Kinase-Modulated Bioluminescent Indicators Enable Noninvasive Imaging of Drug Activity in the Brain

Abstract

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. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. 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. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. 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. 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. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

Figure 1

Development and characterization of KiMBIs. (a) Proposed mechanism of KiMBI. With active kinase, phosphorylated kinase substrate binds to phosphopeptide-binding domain (PBD), outcompeting LgBiT-SmBiT reconstitution (left). Kinase inhibition allows dephosphorylation and LgBiT-SmBiT reconstitution (right). (b) Bioluminescence of cells expressing putative PKA KiMBIs with different linker lengths between PBD and substrate. All variants are inhibited by PKA activators Fsk and IBMX. (c) Domain arrangement and model of an ERK KiMBI. (d) bKiMBI signals with or without caMEK cotransfection or MEK inhibitor mirdametinib. U87E is U87-EGFRvIII. **, p < 0.01; ***, p < 0.001; **** p < 0.0001 by unpaired two-tailed Student’s test. Error bars show SD. (e) Mean bioluminescence of HEK293A cells coexpressing caMEK and tKiMBI or tKiMBImut negative control in response to inhibitors Vx-11e (ERK, 1 μM), U0126 (MEK, 10 μM), mirdametinib (MEK, 1 μM), trametinib (MEK, 1 μM), SCH772984 (ERK, 1 μM), SP600125 (JNK,10 μM), and PD169316 (p38, 10 μM). One-way ANOVA (p < 0.0001) was followed by Tukey’s posthoc test; ns, p > 0.05; *, p < 0.05. (f) Top, domain arrangements of orangeKiMBI and tomatoKiMBI. Left, bioluminescence spectra of KiMBI color variants with caMEK cotransfection, normalized to the 450 nm peak. Right, responses of color variants to mirademetinib. ****, p < 0.0001 by unpaired two-tailed Student’s t test. Error bars show SD. ALU is arbitrary luminescence units.

Figure 2

Molecular imaging of Ras-ERK pathway inhibition in a subcutaneous tumor model. (a) Scheme of the experimental design. U87-EGFRvIII reporter cells expressing tKiMBI (right) and tKiMBImut (left) were implanted subcutaneously to establish tumors in J:NU mice. AkaLuc imaging with AkaLumine injection was used to monitor tumor growth. To visualize ERK inhibition, the indicators were imaged with FFz injection, 2 h before and 2 h after treatment with MEK-ERK inhibitors. All injections were performed intraperitoneally (i.p.). (b) Scheme of ERK signaling pathway and MEK inhibition. (c, d) U87-EGFRvIII tumor-bearing mice were sequentially treated (2 days apart) with MEK inhibitors mirdametinib (c) and trametinib (d) and were imaged with FFz injection 2 h after inhibitor injection. Left, representative bioluminescent images collected before and after inhibitor treatment. Right, the percentage change of bioluminescence signals collected before and after inhibitor treatment. Each line represents an individual mouse. P values, paired two-tailed Student’s t test. L is luminescence.

Figure 3

Molecular imaging of ERK inhibition by MEK inhibitors in mouse brain. (a) Scheme of AAV infection for KiMBI expression in the mouse striatum and pathway schematic with position of MEK inhibitors. (b) 4 weeks after AAV infection in the striatum, tKiMBI- or tKiMBImut-expressing J:NU mice were sequentially treated (2 days apart) with mirdametinib or trametinib and were imaged with CFz injection. Representative bioluminescence images collected 2 h before and 2 h after inhibitor treatment. (c) The percentage change of bioluminescence signals collected before and after inhibitor treatment. P values, one-way ANOVA analysis (p = 0.006) was followed by Holm-Sidak’s posthoc test. Each line represents an individual mouse. (d) Design of the tKiMBI-expressing brain tumor xenograft model. (e) J:NU Mice with tKiMBI- or tKiMBImut-expressing U87-EGFRvIII tumor engrafted in the striatum were sequentially treated (2 days apart) with mirdametinib or trametinib and imaged with CFz injection. Representative bioluminescence images were collected before and after inhibitor treatment. (f) The percentage change of bioluminescence signals collected before and after inhibitor treatment. P values, one-way ANOVA analysis (p = 0.0025) was followed by Holm-Sidak’s posthoc test. Each line represents an individual mouse.

Figure 4

Molecular imaging of pharmacodynamics of ERK inhibition by mirdametinib in multiple disease models. (a) Design of experiments to measure ERK inhibitor pharmacodynamics. Initial bioluminescence L₀ from reporter-expressing cells was measured with substrate (FFz for subcutaneous and intramuscular tumors, CFz for brain tumor and AAV-transduced brain model) injection 2 h before inhibitor treatment. After inhibitor administration, CFz was reinjected and bioluminescence L was measured at various time points. (b) Mirdametinib pharmacodynamics in extracranial tumor models. Top, representative bioluminescence images collected at indicated time points after inhibitor. Bottom, pharmacodynamics time-course of tKiMBI/tKiMBImut ratio changes (ΔR/R₀). (c) Mirdametinib pharmacodynamics in tKiMBi-expressing intracranial xenografts or brains transduced with reporter and caMEK. (d) Summary of pharmacodynamic time-courses of ERK inhbition by mirdametinib in different tissues.

Figure 5

Molecular imaging of ERK inhibitors in AAV-infected mouse brain. (a) Pathway schematic with position of ERK inhibitors. (b–d) After AAV infection in the striatum, tKiMBI- or tKiMBImut-expressing J:NU mice were imaged 2 h after ERK inhibitor treatment. Above, representative bioluminescence images collected before and after each ERK inhibitor treatment. Below, percentage change of bioluminescence signals collected before and after inhibitor treatment. P values, unpaired two-tailed Student’s t test.