Genetic Engineering & Biotechnology News

JAN15 2018

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12 | JANUARY 15, 2018 | | Genetic Engineering & Biotechnology News Kristin Huwiler, James Vasta, Ph.D., Cesear Corona, Ph.D., Chad Zimprich, Jennifer Wilkinson, Morgan Ingold, and Matthew Robers Kinases constitute a family of more than 500 enzymes and play critical roles in cellular signal- ing, both in normal and diseased cells. As a re- sult, kinases continue to be key drug targets, es- pecially for cancer and inflammatory diseases. Traditional biochemical assays with re- combinant kinases are commonly used to characterize small-molecule effects on kinase activity or affinity. Biochemical-kinase assays, however, can fail to predict how kinase in- hibitors might function inside cells. Reasons for failing include: differences in ATP con- centration, use of truncated kinase domains, absence of appropriate cellular cofactors, and differences in kinase activation states. Conse- quently, a biochemical assay often does not recapitulate the physiological complexity that a kinase encounters inside an intact cell. There is a growing need for quantitative kinase-inhibitor cellular target engagement (TE) methods. Several methods exist to assess compound TE to kinases expressed in human cells. Some methods use affinity-based che- moproteomics with cell lysates, which disrupt the cell membrane, and cellular cofactors, which affect affinity. Other methods, such as cellular thermal shift assays (CETSAs), do not require cell lysis during compound incu- bation, but depend on a downstream event such as protein aggregation. CETSAs, there- fore, do not provide a quantitative measure of drug affinity. CETSAs are also prone to misreporting on certain TE events, as not all compounds that bind the target kinase will stabilize it. Most of these techniques fail to meet the de- sirable TE assay qualities of quantitating small- molecule inhibitor affinity in live cells via a di- rect binding event, rather than in lysates or via a downstream event that can be less specific. Live-Cell Quantitative Target Engagement Kinase Assays Here we describe a novel TE platform, which is the first to quantitatively measure compound binding to kinases in live cells without disrupting the cell membrane. These assays are target kinase specific, use full-length kinases, and are performed in multiwell plates. The Promega target engagement assay platform uses a bioluminescence resonance energy transfer technology, NanoBRET™. There are four key components of the as- say: 1) cellular-expressed full-length kinase protein fused to the very bright, small Na- noLuc ® luciferase; 2) cell-permeable energy- transfer probe (or fluorescent tracer) that reversibly binds the active site of the kinase; 3) substrate for NanoLuc luciferase; and (4) extracellular NanoLuc inhibitor to ensure signal originates from live cells. Figure 1 is a schematic of the NanoBRET TE kinase assays, which are direct competi- tive binding assays conducted in live cells. A cell-permeable energy-transfer probe (or trac- er) reversibly binds the active site of a kinase- NanoLuc fusion protein expressed in cells. Energy transfer from NanoLuc to the fluorescent tracer bound to the kinase occurs due to close proximity and results in a BRET signal. It is this proximity that results in the target kinase specificity of the BRET signal. In the presence of a competitive compound, binding to the kinase-NanoLuc fusion pro- tein results in displacement of the probe and decrease in the BRET signal. The NanoBRET-based competitive bind- ing format allows quantitative measures of unmodified compound affinity against the selected target kinase protein in live cells. As shown in Figure 2A, a recommended tracer concentration has been determined for each kinase using competitive binding experi- ments with varying tracer concentrations. At tracer concentrations ≤K D , this assay should measure apparent occupancy of a compound for the selected kinase in live cells. Further confirmation of test compound apparent intracellular K D can be obtained by Cheng-Prousoff analysis of the test com- pound IC 50 vs. tracer concentration (Figure 2B). We have shown that target kinase expression levels do not impact assay results. As Nano- Luc is very bright, we have demonstrated that low levels of kinase-NanoLuc fusion protein expression are detectable with NanoBRET, even from kinases expressed from endog- enous genetic loci (data not shown). Broad Coverage and Scalable Assays Broad-coverage cell-permeable Nano- BRET kinase tracers have been developed and are commercially available in separate Using a diverse panel of 20 different hu- man tissues, Dr. Najafabadi and colleagues looked at the mRNA stability landscape in different cell types. 