Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

We describe a novel, potent peptide substrate mimetic inhibitor of protein kinase B (PKB/Akt). The compound selectively kills prostate cancer cells, in which PKB is highly activated, but not normal cells, or cancer cells in which PKB is not activated. The inhibitor induces apoptosis and inhibits the phosphorylation of PKB substrates in prostate cancer cell lines and significantly increases the efficacy of chemotherapy agents to induce prostate cancer cell death, when given in combination. In vivo, the inhibitor exhibits a strong antitumor effect in two prostate cancer mouse models. Moreover, treated animals develop significantly less lung metastases compared to untreated ones, and the effect is accompanied by a significant decrease in blood PSA [prostate-specific antigen] levels in treated animals. This compound and its potential analogues may be developed into novel, potent, and safe anticancer agents, both as stand-alone treatment and in combination with other chemotherapy agents.

RX-3117 (fluorocyclopentenylcytosine) is a novel small-molecule chemotherapeutic agent that belongs to the class of cytotoxic antimetabolite cytidine analogs. Cytidine analogs, including gemcitabine, have been widely used for the treatment of various types of cancer, both hematologic as well as solid. However, despite the success of gemcitabine, there is no oral formulation of gemcitabine and drug resistance is common. Thus, RX-3117 was synthesized as a oral formulation to overcome gemcitabine resistance with a better pharmacologic profile. In this study, the efficacy of RX-3117 was examined in 12 different human tumor (colon, non-small cell lung, small cell lung, pancreatic, renal, ovarian and cervical) xenograft models, grown subcutaneously in athymic nude mice. Not only has RX-3117 demonstrated potent efficacy in several cancer xenograft models but also oral treatment with RX-3117 results in dose-dependent tumor growth inhibition (TGI), even in tumors that are only moderately sensitive or resistant to gemcitabine. In the Colo-205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 14%, 25%, and 0% TGI, respectively, whereas oral treatment with RX-3117 induced 93%, 91%, 62%, and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. In order to extend the results established in cell line xenograft models, and to test efficacy in a potentially more clinically relevant system, RX-3117 was evaluated in a single primary low passage human pancreatic Tumorgraft™CTG-0298, which is resistant to gemcitabine and has a favorable RX-3117 activating enzyme profile. Treatment with RX-3117 resulted in dose dependent TGI and was superior to the standard of care agent gemcitabine. This study successfully demonstrated preliminary efficacy and therapeutic potential of RX-3117. Citation Format: Mi Young Yang, Young Bok Lee, Deog Joong Kim, Chang-Ho Ahn, Joel Kaye, Tania Fine, Rina Kashi, Osnat Ohne. A novel small molecule cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to treatment with gemcitabine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2014-819