A series of chromone-based small molecules were identified, by high-throughput in silico screening81 and subsequent SAR analysis, to suppress activity of the SCFSKP2 E3 ligase and trigger p53-independent cellular senescence and apoptosis.82 Using
HiPCDock software, over 120,000 compounds were screened, of which 25 were
predicted to bind with SKP2. One of these compounds (26a) was subsequently shown to bind to SKP2 in vivo and inhibit the SKP2-SKP1 interaction, thus attenuating E3 ligase activity.
Structural analysis of the SCFSKP2 complex (Figure 18)83 had revealed the SKP2 protein interacts directly with SKP1 via its F-box domain. Along the SKP2-SKP1 interface, several residues were shown to contribute significantly to this binding interaction and were termed hot-spot residues. The authors concluded from their hot spot analysis that 19 SKP2 residues made direct contact with SKP1, forming two distinct pocket-like regions.82 Pocket 1 was located at the N-terminus of SKP2, within the F-box motif, and included residues W97, F109, E116, K119 and W127. Pocket 2 was close to the SKP2 C-terminus, formed by the leucine-rich repeat (LRR) region and several residues from the F-box.
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Figure 18: Quaternary structure of the SCFSKP2 complex, with the SKP2, SKP1, Cul-1 and RBX-1 proteins coloured in magenta, blue, green and red respectively. Labels A-E represent the 5 α-helices which make up a repeat unit in the cullin protein and three such repeats exist; adapted from83
Modelling of 33a in these binding regions of SKP2 indicated efficient binding in pocket 1 (Figure 19). The benzothiazole ring was predicted to make polar contacts and
aromatic stacking interactions with W97. The chromone moiety was extended through a pocket, formed by residues D98 and W127, and was predicted to make hydrogen bonds or hydrophobic/aromatic interactions with these amino acids. The ethyl group extended into the region where SKP1 normally bound, therefore inhibiting the interaction with SKP2 and its natural substrate. Finally, the piperidine ring was shown to interact closely with D98 and W127 and so was also essential for binding.82
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Figure 19: Predicted docking of 33a in pocket 1 of the SKP2 protein. Yellow dashed lines represent
hydrogen bonds between 33a and the protein. Residues in green form hydrogen bonds/hydrophobic/aromatic stacking interactions with compound 33a.82
In agreement with modelling predictions, analogues of 33a in which the benzothiazole was replaced by a smaller thiazole ring (33n and 33o) lost all binding affinity (Table 9) but the isosteric benzimidazole (33j) retained activity. Removal of the ethyl group (33b) abolished potency, in agreement with the hypothesis that the ethyl group was projecting into the SKP1-binding region and was thus preventing SKP1 from binding. Attempts to insert groups at the 2-position of the chromone scaffold were detrimental to potency (33k, 33l and 33m). The 4-chromone scaffold could be replaced by a 2-chromone and retain affinity (33f), which was predicted to be due to the molecule adopting a different binding conformation. In support of this theory, the ethyl group was no longer essential for activity in the 2-chromone series, as it was shown by modelling that the piperidine ring was occupying the space thought to be where the ethyl group binds in the 4-
chromone series. In the 4-chromone series, the piperidine ring was found to be essential, with ring-opened and substituted ring systems abrogating potency (33e, 33g, 33h and
33i). The model had predicted that the piperidine ring occupied a channel formed by
residues D98 and W127, and was also in close proximity to R126, therefore substituted derivatives would not be tolerated. Site-directed mutagenesis revealed that 33a could not inhibit binding between SKP2 W97A or D98A mutants with SKP1, highlighting the importance of these two residues in binding to 33a.
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Table 9: SAR studies of compound 26 and its derivatives82
ID R1 R2 R3 R4 Activity
33a Benzothiazole Et H -N(CH2)5 ACTIVE
33b Benzothiazole H H -N(CH2)5 Inactive
33c Benzothiazole H Me -N(CH2)6 Inactive
33d Benzothiazole H Me -N(CH2)4 Inactive
33e Benzothiazole H Me 4-N-Me-
piperazine
Inactive
33f See above for structure ACTIVE
33g Benzothiazole H Me 2-Me- piperidine Inactive 33h Benzothiazole Et H 4-Olamine- piperazine Inactive
33i Benzothiazole Et H -NEt2 Inactive
33j Benzimidazole Et H -N(CH2)5 ACTIVE 33k Benzothiazole Et CO2Et -N(CH2)5 Inactive 33l Benzothiazole n-Pr Me 4-N-Me- piperazine Inactive 33m Benzothiazole Et Me -N(CH2)5 Inactive 33n 4-Me-thiazole Et H -N(CH2)4 Inactive
33o 4-Me-thiazole Et H 3-Piperidin-
3-ylpyridine
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In vitro binding assays revealed 5 µM 33a could completely inhibit the SKP2-SKP1 interaction and a similar observation was made in vivo, with 33a impeding this
interaction in a dose-dependent manner (Figure 20a). In vivo p27 ubiquitination assays revealed 33a inhibited SKP2-mediated p27 ubiquitination and induced p27 and p21 expression in prostate cancer cells (Figure 20b).
A) B)
Figure 20: A) In vivo SKP2-SKP1 binding assay with or without #25 (33a) in PC3 cells; B) Treatment of
PC3 cells with either a vehicle or 26 for 24 h before harvesting by immunoblotting assay82
Compound 33a inhibited SKP2-mediated ubiquitination of known SKP2 substrates, including p27, p21 and Akt, however it showed no inhibiting effects on ubiquitination mediated by other SCF E3 ligases, including Fbw-7 and β-TrCP. Consequently, levels of c-Jun, MCL-1 (both Fbw-7 substrates), Snail and IκBα (both β-TrCP substrates) were unaffected upon treatment with 33a. Compound 33a was also shown to significantly reduce cell viability in PC3 cells, in contrast to normal prostate PNT1A cells, where cell viability was only slightly affected (Figure 21).
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Figure 21: Prostate cancer PC3 and LNCaP cells and normal prostate PNT1A cells were treated with
various doses of #25 (33a) before analysis by cell survival assay82
Compound 33a caused cell-cycle arrest at the G2/M phase of the cell cycle, an effect that was attenuated in SKP2-silenced cells. The evidence from these studies conclude that 33a can suppress cell viability of prostate cancer cells by inhibiting SKP2-mediated ubiquitination of cell cycle regulators, including p27, without affecting levels of other E3 ligase substrates and with only limited cytotoxicity in healthy prostate cells.