Design and Characterization of Libraries of Molecular Fragments for Use in NMR Screening against Protein Targets

Abstract

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.

Opportunity Knocks: Organic Chemistry for Fragment‐Based Drug Discovery (FBDD)

“…[7] Properties that we find useful are listed in Table 1. Many of these are easily calculated or predicted prior to synthesis and we always calculate lipophilicity (clogP) and MW before deciding which fragments to add to our library.…”

Structural genomics of protein phosphatases

“…Various approaches to fragment binding detection have been adopted, among them mass spectrometry [81], NMR [82,83], crystallography [84,85] and combinations there-from [86,87]. Special considerations for fragment based discovery using X-ray crystallography include the necessity for obtaining a well characterized crystal form of the target protein which diffracts well (d min < 2.5Å), possesses a lattice amenable to soaking experiments (i.e., without occlusion of the target site), and is able to withstand exposure to modest quantities of organic solvents (e.g., ethanol and DMSO are popular solvents for chemical fragments).…”

Targeting Norovirus Infection—Multivalent Entry Inhibitor Design Based on NMR Experiments

“…[29] In the case of VLPs as target, substantially higher hit-rates are observed than reported before for smaller protein targets [29,30] because the sensitivity of the NMR readout is much higher in the case of very large protein targets (VLPs). Also, we have chosen the experimental conditions for STD NMR experiments such that identification of hits is "safe" even in mixtures with severe signal overlap.…”