Small molecule screening reveals a transcription-independent pro-survival function of androgen receptor in castration-resistant prostate cancer.

In prostate cancer (PCa) patients, initial responsiveness to androgen deprivation therapy is frequently followed by relapse due to development of treatment-resistant androgen-independent PCa. This is typically associated with acquisition of mutations in AR that allow activity as a transcription factor in the absence of ligand, indicating that androgen-independent PCa remains dependent on AR function. Our strategy to effectively target AR in androgen-independent PCa involved using a cell-based readout to isolate small molecules that inhibit AR transactivation function through mechanisms other than modulation of ligand binding. A number of the identified inhibitors were toxic to AR-expressing PCa cells regardless of their androgen dependence. Among these, some only suppressed PCa cell growth (ARTIS), while others induced cell death (ARTIK). ARTIK, but not ARTIS, compounds caused disappearance of AR protein from treated cells. siRNA against AR behaved like ARTIK compounds, while a dominant negative AR mutant that prevents AR-mediated transactivation but does not eliminate the protein showed only a growth suppressive effect. These observations reveal a transcription-independent function of AR that is essential for PCa cell viability and, therefore, is an ideal target for anti-PCa treatment. Indeed, several of the identified AR inhibitors demonstrated in vivo efficacy in mouse models of PCa and are candidates for pharmacologic optimization.

Small-molecule multidrug resistance-associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma.

The multidrug resistance-associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy.

Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-kappaB-dependent mechanism of p53 suppression in tumors.

Renal cell carcinomas (RCC) commonly retain wild-type but functionally inactive p53, which is repressed by an unknown dominant mechanism. To help reveal this mechanism, we screened a diverse chemical library for small molecules capable of restoring p53-dependent transactivation in RCC cells carrying a p53-responsive reporter. Among the compounds isolated were derivatives of 9-aminoacridine (9AA), including the antimalaria drug quinacrine, which strongly induced p53 function in RCC and other types of cancer cells. Induction of p53 by these compounds does not involve genotoxic stress and is mediated by suppression of NF-kappaB activity. In contrast to agents that target IkappaB kinase 2, 9AA and quinacrine can effectively suppress both basal and inducible activities of NF-kappaB, representing inhibitors of a previously undescribed type that convert NF-kappaB from a transactivator into a transrepressor, leading to accumulation of inactive nuclear complexes with unphosphorylated Ser-536 in the p65/RelA subunit. p53 function in RCC can be restored by ectopic expression of a superrepressor of IkappaB as effectively as by 9AA-derived compounds. These findings suggest that the complete or partial repression of p53 observed in many tumors can be the result of constitutive activation of NF-kappaB. The results demonstrate, in principle, the possibility to kill cancer cells selectively through simultaneous inhibition of NF-kappaB and activation of p53 by a single small molecule and suggest anticancer applications for the well known antimalaria drug quinacrine.