Beruflich Dokumente
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Ameeta Agarwal Senior Scientist National Center for Natural Products Research University of Mississippi
Overview
First Half:
Second Half: Example of how DNA microarray technology has been used in investigating the mechanism of action of an antifungal natural product compound
~7.5 cm ~2.5 cm
(~20,000 genes)
~7.5 cm ~2.5 cm
(~20,000 genes)
~7.5 cm ~2.5 cm
~7.5 cm ~2.5 cm
(~20,000 genes)
(~20,000 genes)
cDNA Arrays
cDNA samples are spotted
Oligonucleotide Arrays
Oligonucleotide samples are spotted (consisting of a stretch of 60-70 nucleotides)
Gene
Transcription mRNA
Exon 1 Exon 2 Exon 3
Gene
Either cDNA or an oligonucleotide is placed on an array: - cDNA is made from mRNA using the enzyme Reverse Transcriptase. - An oligonucleotide is synthesized chemically.
Isolate RNA
Prepare cDNA
Label
Cy3
Isolate RNA
Prepare cDNA
Label
Gene expression changes in response to drug treatment are reflected in the RNA populations The two fluorescent dyes allow simultaneous detection of gene expression in the two samples Amount of hybridization to the DNA microarray slide corresponds to level of gene expression Differentially expressed genes are identified using a database from the microarray manufacturer
Data Analysis
Is the signal real? Is the data comparable across arrays (normalization)? Comparison between untreated and treated classes (modified t-tests) Data filtering (is useful data being compared?) - Remove genes with missing signal values - Remove genes with very low signal values - Remove genes with similar signal values across the arrays (unchanged genes)
Final result: Gene expression change = Signal in treated sample Signal in untreated sample
Treated (Cy5)
Untreated (Cy3)
Treated (Cy5)
Treated (signal)
Treated (signal)
Untreated (signal)
Untreated (signal)
Normalization corrects for hybridization differences between Cy3 and Cy5 samples (majority of genes should give equal hybridization) Global normalization (total intensity normalization) Use of house-keeping genes Lowess normalization (based on linear regression)
Statistical Analysis of Data: Class comparison between untreated class and treated class (probability test for difference between the two classes, i.e., deviation from 1.0)
B B
Control Cells (solvent-treated) Isolate RNA Prepare cDNA
B B
Hybridize target samples to DNA probes on a GeneChip
Biotin-labeled cRNA
B
Sample Cells
(compound-treated)
B B
Hybridize target samples to DNA probes on a GeneChip
B
Isolate RNA Prepare cDNA
Biotin-labeled cRNA
Wash chip Stain with fluorescent antibody Detect fluorescence signal Quantitate signals Identify differentially expressed genes
Each gene is represented ~32 times on the array Some gene elements are designed to give greater specificity Some gene elements are designed to give weaker specificity The Affymetrix software (AGCC) performs a modified t-test to determine if the signal is specific Result: One computed signal value per gene P-value to indicate significance Detection call P=Present (very strong specific signal) M=Marginal (weaker signal) A=Absent (very weak non-specific signal) Genes with P calls, strong signal values and low p-values are considered useful
Data Analysis: Class comparison between untreated & treated classes (probability test for difference between the two classes) Software used at NCNPR for statistical analysis of microarray data: BRBArrayTools
Initial data analysis identifies a list of genes that are differentially expressed under a given experimental condition, i.e., which genes are induced and which genes are repressed by a compound.
Further data analysis involves identifying the genes (gene annotation), determining what biochemical pathways they belong to, determining which biochemical pathways are over-represented in the data set, and interpreting the data.
Advanced data analysis involves comparing the data to previously described genomic profiles (searching databases, cluster analysis), and network analysis (is there cross-talk between the identified pathways).
Signal transduction, 3.1% Protein translocation, 1.5% Transcription, 3.1% Other function, 10.8%
Ergosterol
Classification of Compounds
Known MOA
Compound A Compound B
Unknown MOA
Compound X Compound Y Compound Z
(Membrane-disrupter) (DNA-damager)
DNA-damager
Are the appropriate tools available or accessible to me for DNA microarray studies?
- Are DNA microarrays available for my target organism? Is a model organism more suitable? - Is the appropriate instrumentation available to me? Can I find a collaborator who has the instrumen - Do I have the funding to conduct DNA microarray studies?
Cost of a drug MOA study with DNA microarrays GeneChip Cost: $1,380 (One chip = $230, need 6 chips) Reagents for target preparation: $600 (One target = $100, need 6 targets) Reagents for hybridization, staining, etc : $600 ($100 per chip) Miscellaneous supplies: $300 (media, plasticware, RNA isolation kits) Total: ~$3,000
Statistical considerations
- At least triplicates - Do I have access to appropriate statistical analysis software? Do I need training? Collaborator? - Are there appropriate databases available for downstream analyses?
Example of how DNA microarray technology has been used by our group to identify the MOA of the antifungal compound sampangine
N
N O
References: Agarwal AK, Xu T, Jacob MR, Feng Q, Lorenz MC, Walker LA, Clark AM (2008) Role of heme in the antifungal activity of the azaoxoaporphine alkaloid sampangine. Eukaryot Cell. 7:387-400. Huang Z, Chen K, Xu T, Zhang J, Li Y, Li W, Agarwal AK, Clark AM, Phillips JD, Pan X (2011) Sampangine inhibits heme biosynthesis in both yeast and human. Eukaryot Cell. 10:1536-1544.
