6 research outputs found
Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances
Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery
Identification of Differential Protein Binding Affinities in an Atropisomeric Pharmaceutical Compound by Noncovalent Mass Spectrometry, Equilibrium Dialysis, and Nuclear Magnetic Resonance
Identification of Differential Protein Binding Affinities in an Atropisomeric Pharmaceutical Compound by Noncovalent Mass Spectrometry, Equilibrium Dialysis, and Nuclear Magnetic Resonance
Atropisomerism of pharmaceutical
compounds is a challenging area
for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398−6401). Strategies
for dealing with these compounds include raising the energy barrier
to atropisomerization in order to develop the drug as a single isomer
(Tetrahedron 2004, 60, 4337−4347) or reducing the barrier to rotation and
developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364−371). Commonly, however, the atropisomers will be differentiated
in terms of their affinity for a given protein target, and it is therefore
important to rapidly identify the most active component prior to further
compound development. We present equilibrium dialysis and saturation
transfer difference NMR (STD-NMR) as techniques for assessing relative
affinities of an atropisomeric mixture against antiapoptotic protein
targets Bcl-2 and Bcl-x<sub>L</sub>. These techniques require no prior
separation of the mixture of compounds and are therefore rapid and
simple approaches. We also explore the use of noncovalent mass spectrometry
for determining <i>K</i><sub>D</sub> values of individual
atropisomers separated from the equilibrium mixture and compare the
results to solution-phase measurements. Results from equilibrium dialysis,
STD-NMR, and noncovalent mass spectrometry are all in excellent agreement
and provide complementary information on differential binding, amplification
of the strongest binders, and <i>K</i><sub>D</sub> values
Automated Protein–Ligand Interaction Screening by Mass Spectrometry
Identifying protein–ligand binding interactions
is a key
step during early-stage drug discovery. Existing screening techniques
are often associated with drawbacks such as low throughput, high sample
consumption, and dynamic range limitations. The increasing use of
fragment-based drug discovery (FBDD) demands that these techniques
also detect very weak interactions (mM <i>K</i><sub>D</sub> values). This paper presents the development and validation of a
fully automated screen by mass spectrometry, capable of detecting
fragment binding into the millimolar <i>K</i><sub>D</sub> range. Low sample consumption, high throughput, and wide dynamic
range make this a highly attractive, orthogonal approach. The method
was applied to screen 157 compounds in 6 h against the anti-apoptotic
protein target Bcl-x<sub>L</sub>. Mass spectrometry results were validated
using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques
suggests that mass spectrometry offers a powerful, complementary approach
for screening