KRAS is the most frequently mutated oncogene in human cancers. Clinically effective therapy targeting KRAS, however, has not been developed to date despite the decades of intensive efforts. Among various therapeutic approaches taken, blocking the interaction between KRAS and the effector proteins is one of the effective strategies to inhibit KRAS signaling in diseases. Here we have developed the highly-sensitive split ELuc (Emerald Luc, an enhanced luciferase from click beetle) probes which detect the interaction between KRAS (wild-type, G12C and G12D active mutants) and the effector proteins such as BRAF, CRAF, and three RalGEFs (RalGDS, RGL2, and RGL3). The probe pairs transiently co-transfected into 293T cells yielded significantly strong luminescence signals in all combination of KRAS and effector probes but not with negative control probes, indicating the specificity and the sensitivity of the probes. As expected, KRAS G12C and G12D active mutant probes yielded higher signals than KRAS wild-type probe in any combination with effector probes. The novel probe systems described here will be useful tools for the discovery of KRAS genotype- or effector-specific inhibitors.
Propofol, known as “milk of anesthesia”, is used for the induction and maintenance of anesthesia. Recently, propofol has attracted increasing concerns about its safety and abuse potential because of its psychostimulant effects such as euphoria and sexual hallucinations. Previous reports focused on the effects of postoperative and neonatal exposure to propofol. However, the lasting effects of repetitive propofol administration during adulthood have not been well investigated. It is conceivable that prolonged use of propofol affects brain function and the behavioral characteristics of the abused patient. Thus, we performed a comprehensive behavioral analysis of mice exposed to propofol. Adult male C57BL/6J mice were repeatedly administered with propofol (20 or 80 mg/kg/day i.p.), intralipos (vehicle control), or saline only once a day for seven days. We then performed a behavioral test battery to evaluate various behaviors. Afterwards, we resumed the propofol treatment for three days and subsequently conducted contextual and cued fear conditioning tests. In the three‐chamber social approach test, propofol treatment attenuated social novelty preference in mice. In the fear conditioning test, high dose-treated mice exhibited impaired long-term cued-dependent memory retention. In the rotarod test, propofol- and intralipos-treated mice tended to have decreased motor coordination than the saline-treated mice. Our results demonstrated that repetitive propofol treatment has the potential to induce some behavioral changes in mice. Additionally, the solvent itself might have effects different from that of propofol. Our findings provide basic data on the safe use and risk of propofol abuse. Highlights Propofol, known as “milk of anesthesia”, has attracted increasing concerns about its safety and abuse potential. The lasting effects of repetitive propofol administration during adulthood have not been well investigated. To clarify the effects of repetitive propofol use on brain function and behavioral characteristics, we performed a comprehensive behavioral analysis of mice exposed to propofol. In this study, propofol treatment attenuated the social novelty preference and the performance of the cued long-term memory task in mice. Additionally, treatment with propofol and intralipos tended to induce decreased motor coordination. Our results demonstrated that repetitive propofol treatment has the potential to induce some behavioral changes in mice. Furthermore, the solvent itself might have effects different from that of propofol. Our findings provide basic data for the safe use and risk of propofol abuse.
In recent years, it has become clear that the interstitial tissue structure varies markedly between the normal healthy state and various disease states. Focusing on differences in interstitial structure is expected to lead to the discovery of new targets for drugs. Considerable attention is currently focused on approaches that utilize organoids. The organoids retain the structure of the tissue, thereby facilitating identification of drugs that target interstitial tissues including neovascular vessels. Furthermore, protein expression analysis techniques have been developed that focus on the tissue structure based on an approach using chemical proteomics. In this mini-review, we will summarize the latest technological innovations aimed at identifying target molecules that are useful for the development of biopharmaceuticals, including antibody drugs.
EU and US proposals for a paradigm shift in toxicity testing have brought about a worldwide evolution in the safety assessment of chemicals by means of novel approach methods (NAM) that do not use animals. In Japan, as well, despite the fact that there have been no changes in regulation of cosmetics, pharmaceuticals, and other chemical substances, a number of manufacturers have voluntarily discontinued the use of animal testing, and we must now give serious consideration to means for ensuring the safety of chemicals without the use of animal testing in accordance with the international harmonisation. The expanding use of non-animal test methods in the safety assessment of chemicals continues to merit close scrutiny.