Transcript Biol518Lec2final - 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



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



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


Target-specific sensitized cell-based
assays (antisense expression)
Cell growth inhibition followed by rapid
target identifications (e.g., overexpression of essential genes)
Antisense RNA

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
FabF Clone – antibiotic profile
IleS Clone – antibiotic profile
Microbiological profiles
Molecular Interaction
Over-expression of Essential
genes



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