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Proceedings -Thursday, October 11, 2001
ThOA1
Predictive Method for Drug Discovery:
Screening Using Fourier Transform Mass Spectrometry
Steven Hofstadler, Ibis Therapeutics
Background
DNA is transcribed into RNA and RNA is translated into proteins.
Most therapeutic efforts focus on binding small molecules to these
proteins. At Isis, RNA is seen as a therapeutic target. Isis makes a
synthetic RNA molecule which has a complementary sequence; it
prevents a specific protein from being made. Mass spectrometry is
used to find these binders. Structured RNAs have thus emerged as an
exciting new target for small molecule therapeutics.
Premise:
With the advent of mild ionization techniques, such as ESI, it
became possible not only to obtain low fragmentation mass spectra of
individual mixture compounds but also to detect and analyze their
non-covalent complexes formed in solution. The Fourier transform ion
cyclotron resonance (FTICR) mass spectrometry offers particular
advantages for the study of non-covalent complexes. Besides high
sensitivity, FTICR MS provides mild interface conditions that can be
used to obtain intact non-covalent complex ions with a good signal
to noise ratio. Another unique feature of the method is its ability
to selectively accumulate certain ions in the trap allowing one to
study minor mixture components.
High throughput screening mode is used to identify small molecules
that bind RNA targets; FTICR is seen as an alternative to classical
high-throughput screening of combinatorial libraries. The MASS
(Multitarget Affinity/Specificity Screening) assay takes advantage
of the "intrinsic mass" label of each compound and target RNA by
employing high resolution, high precision mass measurements. The
ability to analyze complex mixtures allows large mixtures to be
screened in the presence of multiple RNA targets simultaneously. The
identity of the small molecule(s) which bind, the RNA target to
which it binds, the compound-specific binding affinity, and the
location of the binding site on the RNA can be determined in one set
of rapid experiments. The MASS assay detects noncovalent complexes
with dissociation constants of < ˜5 mM, with high sensitivity and is
presently being employed to screen large compound collections
against multiple RNA targets.
Some throughput numbers reported are as follows:
- Rapidly evaluate the affinity of compounds for targets
- 55 sec / well
- 3 targets x 10 compounds / well
- 16 plates / day
- 16,000 samples per day or 48,000 targets / day
- Run nonstop for 4 days then give MS a break for cleaning
- Injection volume is 7.5 uL / well
- Can detect 18 pmol RNA/target/well
A ligand-based drug discovery strategy based on SAR was also
presented. Briefly, the strategy is based on using a mass
spectrometry to measure the affinity of chemical motifs for a
target; performing competitive binding experiments and molecular
modeling with the motifs to determine the binding site(s) of the
ligands; fusing binding motifs into a more complex structure to
afford higher affinity compounds, and identifying the appropriate
linker group using MS. Using this approach, ligand binding is
independent of other ligands in solution. Throughput was expressed
at 3 targets analyzed with 25 compounds/well, and each compound at a
concentration of 50 uM.
Links
Steven Hofstadler and Richard Griffey, "Mass spectrometry as a drug
discovery platform against RNA targets." Current Opinion in Drug
Discovery & Development 3(4), 423-431 (2000).
JE Bruce, GA Anderson, R Chen, X Cheng, DC Gale, SA Hofstadler, BL
Schwartz and RD Smith, "Bio-affinity characterization mass
spectrometry." Rapid Commun Mass Spectrom 9, 644-650 (1995).
A general overview is provided for Fourier-transform ion cyclotron
resonance mass spectrometry that, in combination with ESI, allows
the investigation of specific non-covalent complexes formed in
solution.
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