The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

In vitro and in vivo antifungal activities of TAK-456, a novel oral triazole with a broad antifungal spectrum.

TAK-456 is a novel oral triazole compound with potent and broad-spectrum in vitro antifungal activity and strong in vivo efficacy against Candida albicans and Aspergillus fumigatus. TAK-456 inhibited sterol synthesis of C. albicans and A. fumigatus by 50% at 3 to 11 ng/ml. TAK-456 showed strong in vitro activity against clinical isolates of Candida spp., Aspergillus spp., and Cryptococcus neoformans, except for Candida glabrata. The MICs at which 90% of the isolates tested were inhibited byTAK-456, fluconazole, itraconazole, voriconazole, and amphotericin B were 0.25, 4, 0.5, 0.13, and 0.5 microg/ml, respectively, for clinical isolates of C. albicans and 1, >64, 0.5, 0.5, and 0.5 microg/ml, respectively, for clinical isolates of A. fumigatus. Therapeutic activities of TAK-456 and reference triazoles against systemic lethal infections caused by C. albicans and A. fumigatus in mice were investigated by orally administering drugs once daily for 5 days, and efficacies of the compounds were evaluated by the prolongation of survival. In normal mice, TAK-456 and fluconazole were effective against infection caused by fluconazole-susceptible C. albicans at a dose of 1 mg/kg. In transiently neutropenic mice, therapeutic activity of TAK-456 at 1 mg/kg of body weight against infection with the same strain was stronger than those at 1 mg/kg of fluconazole. TAK-456 was effective against infections with two strains of fluconazole-resistant C. albicans at a dose of 10 mg/kg. TAK-456 also expressed activities similar to or higher than those of itraconazole against the infections caused by two strains of A. fumigatus in neutropenic mice at a dose of 10 mg/kg. These results suggest that TAK-456 is a promising candidate for development for the treatment of candidiasis and aspergillosis in humans.

Efficacy of TAK-457, a novel intravenous triazole, against invasive pulmonary Aspergillosis in neutropenic mice.

TAK-457 is an injectable prodrug of TAK-456, which is a novel oral triazole compound with potent antifungal activity. The in vivo efficacy of TAK-457 was evaluated in two models of invasive pulmonary aspergillosis with CDF(1) mice and CBA/J mice with transient neutropenia induced by cyclophosphamide. Against the infection in CDF(1) mice, treatment with 10 mg of TAK-457 and 1 mg of amphotericin B/kg reduced the fungal burden in lungs and rescued all mice. In the infection model with CBA/J mice, TAK-457 at a dose of 10 mg/kg significantly prolonged the survival time of mice, showing significant reduction of lung chitin levels and the plasma beta-D-glucan levels. On the other hand, amphotericin B at 1 mg/kg which was a maximum tolerable dose showed slight but not significant prolongation of survival time of mice, although it also reduced the lung chitin levels and the plasma beta-D-glucan levels to a lower extent but still significantly. These results suggest that TAK-457 is a promising candidate for development for the treatment of invasive aspergillosis in humans.