Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be ‘undruggable.’ A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.

Potassium cyanide (KCN) is an inhibitor of cytochrome C oxidase causing rapid death due to hypoxia. A well-characterized model of oral KCN intoxication is needed to test new therapeutics under the Food and Drug Administration Animal Rule. Clinical signs, plasma pH and lactate concentrations, biomarkers, histopathology, and cyanide and thiocyanate toxicokinetics were used to characterize the pathology of KCN intoxication in adult and juvenile mice. The acute oral LD50s were determined to be 11.8, 11.0, 10.9, and 9.9 mg/kg in water for adult male, adult female, juvenile male, and juvenile female mice, respectively. The time to death was rapid and dose dependent; juvenile mice had a shorter mean time to death. Juvenile mice displayed a more rapid onset and higher incidence of seizures. The time to observance of respiratory signs and prostration was rapid, but mice surviving beyond 2 hours generally recovered fully within 8 hours. At doses up to the LD50, there were no gross necropsy or microscopic findings clearly attributed to administration of KCN in juvenile or adult CD-1 mice from 24 hours to 28 days post-KCN challenge. Toxicokinetic analysis indicated rapid uptake, metabolism, and clearance of plasma cyanide. Potassium cyanide caused a rapid, dose-related decrease in blood pH and increase in serum lactate concentration. An increase in fatty acid-binding protein 3 was observed at 11.5 mg/kg KCN in adult but not in juvenile mice. These studies provide a characterization of KCN intoxication in adult and juvenile mice that can be used to screen or conduct preclinical efficacy studies of potential countermeasures.

Chimeric transcription factors have long been considered ideal anti-cancer targets since they are only present in tumor cells, however, their lack of enzymatic activity has placed them in a category of ‘undruggable’ proteins. The EWS-FLI1 fusion protein of Ewing Sarcoma (ES) has been validated as an anticancer target both alone and as a partner of RNA Helicase A (RHA). Our prior work identified the small molecule YK-4-279 as a specific inhibitor of EWS-FLI1 by blocking the interaction with RHA. Blocking this interaction leads to apoptosis. Data suggests that relatively long exposures of drug are necessary to keep EWS-FLI1 and RHA apart. We have modeled YK-4-279 treatment using cell lines (TC71, TC32, A4573, SK-ES, and RD-ES) to establish the duration of YK-4-279 exposure that leads to apoptosis. Apoptosis was measured by caspase-3 cleavage. A validated plasma detection method allows for HPLC measurement of YK-4-279. Pharmacokinetic (PK) models of YK-4-279 for both intraperitoneal (IP) and intravenous (IV) administration were developed in SD rats and BL6 mice. ES cell lines were exposed to YK-4-279 over a time-course, followed by a caspase-3 activity assay that demonstrated a minimum of 16 hours exposure was necessary to achieve maximal apoptosis using either 3 or 10 microM compound. A dose-dependency assay demonstrated that while apoptosis could be achieved with 30 - 80 microM YK-4-279 after 4 hours of treatment, caspase-3 activity was less than or equal to 3 microM for 16 hours. Rat PK modeling demonstrated a t1/2 of 30 minutes following IV administration. BL6 mice demonstrated similar kinetics to the rat. SCID/bg mice, which are necessary for tumor efficacy studies, demonstrated approximately 50% faster clearance than either rat or BL6 mice. Absolute bioavailability for IP administration was 41%. Models that use cell culture based targets for plasma levels and duration of exposure will be created to optimize IP dosing regimen. The optimized IP dosage and dosing intervals will be evaluated in tumor bearing animals in order to advance development of YK-4-279. The results of these studies will be used to guide toxicology studies in animals. In addition, pharmacodynamics models are being developed to compare YK-4-279 levels with functional activity. The combined results of these investigations will lead to human clinical trials for this first-in-chemical class, first-in-mechanism drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-316. doi:1538-7445.AM2012-LB-316

9575 Background: Chimeric transcription factors are ideal anti-cancer targets; however, they lack enzymatic activity thus rendering them ‘undruggable’ proteins. The EWS-FLI1 fusion protein of Ewing sarcoma (ES) has been validated as an anticancer target both alone and as a partner of RNA Helicase A (RHA). Our prior work identified the small molecule YK-4-279 as a specific inhibitor that blocks RHA from binding to EWS-FLI1. Blocking this interaction leads to apoptosis. We have modeled YK-4-279 treatment using ES cells to establish the duration of YK-4-279 exposure that leads to apoptosis. Methods: Apoptosis was measured by caspase-3 cleavage. A validated plasma detection method allows for HPLC measurement of YK-4-279. Pharmacokinetic (PK) models of YK-4-279 for both intraperitoneal (IP) and intravenous (IV) administration were developed in SD rats and BL6 mice. Results: ES cell lines were exposed to YK-4-279 over a time-course, followed by a caspase-3 activity assay that demonstrated a minimum of 16 hours exposure was necessary to achieve maximal apoptosis using either 3 or 10 microM compound. A dose-dependency assay demonstrated that while apoptosis could be achieved with 30 – 80 microM YK-4-279 after 4 hours of treatment, caspase-3 activity was less than or equal to 3 microM for 16 hours. Rat PK modeling demonstrated a t1/2 of 30 minutes following IV administration. BL6 mice demonstrated similar kinetics to the rat. SCID/bg mice, which are necessary for tumor efficacy studies, demonstrated approximately 50% faster clearance than either rat or BL6 mice. Absolute bioavailability for IP administration was 41%. Conclusions: Models will be created to optimize IP dosing regimen. The optimized IP dosage and dosing intervals will be evaluated in tumor bearing animals in order to advance development of YK-4-279. The results of these studies will be used to guide toxicology studies in animals. In addition, pharmacodynamics models are being developed to compare YK-4-279 levels with functional activity. The combined results of these investigations will lead to human clinical trials for this first-in-chemical class, first-in-mechanism drug candidate.