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  • Screening a selection of analogues from this

    2024-06-05

    Screening a selection of analogues from this library against the panel of 26 kinases (16, 22, and 24 were screened at 30 μM due to poor solubility; 17 tested at 100 μM) generally demonstrated an increase in activity compared to DL-α-Difluoromethylornithine hydrochloride hydrate mg 3 (Fig. 3, Fig. 6). The benzamide analogue 16 demonstrated >50% inhibition against eight members of the panel including MPS1 and Aurora A (S(50%) = 0.31; Supplementary data Table 3), and the 3-methoxybenzamide analogue 17 also exhibited a selectivity score S(50%) of 0.31 (Fig. 6, Supplementary data Table 3). The N-methylpiperazine analogue 22 displayed higher inhibitory potencies compared to 16 while maintaining a selectivity score S(50%) of 0.38 (Fig. 6, Supplementary data Table 3). Analogue 24 showed high percentage inhibition against a large number of kinases in the panel (Fig. 6) with a S(50%) of 0.77 (Supplementary data Table 3). This trend is in agreement with the poor selectivity observed with the structurally related fragments 7 and 8 (Fig. 2, Fig. 3). The decrease in selectivity seen with analogue 24 relative to that observed with 16, 17, and 22 may be attributable to the aminopyrimidine system making additional or stronger interactions with the protein or acting as an alternative hinge binding motif. As compounds 16, 17, 22, and 24 often demonstrated > 50% inhibition against Aurora A, Aurora B and MPS1, inhibitory activities against these three kinases were again determined for the expanded compound library (Table 2). The benzamide analogue 16 displayed a 15-fold increase in inhibitory activity against MPS1 relative to that seen with 3, and also showed significantly higher inhibitory activity against Aurora A compared to that observed for compound 3 (Table 1, Table 2). Mono- or disubstitution at the meta-position of the benzamide 16 with methoxy groups (compounds 17 and 18) was not well tolerated and equivalent fluorine substitutions (compounds 19 and 20) led to significant loss of activity (Table 2). The selectivity of compound 17 inhibiting MPS1 over Aurora A and Aurora B could possibly be explained by differences in the main chain conformation along the hydrophobic channel leading to the solvent exposed surface. Introduction of solubilising groups such as N-methylpiperazine (compound 22) and piperidine (compound 23) retained good activity against all three kinases but resulted in a reduction in ligand efficiency due to the increase in the number of heavy atoms (Table 2). It should be noted that compounds 16 and 23 have been reported by Charrier et al. as Itk inhibitors. In order to gain further structural insights into the binding modes of active members of this library, we successfully co-crystallised compounds 22 and 23 with MPS1 (Fig. 7, Fig. 8) with both structures showing a hydrogen bonding pattern to the amino acid backbone of the hinge region identical to that seen with fragment 2 bound to MPS1 (Fig. 4). A further weak hydrogen bond between the nitrogen of the pyridyl ring in 23 and K553 was observed (2.6 Å) as shown in Fig. 7. The piperidine and N-methyl piperazine groups present in compounds 23 and 22, respectively, pointed out towards the solvent exposed surface as hypothesised (Fig. 7, Fig. 8). The pyridyl ring is also involved in hydrophobic packing between V539 and I663 as shown in Fig. 7. Further study of the protein environment of the pyridyl ring system may provide greater understanding of selectivity trends observed across the initial fragment library. Regarding MPS1 and Aurora kinases, it should be noted that MPS1 possesses a gatekeeper residue which is different to that seen in Aurora A and B kinases. Where MPS1 has a flexible methionine residue (M602), Aurora A and B have a leucine residue (L210 in Aurora A) as shown in Fig. 8. In addition, MPS1 possesses an isoleucine (I663) residue that is involved in hydrophobic packing, the corresponding residue in Aurora A and B is alanine (A273 in Aurora A) (Fig. 8). As previously stated, introduction of a benzamide or substituted benzamide group resulted in a loss in selectivity, both between MPS1 and Aurora A/B, and against the panel of 26 kinases. The hydrophobic channel, into which these groups project, is a common feature observed in kinases. Although the amino acid sequence of the hydrophobic channel is not as highly conserved as that of the hinge region, features of this channel are relatively similar. Since the benzamide analogues 16, 17, 22 and 24 show greater percentage inhibition against a large number of the kinases in the panel, it could be assumed that they form non-directional hydrophobic interactions in this region. These findings are in agreement with previous reports suggesting that elaboration of selective fragments does not always result in selective compounds.