Expanding
the repertoire of electrophiles with unique reactivity
features would facilitate the development of covalent inhibitors with
desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane
(BCB) carboxylic amide as a new class of thiol-reactive electrophiles
for selective and irreversible inhibition of targeted proteins. We
first streamlined the synthetic routes to generate a variety of BCB
amides. The strain-driven nucleophilic addition to BCB amides proceeded
chemoselectively with cysteine thiols under neutral aqueous conditions,
the rate of which was significantly slower than that of acrylamide.
This reactivity profile of BCB amide was successfully exploited to
develop covalent ligands targeting Bruton’s tyrosine kinase
(BTK). By tuning BCB amide reactivity and optimizing its disposition
on the ligand, we obtained a selective covalent inhibitor of BTK.
The in-gel activity-based protein profiling and mass spectrometry-based
chemical proteomics revealed that the selected BCB amide had a higher
target selectivity for BTK in human cells than did a Michael acceptor
probe. Further chemical proteomic study revealed that BTK probes bearing
different classes of electrophiles exhibited distinct off-target profiles.
This result suggests that incorporation of BCB amide as a cysteine-directed
electrophile could expand the capability to develop covalent inhibitors
with the desired proteome reactivity profile.
Vancomycin (VCM) is a first-line antibiotic for serious infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is one of the most complaint in VCM therapy. We previously reported that VCM induced apoptosis in a porcine proximal tubular epithelial cell line (LLC-PK1), in which mitochondrial complex I may generate superoxide, leading to cell death. In the present study, VCM caused production of mitochondrial reactive oxygen species and peroxidation of the mitochondrial phospholipid cardiolipin that was reversed by administration of the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). FCCP also significantly suppressed VCM-induced depolarization of the mitochondrial membrane and apoptosis. Moreover, the lipophilic antioxidant vitamin E and a mitochondria-targeted antioxidant, mitoTEMPO, also significantly suppressed VCM-induced depolarization of mitochondrial membrane and apoptosis, whereas vitamin C, n-acetyl cysteine, or glutathione did not provide significant protection. These findings suggest that peroxidation of the mitochondrial membrane cardiolipin mediated the VCM-induced production of intracellular reactive oxygen species and initiation of apoptosis in LLC-PK1 cells. Furthermore, regulation of mitochondrial function using a mitochondria-targeted antioxidant, such as mitoTEMPO, may constitute a potential strategy for mitigation of VCM-induced proximal tubular epithelial cell injury.
Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and is used for identification and isolation of CSC. Here, we show that the population of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells were generated by time-dependent increases and decreases in the expression of Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of in ALDH-positive cells was dependent on the time-dependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, antitumor and antimetastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. This seminal report reveals that circadian dynamics of CSC are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy in TNBC. .
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