Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies.
Bottom Line: The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors.Select purine scaffolds showed good affinities (K(D), 0.8-12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 μM.Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.
To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure-activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (K(D), 0.8-12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 μM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.
No MeSH data available.
Related in: MedlinePlus
Mentions: Our study shows that 2,6,9-trisubstituted purine scaffolds interact with Stat3, presumably at the three subpockets on the Stat3 SH2 domain surface (Figures 1A and 1D). The interaction with Stat3 is expected to disrupt Stat3 binding to its cognate pTyr peptide and hence, Stat3:Stat3 dimerization.6−8,16 Further evaluation of purine-scaffold molecules in in vitro Stat3 DNA-binding assay/electrophoretic mobility shift assay (EMSA) analysis, as previously reported,16 demonstrated that the select agents, S3I-V3-29, S3I-V3-30, S3I-V3-31, S3I-V3-32, S3I-V3-34, S3I-V4-01, and S3I-S2-21, which showed good binding affinity for Stat3 (Table 1, SPR), also strongly inhibited Stat3 activity, with IC50 values of 27−84 μM (Table 1, EMSA). Data from the EMSA analysis did not always show a good correlation to the SPR analysis. We note that although Stat3 dimerization disruptors may exhibit high potency in inhibiting dimerization, the activities in DNA-binding assay/EMSA analysis have generally been weaker.16 The reasons for this difference may be the presence of significantly large number of protein “targets” in the nuclear extract preparations utilized in the DNA-binding assay and the potential that disrupting preformed Stat3:Stat3 dimers, as in the DNA-binding assay is a more challenging task, as has previously been suggested.5,6 Immunoblotting analysis showed a moderate to strong inhibition by S3I-V3-31, S3I-V3-32, S3I-V3-34, and S3I-V4-01 of constitutive Stat3 phosphorylation in the v-Src transformed mouse fibroblasts (NIH3T3/v-Src) and the human breast cancer line, MDA-MB-231 that harbor aberrant Stat3 activity3,4 following treatments for 12, 24, or 48 h (Figure 2A(i) and Figure 2A(ii), pY705, lanes 2, 3, 5, 6, 8, 9, 11, 12, 15, and 18, compared to lanes 1, 4, 7, 10, 13, and 16, and data not shown). By contrast, same treatments have no effect on the constitutive levels of phospho- ErkMAPK (pErk1/2) or Src (pSrc) (Figure 2A). The inhibition of Stat3 phosphorylation, however, showed different kinetics among the purine compounds studied. Thus, additional time-course study was performed to provide more insight into the effects of the inhibitors. In NIH3T3/v-Src fibroblasts, treatment with S3I-V3-31 or S3I-V3-32 consistently inhibited Stat3 activity as early as 15−60 min (Figure 2A(iii), right two panels). Data also shows the Stat3 phosphorylation, which was inhibited early, appeared to be temporarily restored during prolonged treatment (Figure 2A(iii), NIH3T3/v-Src). Similarly, in the MDA-MB-231 line, Stat3 phosphorylation was consistently inhibited by S3I-V3-31 as early as 15−30 min following treatment, with evidence of restoration during treatment for 6 h (Figure 2A(iii), left, top two panels), while inhibition of Stat3 activation by S3I−V3−32 was strong at 6 h following treatment (Figure 2A(iii), left, bottom two panels). While data together suggests different kinetics of inhibition of intracellular constitutive Stat3 phosphorylation by purine scaffolds, data shows that, by 48 h following treatment, Stat3 phosphorylation remains low.
No MeSH data available.