DNA Microarray Experiment with Sampangine using the Model Yeast Saccharomyces cerevisiae
DMSO (0.25%)
Isolate RNA
Response to Stimulus
Cellular Process
Homeostasis
Transport Generation of Precursor Metabolites and Energy Ion Transport Cellular Respiration and Mitochondrial Electron Transport
SRX1 (15.1) AAD4 (6.7) GPX2 (5.1) AAD6 (4.6) GTT2 (4.3) CCP1 (3.9) TSA2 (2.7) CTA1 (2.6) PRX1 (2.6)
IZH4 (14.4) HES1 (11.4) IZH2 (2.9) ATF2 (2.9) UPC2 (2.7) SFK1 (2.7) ATF1 (2.7) ARE1 (2.0) YMR210W (-2.0) ERG20 (-2.1) MCR1 (-2.4) ERG28 (-2.6) ERG5 (-2.8) CYB5 (-3.0)
Sterol Transport
NDI1 (-2.2) COX15 (-2.2) COX6 (-2.3) MCR1 (-2.4) COX5B (-2.4) COX12 (-2.5) QCR7 (-2.6) DLD1 (-2.8) QCR9 (-2.8) CYC7 (-3.0) RIP1 (-3.1) COX13 (-3.4) QCR2 (-3.4) PET10 (-4.0) COX5A (-4.4) QCR10 (-5.2) NDE1 (-5.2) COX4 (-13.2) CYC1 (-13.3)
FRE1 (-2.2) ENB1 (-2.4) FRE4 (-2.6) FRE5 (-2.7) ARN1 (-3.3) SIT1 (-3.6) FIT3 (-5.2) FTR1 (-6.0) ARN2 (-9.0) FET3 (-11.0) FIT2 (-14.3)
DAN1 (67.5) TIR1 (9.9) DAN2 (3.7) TIR4 (3.0) DAN4 (2.5) TIR3 (2.5) DAN3 (2.5)
YLR126C (-3.3) SIT1 (-3.6) CCC2 (-5.0) HMX1 (-5.8) TIS11 (-8.6) ARN2 (-9.0) Other Processes
ANB1 (6.2) HEM13 (5.6) PAU1 (4.3) AAC3 (3.5) YLR413W (3.5) PAU6 (3.5) PAU4 (3.4) PAU3 (2.9) YGL039W (2.9) PAU7 (2.6) YJR116W (2.5) YGR131W (2.4) YLR437C (2.4) YMR002W (-2.0) YBR230C (-2.3) YOR215C (-2.5) YDL110C (-2.8) YHB1 (-3.7)
Cell wall mannoproteins Induced in anaerobic conditions Some are involved in sterol uptake
Induced in anaerobic conditions Involved in sterol uptake Transcription factor Regulates anaerobic genes Induced in anaerobic conditions
HEM13
Downregulated genes
COX12, COX13, COX15, COX4, COX5A, COX5B, COX6, CYC1, CYC7, QCR10, QCR2, QCR7, QCR9 ARN1, ARN2, FET3, FIT2, FIT3, FRE1, FRE4, FRE5, FTR1, HMX1, SIT1 Cytochrome c oxidases/reductases (mitochondrial electron transport) Iron uptake and homeostasis
ALA
Hem2
- Does SMP inhibit the heme synthesis pathway? - Does this inhibition cause accumulation of pathway intermediates? - Does this inhibition cause reduction in heme production? - Will exogenous heme rescue cells from SMP sensitivity? - Will mutants with deletions in the pathway genes show hypersensitivity to SMP? - Does SMP interfere with the activity of a specific enzyme in the pathway?
Porphobilinogen
Hem3
Preurogen
Hem4
Urogen III
Hem12
Coprogen III
Hem13
Protogen IX
Hem14
Protoporphyrin
Hem15
Heme
Mutant Analysis
-SMP
Wild-type
Checkerboard Assays
Biochemical Analysis
120
hem1
% Growth
100
+ 80 Heme
60 0.001 0.01 0.1 40 20 0 1 10
+SMP
Wild-type
15
7.5
-20
70 60 50 40 30 20 10 0
% Growth
hem1
120 100
+ SMP - SMP
+ Ergo
60 0.001 0.01 0.1 40 1 20 0 -20 10
80
Tr
Un
Uro
Uro po rphyrin
Copro III
Proto IX
Yeast cells
Human cells
SMP does not inhibit Hem12 activity SMP induces Hem3 and Hem4 activity
ALA
Hem2
Porphobilinogen
Hem3
Preurogen
Hem4 Hem3 Hem4 Hem12
Urogen III
Hem12
SMP
Coprogen III
Hem13
Protogen IX
Hem14
Protoporphyrin
Hem15
Xuewen Pan, Baylor College of Medicine (now at Novartis)
Heme
DNA microarrays are a starting point in a MOA study. Followup studies are necessary to identify the precise MOA of a drug. DNA microarrays only measure mRNA abundance, and dont account for changes in protein levels or metabolite levels
Use of proteomics and metabolomics technologies allow the measurement of proteins and metabolites respectively
Combining genomics, proteomics and metabolomics would be a powerful way of determining the global effects of drugs