4 "The mRNA stability landscape in the brain was quite different from what we saw in other human tissues," reports Dr. Najafabadi. This observation pointed toward the contribution of post-transcriptional pro- grams to shaping the brain transcriptome, reinforcing similar observations that were made earlier using several different experi- mental approaches. From RNA-seq measurements in the brain tissue, Dr. Najafabadi and colleagues identified four microRNAs and two RNA- binding proteins that were critical determi- nants of mRNA stability. One protein found during this work, RBFOX1, is involved in regulating the stability of mRNAs that en- code synaptic transmission proteins. "Loss of synaptic function is a major change in Alzheimer's disease," notes Dr. Najafabadi. "Accordingly, we surmised that in Alzheimer's disease, there is a good chance that RBFOX1 is involved." Dr. Najafabadi and colleagues found that the mRNAs encoding synaptic transmis- sion proteins degraded much more rapidly in the brains of individuals with Alzheimer's disease. These investigators also determined that RBFOX1 inhibition in neurons leads to a transcriptome remodeling pattern like the one seen in Alzheimer's disease, and that RBFOX1 overexpression in deficient cells shifts the transcriptome profile back toward its normal state. In their studies on mRNA stability, in- vestigators in Dr. Najafabadi's lab have used bulk mRNA, which provides an average measurement of the cellular events but does not capture mRNA degradation differences at the single-cell level, which could be infor- mative about the heterogeneity of mRNA stability across cell types. "We hope that as single-cell RNA-seq technology moves forward, we can have enough coverage from single cells to look at both mRNA and pre-mRNA to study the rate of RNA degradation at the single-cell level," states Dr. Najafabadi. "We are cur- rently working on that." References available online. Live-Cell Platform for Studies Exploring Kinase-Inhibitor Binding Quantitative Kinase Target Engagement Taming the Transcriptome Continued from page 11 Drug Discovery Tutorial Kristin Huwiler (kristin.huwiler@promega. com) is a global strategic manager; James Vasta, Ph.D., is a research scientist; Cesear Corona, Ph.D., is a staff scientist; Chad Zimprich is a research scientist; Jennifer Wilkinson is a research scientist; Morgan Ingold is a research scientist; and Matthew Robers is a senior research scientist and group leader at Promega. Website: Figure 1. Schematic of Live-Cell Kinase NanoBRET® Target Engagement Assay. A cell- permeable energy transfer probe (or tracer) reversibly binds the kinase active site, generating a BRET signal in live cells. Unlabeled test compound binding to the selected kinase results in a decrease in BRET signal. 1 Table. Partial List of Kinases Assayed Using NanoBRET TE Intracellular Kinase Assays Kinase Name Gene Name Official Full Name Also Known As NCBI Gene ID Vector Cat.# Vector TE Assay Data Sheet AAK1 AAK1 AP2-associated kinase 1 22848 NV1001 NanoLuc-AAK1 Fusion Vector NanoBRET TE Intracellular Kinase Assays, K-5 PDF ABL1 ABL1 ABL proto-oncogene ABL, JTK7, p150, c-ABL 25 NV1011 NanoLuc-ABL1 Fu- sion Vector NanoBRET TE Intracellular Kinase Assays, K-4 PDF ALK4 ACVR1B Activin A receptor type 1B ALK4, SKR2, ACTRIB, ACVRLK4 91 NV1021 ACVR1B-NanoLuc Fusion Vector NanoBRET TE Intracellular Kinase Assays, K-5 PDF AKT2 AKT2 AKT serine/threonine kinase 2 PKBB, PRKBB, PKBBETA 208 NV1031 AKT2-NanoLuc Fu- sion Vector NanoBRET TE Intracellular Kinase Assays, K-5 PDF The table shows the top few rows from a list of kinases assayed at Promega using NanoBRET TE Intracellular Kinase Assays. The complete searchable table is available at and includes >120 kinases from all groups (AGC, CAMK, CK1, CMGC, STE, TK, TKL, other, and atypical). Each kinase in the complete table is available as a kinase-NanoLuc fusion vector from the company, as a PDF downloadable technical data sheet for each kinase.

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