Finally, FLuc appears especially prone to catalytic inhibition or protein stabilization by a wide variety of drugs. (20) Thus, a FLuc-based kinase reporter may respond to both long-term effects of kinase inhibition on ATP, in addition to responding to kinase inhibition directly. Even on-target effects on kinases could produce artifactual responses from FLuc-based reporters, as suppression of kinase activity can induce either feedback activation of glucose metabolism over hours (19) or reduced metabolism over days. (18) Thus, a FLuc-based kinase reporter could respond artifactually to an off-target effect of a test compound on ATP levels via either a different kinase or a nonkinase pathway. (17) Second, FLuc catalysis requires ATP, and photon emission from FLuc in animals has been shown to vary with conditions that alter ATP levels. First, FLuc produces an order of magnitude less light in vivo compared to modern luciferases. However, past bioluminescent reporters of kinase activity were based on firefly luciferase (FLuc), (15,16) which introduces three limitations. Given these limitations of current methods to assess pharmacodynamics in the brain, a fast and inexpensive approach to evaluate kinase inhibitor activity within target cells in the brain would be highly desirable. (11) However, this approach is highly time-consuming, resource-intensive, and requires high-quality phosphorylation-specific antibodies that may not exist for every drug target. One solution is to perform immunocytochemistry with phosphorylation-specific antibodies to specific kinase substrates and with cancer-specific markers, allowing some determination of phosphorylation levels specifically in cancer cells. (10) Accurate assessment of kinase inhibition can be especially problematic for brain cancers, where cancer cells can be dispersed among a larger number of normal cells. Cell type-specific drug export or metabolism may limit drug efficacy even with high drug levels in the surrounding tissue. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.Īnother fundamental challenge is that pharmacokinetics can differ from pharmacodynamics, i.e., how the drug target responds over time. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. ERK KiMBI discriminates between brain-penetrant and nonpenetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for noninvasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. This preserves the integrity of the original DNA strand that is passed onto the daughter cells.Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. The DNA is therefore “proofread” by DNA polymerase after it has been copied so that misplaced base pairs can be corrected. These primers are made by another enzyme called primase.Īlthough the function of DNA polymerase is highly accurate, a mistake is made for about one in every billion base pairs copied. Primers are usually composed of RNA and DNA bases, and the first two bases are always RNA. A primer is therefore needed, at which nucleotides can be added. However, DNA polymerase cannot begin forming this new chain on its own and can only add nucleotides to a pre-existing 3'-OH group. This opens up or “unzips” the double-stranded DNA to give two single strands of DNA that can be used as templates for replication.ĭNA polymerase adds new free nucleotides to the 3’ end of the newly-forming strand, elongating it in a 5’ to 3’ direction. In this way, genetic information is transmitted from generation to generation.īefore replication occurs, an enzyme called helicase unwinds the DNA molecule from its tightly woven form. The potential mechanisms of SARS-CoV-2 to induce DNA damageĮvery time a cell divides, DNA polymerase is required to help duplicate the cell’s DNA so that a copy of the original DNA molecule can be passed to each daughter cell.
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