High throughput screening of a small molecule one-bead-one-compound combinatorial library to identify attenuators of p21 as chemotherapy sensitizers

Abstract

Kidney cancer is notoriously difficult to treat when metastatic due to its resistance to conventional chemotherapy. p21 is a cyclin kinase inhibitor which, in many tumor cell lines, conveys an antiapoptotic function through its induction by the DNA damage responsive p53 pathway, such that attenuation of p21 sensitizes several disparate cancer cell lines to DNA-damaging chemotherapy. Since clinical applications with therapeutic antisense and siRNA approaches are problematic, we sought to discover other methods to inhibit p21 which are more readily translatable to the clinic. Utilizing an on-bead enzyme-linked colorimetric binding assay, we screened a diverse one-bead-one-compound combinatorial small molecule library and identified 12 candidate compounds which bind p21. Each of the 12 candidate compounds was synthesized and tested individually and three ligands were found which had the highest p21 binding affinity and yielded similar chemical structure. These three compounds caused dose-dependent cytotoxicity as well as apoptosis when exposed to two RCC cell lines. In addition, these compounds sensitized cells to apoptosis when incubated with doxorubicin such that a lower dose of doxorubicin was required in the presence of the compounds for equivalent cell killing. Interestingly, a representative of the three compounds decreased p21 levels by specific induction of ubiquitin-dependent proteosome degradation. Thus, by high throughput screening of thousands of candidate small molecules, we have identified compounds which attenuate p21, cause RCC cell apoptosis and sensitize RCC cells to DNA-damaging chemotherapy. These compounds are currently being evaluated in in vivo assays as potential novel therapeutic for RCC.

Salinomycin, a p-glycoprotein inhibitor, sensitizes radiation-treated cancer cells by increasing DNA damage and inducing G2 arrest

“…Since it is reported that DOX-or ETO-mediated increased p21 expression is reduced by co-treatment with Sal [14], we also tested whether Sal could reduce increased p21 levels resulting from radiation. Previously, the p21 tumor suppressor protein was shown to exert an antiapoptotic effect, thereby promoting cell survival following DNA damage [25][26][27][28][29]. We found that Sal also reduced p21 levels in radiation-treated cells, suggesting that the mechanism of Sal sensitization is conserved in nonchemotherapeutic DNA damaging drug action.…”

CD4+ T cells from elite controllers resist HIV-1 infection by selective upregulation of p21

“…15 (diluted in DMSO; ref. 24) was added at a concentration of 2 μM 48 hours prior to infection; control samples were treated with DMSO alone.…”

Small-Molecule Inhibitors of p21 As Novel Therapeutics For Chemotherapy-Resistant Kidney Cancer

“…These include butyrolactone I (BL) [11], LLW10 [12], sorafenib [13] and recently reported UC2288 [14]. BL is a competitive inhibitor of ATP for binding and activation of CDK.…”

“…We have previously discovered a small molecule p21 inhibitor, LLW10, using a one-bead one-compound combinatorial chemistry approach [12]. Rather than causing inhibition of p21 activity, LLW10 binds to p21 and induces ubiquitinization and proteosomal degradation of p21 and consequent sensitization of chemotherapy-induced apoptosis in two RCC cell lines.…”