A comparative study of fragment screening methods on the p38α kinase: new methods, new insights

Abstract

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Fig. 1

Calculated physiochemical properties of the BioFocus fragment library: (a) molecular weight, (b) calculated logP, (c) number hydrogen bond donors, (d) number of hydrogen bond acceptors, (e) number of rotatable bonds

Fig. 2

Plot of BioFocus fragment library diversity coverage showing 20,000 clusters compared with clusters representing the ChEMBL database

Fig. 3

Structures fragment hits described in Table 1

Fig. 4

SPR analysis of binding competition between SB203580 and the four fragments analyzed by X-ray crystallography

Fig. 5

Comparison of hits identified in the SPR and LC3000 MSA screens. Two hundred and sixty-six fragments were tested in SPR at 200 μM and in the LC3000 MSA screen at 250 μM and 1 mM. Of those fragments, 102 were primary hits from SPR with TR_max > 50%, 10 were (confirmed) hits in LC3000 (<50% Ar at 1 mM and <70% Ar at 250 μM)

Fig. 6

Comparison of activities obtained for 42 fragments tested in the LC3000 MSA and FLT assays. Fragment concentration was 250 μM in both assays. ATP concentration was 10 μM in the MSA and 150 μM in FLT. % Ar are relative activities with regards to no enzyme (0%) and full reaction (100%) control raw data

Fig. 7

Comparison of the three screen technologies. Twenty-six compounds tested in all technologies were considered. Due to the selection process followed, all were primary hits in the SPR screening. Hit criterion SPR: TR_max > 50% (includes the 24 confirmed hits), Hit criteria LC3000 MSA: > 50% inhibition at 1 mM and >30% inhibition at 250 μM, hit criterion FLT: > 30% inhibition at 250 μM

Fig. 8

Structures fragments described in Table 2

Fig. 9

Structures of compounds (3–6) analyzed by X ray crystallography with binding mode

Fig. 10

Overlay of fragments binding to p38α; hinge (magenta) and activation loop (orange). Compounds 3, 4 and 5 bound to the active site and compound 6 bound to a distal site

Fig. 11

Binding mode of compounds 3–6 in complex with p38α

Scheme 1

Synthesis of triazole analogs. R is defined in Table 4

Fig. 12

Crystal structure of biphenyl 41 bound to the allosteric pocket