Transcript Biol518Lec2final-2 - Cal State LA

Biol518
Lecture 2
HTS and Antibiotic Drug Discovery
Follow-up
Monitoring
Drug Approval
Clinical Trials
Drug Candidate
Selection
Lead
Optimization
HTS
Assay
Development
Target Selection/
Validation
Program
Selection
Modern Drug Discovery
HTS Workflow
Traditional Approach: cell
growth inhibition
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Discovery of most antibiotics and
antifungal drugs was accomplished by
looking for cell growth inhibition by
natural compounds
Once potent compounds are identified,
their targets are discovered through
extensive biochemical and physiological
research
This is also a chemical genomics
approach
Yeast halo assay
Reverse Chemical Genomics
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Now we know many essential genes
(whose products are essential), we can
simply clone the genes and overexpress and purify proteins
Using purified proteins (enzymes), we
can search for compounds inhibiting
enzyme activity
Test compounds on cells to see if cell
growth is inhibited
Purified Potential Drug Targets
1
A
2
kDa
230
1
2
B
130
95
72
56
36
28
1
2
C
130
95
72
56
130
36
28
36
28
17
72
17
11
17
11
11
FabB (A)
kDA
230
Def (B)
FabD (C)
Traditional Paradigm with a twist
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Target-specific sensitized cell-based
assays (antisense expression)
Cell growth inhibition followed by rapid
target identifications (e.g., overexpression of essential genes)
Definitions:
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Essential genes – genes absolutely
required for growth and survival.
Essential proteins – proteins encoded
by essential genes that are required for
growth and survival.
Non essential genes – genes whose
destruction does not lead to significant
growth defects in a cell.
Why study essential genes?
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Essential genes are important for cellular
function and physiology; to study them
will reveal details about microbial
physiology
Practical application: essential genes
encode essential proteins which are
excellent drug targets to develop new
antibiotics
Strategies for Essential ID
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Saturated transposon mutagenesis
Systematic gene knock-out (or inability
to knock-out)
Antisense expression controlling gene
expression
Number of essential genes determined
for various bacterial species.
Species
No. of Essential Genes
Methods Used
Bacillus subtilis
271
Gene disruption
Mycoplasma genitalium
265-350
Transposon Mutagenesis
Streptococcus pneumoniae
113
Gene disruption
Haemophilus influenzae
478
Transposon Mutagenesis
Escherichia coli
620
Transposon Mutagenesis
E. coli (PEC data base)
250
Various methods
Staphylococcus aureus
150
Antisense expression
Staphylococcus aureus
168
Antisense expression
Typical bacterial species
206
Theoretical analyses
Transposition
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Transposons – DNA elements that can hop
(transpose) from one place in DNA to another
Transposons are known to exist in all
organisms on earth
Movement by a transposon is called
transposition, catalyzed by enzymes called
transposases
Transposons usually encode their own
transposases
Transposition
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Many transposons are essentially cut out of
one DNA and inserted into another
Other transposons are copied and then
inserted elsewhere
Donor DNA and target DNA
Structure of Bacterial Transposons
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All contain repeats at their ends, usually
inverted repeats (IR)
Presence of short direct repeats in the target
DNA that bracket the transposon
The sites of insertion are different among
target DNAs
Gene Knock-out
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Gene replacement (knock-out). The
purpose is to remove (knock-out) most
of one gene and see what happens to
the phenotype of the organism. Suicide
vector is used.
Gene Knock-In
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Gene replacement (knock-In): The
purpose is to disrupt the structure of a
gene by inserting a resistance marker
gene and see what happens to the
phenotype of the organism. Suicide
vector is used.
Also known as plasmid insertion
mutagenesis.
Gene Knock-In
A suicide plasmid containing an antibiotic resistance gene (AbR) is constructed to contain a small region of
sequence homology (denoted by the solid box) to orfX (denoted by the wide arrow). When the plasmid is
introduced into wild-type cells (W+), a single cross-over recombination event between these two regions of
homology leads to insertion of plasmid sequences and disruption of the orfX reading frame. The resulting
mutant is antibiotic resistant and defective for orfX (orfX−).
Antisense RNA
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Antisense RNA expression. Random
cloning and expression of short pieces
of genomic DNA on a plasmid in an
microorganism to elucidate the function
of the genes
Conditional Antisense Inhibition
of Protein Synthesis
Plasmid DNA
Protein
mRNA
Inducible
promoter
Antisense RNA
X
mRNA
DNA
Normal cell
No
protein
DNA
Antisense cell
Shotgun Antisense Expression
Determines Essentiality of Genes
Pathogen genome
Millions of random
DNA fragments
Non essential gene
blocked by antisense
Essential Protein
mRNA
DNA
Essential gene
blocked by antisense
No cell growth
Ultra-Rapid Functional Genomics
Antisense
(+ inducer)
No antisense
(- inducer)
Identify >100 essential gene
drug targets per month
Selective Sensitization
GyrA Clone – antibiotic profile
Xu et al, 2010
FabF Clone – antibiotic profile
Xu et al, 2010
IleS Clone – antibiotic profile
Xu et al, 2010
Over-expression of Essential
genes
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Concept: over-expression of a target
protein in a cell renders the cell
resistant to an inhibitor specifically
targeting the protein target
Strategy: create a large collection of cell
clones each over-expressing one
essential protein
Expose cell array to inhibitory
concentration of a compound -> cell
growth conferred by a specific clone
Over-expression of Essential
genes
Triclosan Dose Response
(Xu et al., 2006 BBRC)
Inhibitor-Target Specificity
MurAClone
TrpS Clone
FabI Clone
(Real et al., submitted)
Target Identification Using Mixed Clone
Assay
A
C
B
(Real et al., submitted)
Target Identification Using Individual
Array
(Real et al., submitted)
B
A
ArgS
AsnS
AspS
CysS
Efp
FabA
FbaA
FabD
FabG
FabI
FabZ
FtsE
FtsI
FtsX
FtsY
FtsZ
GyrA
GlnS
GlyS
HisS
LolD
LolE
MrdB
MurA
MurG
NrdB
NadE
PheS
PheT
PlsC
PrfA
PrfB
Ppa
RplE
RplJ
RpsL
RpoD
TrpS
SerS
Rho
MurI
MurD
MurF
PolA
TrmA
ThrS
TmK
ZipA
indolmycin
D
C
phosphomycin
triclosan