DNA

1
Bacteria Must Regulate Gene Expression
Promoter
Operator
Open reading frame
DNA
RBS
mRNA
Protein(s)
2
The glmS Riboswitch-Ribozyme Regulates Gene
Expression
RBS
glmS
mRNA
Riboswitch A Messenger RNA Domain that Binds a
Metabolite and Controls Gene Expression

glucosamine-6-phosphate
Nature (2004) 428281-286
3
The glmS Ribozyme Could be an Antibacterial Drug
Target
Glms
Glucosamine-6-phosphate synthase
Cell wall precursors
fructose-6-phosphate
glucosamine-6-phosphate

• Synthesis of glucosamine-6-phosphate is
  essential for survival in many bacteria

• The glmS ribozyme forms an elaborately
  structured receptor akin to classical drug targets

glucosamine-6-phosphate

• A drug compound that targets the glmS riboswitch
  and repression expression of the glmS gene should
  be lethal

Science (2006) 3131752-1756
4
HTP Screen Design
light
fluorophore
DETECTOR
quencher
5
Addition of GlcN6P Increases the Fluorescence of
the Ribozyme
Cleavage of the bimolecular glmS ribozyme
DETECTOR
GlcN6P ribozyme
fluoresence
- GlcN6P ribozyme
GlcN6P - ribozyme
time (min)
Single tube pH 7.5
RNA Biology (2006) 3 77-81
6
glmS Reaction Requires Detergent in Microplates
microplate
7
HTP Screen Mirrors Biochemical Results
GlcN6P (1)
8
Library Screening Identified 4 Hits
fluoresence
Yales 20,000 compound library
well
glucosamine
9
Glucosamine Could Fit into the Ribozyme Pocket
glucosamine-6-phosphate
Science (2006) 3131752-1756
10
Conclusions

• The glmS riboswitch/ribozyme is amenable to HTS
• The first demonstration of HTS for riboswitches
  and the first screen completed at Yale
• Larger libraries in different chemical space may
  be needed to enable the discovery of novel glmS
  activators

11
The Future of Riboswitch HTS
12
Acknowledgments
Izabela Puskarz Jinsoo Lim Paul
Fletcher YCGS Ron Breaker Breaker Lab DARPA
NIH