biol518
DESCRIPTION
Biol518. Lecture 2 HTS and Antibiotic Drug Discovery. Modern Drug Discovery. Program Selection Target Selection/ Validation Assay Development HTS Lead Optimization Drug Candidate Selection Clinical Trials Drug Approval Follow-up Monitoring. HTS Workflow. - PowerPoint PPT PresentationTRANSCRIPT
Modern Drug DiscoveryP
rog
ram
S
ele
cti
on
Targ
et
Sele
cti
on
/V
alid
ati
on
Assay
Develo
pm
en
t
HTS
Lead
O
pti
miz
ati
on
Dru
g C
an
did
ate
Sele
cti
on
Clin
ical Tri
als
Dru
g A
pp
roval
Follow
-up
M
on
itori
ng
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
Reverse Chemical Genomics
Now we know many essential genes (whose products are essential), we can simply clone the genes and over-express 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
A CBkDa230
130
95
17
1 21 2
72
56
36
28
1 2
11
kDA230
56
28
130 9572
36
17
11
130
17
11
72
28
36
FabB (A) 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., over-expression 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
Antisense cellAntisense cell
Noprotein
XXAntisense RNAAntisense RNA
Inducible promoterInducible promoter
mRNAmRNA
Normal cellNormal cell
Protein
mRNAmRNA
Plasmid DNA
DNADNA
Shotgun Antisense Expression Determines Essentiality of Genes
Shotgun Antisense Expression Determines Essentiality of Genes
Non essential geneblocked by antisenseNon essential geneblocked by antisense
Essential geneblocked by antisenseEssential geneblocked by antisense
Millions of random DNA fragmentsMillions of random DNA fragments
No cell growthNo cell growthmRNAmRNA
Essential Protein
DNA
Pathogen genome
Ultra-Rapid Functional GenomicsUltra-Rapid Functional Genomics
Identify >100 essential gene drug targets per monthIdentify >100 essential gene drug targets per monthAntisense
(+ inducer)Antisense(+ inducer)
No antisense(- inducer)No antisense(- inducer)
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
Inhibitor-Target SpecificityInhibitor-Target Specificity
FabI Clone
MurAClone
TrpS Clone
(Real et al., submitted)
Target Identification Using Mixed Target Identification Using Mixed Clone AssayClone Assay
A BC
(Real et al., submitted)
Target Identification Using Target Identification Using Individual ArrayIndividual Array
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
A B
C D
indolmycin
phosphomycin triclosan
(Real et al., submitted)