Automated Protein–Ligand Interaction Screening by Mass Spectrometry

Abstract

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 K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) 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(L). 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.

Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90

“…This is likely due to the fact that ESI-MS is a direct binding method able to probe all protein surfaces, allowing in this way the discovery of ligands that interact, in addition to the investigated active site, also through other binding sites such as allosteric cavities. However, we cannot exclude that unspecific adducts could be formed in the ion source during ionization process leading to false-positive results, and the formation of non specific complexes was described in previous papers (Maple et al, 2012;Mathur et al, 2007;Peschke et al, 2004) and is a limitation of this approach. Considering these facts, among the hits identified, it is important to discriminate between the different possible mechanisms of inhibition (i.e.…”

“…Compared to the previously described medium to high throughput methods, commonly based on infusion (Gao et al, 1996;Greig and Robinson, 2000;Maple et al, 2012;Vallee et al, 2011;Vivat Hannah et al, 2009;Wigger et al, 2002), our procedure is substantially different and makes use of a flow injection operation mode.…”

“…Some of the more recent methods make use of an automated nano-electrospray ion source, based on a chip platform for sample infusion analysis (Benkestock et al, 2005(Benkestock et al, , 2003Jecklin et al, 2009c;Vivat Hannah et al, 2009;Wortmann et al, 2008), that was also applied to fragment based screening (Maple et al, 2012;Vivat Hannah et al, 2009). Compared to these methods, AFI-MS makes use of a flow injection operation mode; samples are injected in a micro-flow by an autosampler and reach in few seconds the mass spectrometer ion source.…”

Structure-Based Discovery of Small Molecules Binding to RNA

“…The method is sensitive enough to detect ligands with K D -values in the typical range of fragments. However, one of the drawbacks is the difficulty in identifying nonspecific binding [103].…”

Le criblage de fragments

“…En général, le criblage de fragments est réalisé avec une première technique, puis confirmé par une seconde approche. D'autres techniques ont aussi été utilisées, comme la spectrométrie de masse en conditions douces [12], la chromatographie à faible affinité [13], la mesure de la température de dénaturation des protéines, ou encore la calorimétrie [14]. La calorimétrie permet d'obtenir des données cruciales pour développer des molécules spécifiques, en favorisant les interactions dues à l'enthalpie, via des interactions polaires telles que liaison hydrogène ou attraction électrostatique.…”