Abstract
Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (KD) = mM − μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target—cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with KD < 200 μM were detected, including a few highly efficient GAK binders (KD of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.
Highlights
During the last 10 years, the fragment-based drug discovery (FBDD) paradigm, where a lead compound is evolved from small molecular fragments that bind to a biological target, has to an increasing extent been implemented both within academia and the pharmaceutical industry [1,2,3]
Through virtual screening by Glide, 170 fragments were selected from a fragment based library of 3,200 substances on the basis of their average internal rank towards the ATPbinding site of five crystal structures of four kinases of different types and one chaperone (MST3, heat shock protein 90 (Hsp90), Jun N-terminal kinase 3 (JNK3), protein kinase A (PKA), and vascular endothelial growth factor (VEGFR)). The structures of these randomly selected proteins with an ATP-binding site were used for virtual screening as there were no crystal structures available for G-associated kinase (GAK) with a docked inhibitor that could be used for ligand docking by Glide
The object was to find fragments with affinity to the ATP-binding site, which would produce a set of fragments that were likely to bind to GAK or other kinases due to the conserved nature of the ATP-binding site
Summary
During the last 10 years, the fragment-based drug discovery (FBDD) paradigm, where a lead compound is evolved from small molecular fragments that bind to a biological target, has to an increasing extent been implemented both within academia and the pharmaceutical industry [1,2,3]. It is believed that FBDD enables the development of inhibitors against difficult targets where HTS has failed [5]. The concept of FBDD was presented by Jencks in 1981 [6]. Subsequent linking of the two fragments to each other resulted in a compound with nanomolar affinity to the target, demonstrating the concept of FBDD in practice [7]
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