Catarina Malta

HDX-MS as a tool for the characterization of low-affinity fragments binding to human Cyclophilin D

Catarina F. Malta1,2*, Tiago M. Bandeiras1,2, Joerg Bomke3, Diana O. Silva1,2, Ulrich Graedler4, Pedro M. Matias1,2, Pedro M. F. Sousa1, Daniel Schwarz3, Alessio Bortoluzzi1*

1. iBET, Instituto de Biologia Experimental e Tecnológica, Av. da República, 2781-901 Oeiras, Portugal.
2. ITQB, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
3. Merck Healthcare KGaA – Discovery Pharmacology, Frankfurter Strasse 250, 64293 Darmstadt, Germany.
4. Merck Healthcare KGaA – Molecular Interaction and Biophysics, Frankfurter Strasse 250, 64293 Darmstadt, Germany.
* To whom correspondence should be addressed.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is emerging as a powerful biophysical technique for probing protein interactions, structure, and conformational dynamics. While HDX-MS is well-established for the characterization of potent compounds binding to their target proteins there are only few examples in the literature regarding the application of HDX-MS to the study of low-affinity fragments and none describing the usage of HDX-MS for molecules with affinities in the mM range.

Fragment-based drug design (FBDD) has emerged as an effective alternative to high throughput screening for the identification of potent compounds in drug discovery. Fragments are small molecules with a molecular weight typically below 300 Da. The fragment-based drug design starts with library screensthat generally result in fragment hits with weak affinities to their protein target. Then, the most promising hits are grown to more potent and selective compounds in a process that is often challenging and laborious. In this process structural information on the fragments binding mode is key for a successful optimization. HDX-MS is an attractive option to obtain this information, especially in cases where other high-resolution techniques, such as x-ray crystallography or NMR, do not work or are not an option.

In our work, we use human Cyclophilin D (CypD) as system to explore the possibility of using HDX-MS to characterize the binding of fragments with mM binding affinities. CypD is the mitochondrial isoform of Cyclophilins which plays an important role in the execution of cell death by regulating the mitochondrial permeability transition pore. Mitochondrial dysfunction has been implicated in a cascade of cellular processes related to several diseases as multiple sclerosis and cardiovascular disease, making CypD an interesting target for therapeutic intervention. Colleagues at Merck Healthcare KGaA synthesized fragments targeting the different binding sites of CypD: the aniline, the proline, and the pyrrole pockets. In our work we will show that HDX-MS can be used to monitor the response of CypD to these three different series of fragments with millimolar (mM) affinities. Initially, fragments with crystal structure information available were tested in order to validate the HDX-MS method. Then, fragments with no structural information were tested. These experiments revealed a reduction in deuterium uptake in the different regions of the protein, allowing the mapping of the different binding sites. Altogether our results show that HDX-MS could be a valuable tool in fragment-based drug design projects also when the initial hits present weak affinities. Additionally, we will provide technical information to help the design of HDX experiments with this class of challenging ligands.

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