Ketamine analgesia is not related to an opiate action in the periaqueductal gray region of the rat brain.

Ketamine is an injectable anesthetic agent that has been shown to interact as an agonist at opiate receptors. In addition, its antinociceptive action in rats is antagonized by the narcotic receptor antagonist naloxone. Thus it was assumed that the anesthetic may activate the pain inhibitory pathway, originating in the periaqueductal gray (PAG) and descending into the spinal cord, in a manner similar to that of narcotics like morphine. In the present study, it was verified that the systemic administration of naloxone (3 mg/kg i.p.) antagonized the elevation in tail-flick latency produced by an anesthetic dose of ketamine (160 mg/kg i.p.), but did not alter the duration of anesthesia (defined as duration of the loss of the righting reflex). However, when naloxone (3 micrograms/0.5 microliter/30 sec) was given by microinjection into the PAG it was found to be ineffective against the ketamine-induced elevation of the tail-flick latency. In contrast, the microinjection of the antagonist significantly attenuated (halved) the elevated latency in response to systemically administered morphine (4 mg/kg s.c.). It was also shown that ketamine was unable to elicit an increase in the latency of the tail-flick reflex when administered directly into the PAG over a wide range (0.10-100 micrograms) of doses. On the other hand, a local anesthetic-like effect of ketamine, known to occur when the drug is used in high concentration, was observed when doses exceeding 0.1 microgram were injected into the PAG. This action interfered with opiate actions in the PAG and made data from the microinjection studies difficult to interpret. The descending, pain inhibitory neuronal system originating in the PAG does not appear to participate in the antinociceptive action of ketamine measured by the tail-flick reflex. Perhaps the drug's effects are associated with alternative opiate mechanisms and/or opiate receptor subtypes not present on the cells of origin of the descending nerves within the PAG.

Single enantiomer of YK-4-279 demonstrates specificity in targeting the oncogene EWS-FLI1.

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.