No abstract

Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

As part of our program to explore the influence of small structural modifications of our drug candidate, 3β-(hydroxy)-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene (galeterone, 5) on the modulation of the androgen receptor (AR), we have prepared and evaluated a series of novel C-3, C-16 and C-17 analogs. Using structure activity analysis, we established that the benzimidazole moiety at C-17 is essential and optimal and also that hydrophilic and heteroaromatic groups at C-3 enhance both anti-proliferative (AP) and AR degrading (ARD) activities. The most potent anti-proliferative compounds were 3β-(1H-imidazole-1-carboxylate)- 17-(1H-benzimidazol-1-yl)-androsta-5,16-diene (47), 3-((EZ)-hydroximino)-17-(1Hbenzimidazol- 1-yl)-androsta-4,16-diene (36), 3β-(pyridine-4-carboxylate)-17-(1H-benzimidazol- 1-yl)-androsta-5,16-diene (43), with GI50 values of 0.87, 1.91 and 2.57 μM, respectively. Compared to 5, compound 47 was 4- and 8-fold more potent with respect to AP and ARD activities, respectively. Importantly, we also discovered that our compounds, including 5, 36, 43 and 47 could degrade both full-length and truncated AR in CWR22rv1 human prostate cancer cells. With these activities, their potential for development as new drugs for the treatment of all forms of prostate cancer.

Some retinoic acid metabolism blocking agents (RAMBAs) are known to exhibit a wide range of anticancer activities by mechanisms that are still not completely resolved. This study investigated the anticancer efficacy and mechanism(s) of novel RAMBA retinamides (RRs) in triple negative and Her-2 overexpressing breast cancer cells. Specifically, we examined the possibility that RRs affect the translational machinery in these breast cancer (BC) cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine209 phosphorylation by upstream MAPK-interacting kinases (Mnks). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating BCs, particularly for Her2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. We report for the first time that the degradation of Mnk1 by RRs in BC cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, colonization, invasion, and migration and induce apoptosis. Most importantly, the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (cercosporamide and CGP57380). Furthermore, RRs potencies on peIF4E down-regulation and growth inhibition were superior to those of two clinically relevant retinoids and the Mnk inhibitors. Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk/eIF4E based therapeutic treatment of breast cancers.

Galantamine, a drug used to treat Alzheimer's disease, is a nicotinic allosteric potentiating ligand, and kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is an endogenous noncompetitive inhibitor of ␣7* nicotinic receptors (nAChRs) [the asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Pharmacol Rev 51:397-401, 1999)]. Here, possible interactions between KYNA and galantamine at ␣7* nAChRs were examined in vitro and in vivo. In the presence of tetrodotoxin (TTX), approximately 85% of cultured hippocampal neurons responded to choline (0.3-30 mM) with ␣7* nAChR-subserved whole-cell (type IA) currents. In the absence of TTX and in the presence of glutamate receptor antagonists, choline triggered inhibitory postsynaptic currents (IPSCs) by activating ␣7* nAChRs on GABAergic neurons synapsing onto the neurons under study. Galantamine (1-10 M) potentiated, whereas KYNA (10 nM-1 mM) inhibited, choline-triggered responses. Galantamine (1 M), applied before KYNA, shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC 50 of KYNA from 13.9 Ϯ 8.3 to 271 Ϯ 131 M. Galantamine, applied before or after KYNA, antagonized inhibition of choline-triggered IPSCs by KYNA. Local infusion of KYNA (100 nM) in the rat striatum reduced extracellular dopamine levels in vivo. This effect resulted from ␣7* nAChR inhibition and was blocked by coapplied galantamine (1-5 M). It is concluded that galantamine competitively antagonizes the actions of KYNA on ␣7* nAChRs. Reducing ␣7* nAChR inhibition by endogenous KYNA may be an important determinant of the effectiveness of galantamine in neurological and psychiatric disorders associated with decreased ␣7* nAChR activity in the brain.

We have developed a procedure for the synthesis of N-hydroxy-N(1)-phenyloctanediamide (suberoylanilide hydroxamic acid (SAHA)), providing the product in 79.8% yield. SAHA is a potent inhibitor of histone deacetylase, induces differentiation and/or apoptosis in certain transformed cells in culture, and suppressed the growth of human prostate cancer LNCaP and PC-3 cell lines. The combination of SAHA with other compounds inhibited cell proliferation of LNCaP cells in additive fashion and resulted in synergistic growth inhibition.

Bypassing tyrosine kinases responsible for Stat5a/b phosphorylation would be advantageous for therapy development for Stat5a/b-regulated cancers. Here, we sought to identify small-molecule inhibitors of Stat5a/b for lead optimization and therapy development for prostate cancer (PC) and Bcr-Abl-driven leukemias. In silico screening of chemical structure databases combined with medicinal chemistry was used for identification of a panel of small-molecule inhibitors to block SH2-domain-mediated docking of Stat5a/b to the receptor-kinase complex and subsequent phosphorylation and dimerization. We tested the efficacy of the lead-compound, IST5-002, in experimental models and patient samples of two known Stat5a/b-driven cancers, prostate cancer (PC) and chronic myeloid leukemia (CML). The lead compound Inhibitor of Stat5-002 (IST5-002) prevented both Jak2 and Bcr-Abl-mediated phosphorylation and dimerization of Stat5a/b, and selectively inhibited transcriptional activity of Stat5a (IC50 1.5 μM) and Stat5b (IC50 3.5 μM). IST5-002 suppressed nuclear translocation of Stat5a/b, binding to DNA and Stat5a/b target gene expression. IST5-002 induced extensive apoptosis of PC cells, impaired growth of PC xenograft tumors and induced cell death in patient-derived PCs when tested ex vivo in explant organ cultures. Importantly, IST5-002 induced robust apoptotic death not only of imatinib-sensitive but also imatinib-resistant chronic myeloid leukemia (CML) cell lines and primary CML cells from patients. IST5-002 provides a lead structure for further chemical modifications for clinical development for Stat5a/b-driven solid tumors and hematological malignancies.

In a continuing study of our clinical candidate 5 (VN/124-1 or TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ~2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.