grouped and color-coded according to the DNA damage response pathway they represent.

  20. Genetics • Scope • The causal relationship between the • genome (genotype) and properties of • the organism (phenotype) • Scope • The causal relationship between the • genome (genotype) and properties of • the organism (phenotype) • Scope • The causal relationship between the • genome (genotype) and properties of • the organism (phenotype) • Method • Observe properties of whole system • (cell, organism) altered in one (several) • genes • (Cf. physiology: observe whole system, • infer relationship between parts; • biochemistry: study gene products in • isolation)

  21. Forward genetics • Aim • - To go from function to gene • Means • Screen populations of mutants for • gain or loss of a particular function Reverse genetics Aim - To define the function(s) of a gene Means - Analyze a particular mutant for gain or loss of a variety of functions

  22. The geneticist's dilemma • Most mutations are recessive, loss-of-function • Most mutations confer sensitivity, not resistance to • a specific condition • Screening for resistance is easy, simply selecting will do • Screening for sensitivity is extremely labor-intensive, • involves replica-plating and visual inspection

  23. Control screen: tunicamycin • Step1: Heterozygous mutations in three loci conferred sensitivity: • ALG7 (Asn-linked glycosyl transferase; • previously known target) • YMR007w (unknown function) • YMR266w (membrane transport) • Step 2: test cognate homozygous mutations: • alg7/alg7 wild-type sensitivity • ymr007w/ymr007w supersensitive • ymr266w/ymr266w supersensitive •  Ymr007w and Ymr266w ruled out as direct drug • targets because of supersensitivity in the absence of • the gene product

  24. Direct drug effect on target (1) Normal situation Drug Output Smaller output

  25. Haploinsufficiency (less gene product) Drug Direct drug effect on target (2) Insufficient output (cell dies) Homozygous mutation (no gene product) Drug Insufficient output (cell dies) Insufficient output (cell dies)

  26. Indirect effects (drug binds to gene product related to the mutated one) Drug Output Output

  27. Indirect effects (2) Haploinsufficiency (heterozygous mutation) Homozygous mutation Drug Drug Insufficient output Insufficient output

  28. Principle of ”molecular bar-codes”: Synthetic DNA sequence (”tag” or ”bar- code”) is inserted adjacent to site of genomic disruption Due to flanking common sequences, the bar-code is PCR-amplifiable and can be hybridized to DNA array

  29. Competition experiments using the ”bar-code” concept

  30. Tunicamycin control • experiment: • array hybridization • quantification

  31. A sensor in yeast for ligand binding • deletion of the yeast’s own DHFR gene (dhr1) from the genome • insertion in mouse DHFR of heterologous amino acids with binding domains • from different proteins • ts variant of DHFR makes the protein extra sensitive to conformational • changes • binding of ligand gives increased stability of DHFR Tucker & Fields, Nature Biotechnology 19:1042 (2001)

  32. Growth is selective for ligand interaction Growth correlates with binding affinity

  33. Yeast-based p53 diagnosis • p53 is the most commonly mutated gene in human tumors • wide mutation spectrum, many mutated alleles • p53 is transcription factor; functional assay time-saving • variation: ADE2 gene as a reporter. Colony-color based readout • (ratio red/white colonies), diagnosis of heterozygous mutations possible

  34. 7-TM receptor in yeast eliminated • mammalian homolog expressed, • interacts with yeast G-protein • large numbers of yeast clones can • be screened efficiently • variety of marker genes makes • possible selection both for and • against interactions • Further improvements: • express human G-protein instead • of yeast homolog • delete FAR1 gene to prevent • cell cycle arrest as a result of • pathway activation

  35. Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeastChristine Klein et al.Nature Biotechnology 16, 1334 - 1337 (1998) • Human formyl peptide receptor like-1 (FPRL-1) receptor expressed in yeast • Expression of random peptides (13-mers) linked to secretion signal • Activation of receptor-coupled pathway linked to expression of HIS3 reporter • Three rounds of selection yielded 5 positive peptides out of 106 initial clones

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