WP1: Design, preparation and validation of PRETI
Designed a caged RET inhibitor (CRETI) using photopharmacology (Figure 1a), PRETI (6) integrates a 7-diethylamino-4-methylcoumarin (DEACM) caging group onto the RET-specific kinase inhibitor, Pralsetinib (BLU-667, 5). Under visible light, 6 undergoes decaging to yield 5 and alcohol 3 that possesses spectroscopic attributes.
Task 1: Introduce the caging group (L) onto the exocyclic amino moiety of the heterocyclic core of the kinase inhibitor 5 by using conventional organic synthesis technics. The synthetic strategy and the decaging process is depicted in Figure 1b. The synthesised compounds (including intermediates) were purified by flash column and high-performance liquid chromatography (HPLC) and characterized by high resolution NMR and LC-MS techniques.
Task 2: Determine the optimal conditions and reaction kinetics of the decaging process using the CRETI (6).
Initially, stability tests showed that the compound 6 was found to be sufficiently stable in various assay buffers and in DMSO at various temperatures. First, optimal conditions and reaction kinetics of the decaging process of 6 in the aqueous buffer was evaluated. Both compounds 3 and 6 showed UV-Vis. absorption maxima at 385 nm in 0.1% DMSO-1x ADP-Glo™ assay buffer. However, upon excitation at the absorption maxima, 6 yielded, blue-shifted fluorescence centred at 472 nm compared to the fluorescence of 3 centred at 486 nm (Figure 2a). The calculated molar absorptivity (ε) and fluorescence quantum yield (ϕf) of 6 and 3 were 10883 M-1cm-1 and 12.97% and 20250 M-1cm-1 and 14.83%, respectively. The fluorescence intensity of 2.0 µM of 6 in 0.1% DMSO-1x ADP GloTM assay buffer increased gradually upon continuous irradiation with 405-nm light (Figure 2b). The variation in fluorescence intensity at the fluorescence maxima as a function of irradiation time is depicted in Figure 2c. This fluorescence intensity pattern with increasing irradiation time was fitted well by a mono-exponential growth function, and the time constant was 16.22 ±0.22 s. Of note, the photostability of 5 in aqueous buffer medium was good with less than 10% degradation.
Task 3: Validation of PRETI in a biochemical assay.
Compound 6 was tested in an ADP-GloTM RET kinase assay following the protocol provided by the supplier (Promega). It was found that the compound 6 was inactive in the assay. However, it undergoes decaging upon irradiation and the decaged product inhibited the enzyme activity. The time constant for decaging of 6 in the assay was found to be 32.00 ±3.00 s. The half maximal inhibition concentration (IC50) of the decaged inhibitor was 29.14.33 ±1.18) nM.
Task 4: Validation of PRETI in a live cell assay.
For the purpose, NanoBRET intracellular RET kinase assay was performed. Briefly, this assay allows to evaluate compounds that bind to RET fused to N-terminal nanoluciferase (NanoLuc) in a competitive format by using a cell-permeable fluorescent NanoBRET tracer in HEK293 cells (human embryonic kidney cells). HEK293 cells in combination with the NanoBRET intracellular kinase assay kit from Promega were used for the live cell assay. It was observed that the caged inhibitor 6 is inactive in the assay. However, compound 6 was cell permeable and undergoes decaging inside cells upon irradiation with 405 nm light, that releases 5. The time constant for decaging of 6 in the live cells was found to be10.61 ± 1.25 s (Figure 3). The IC50 value of the decaged inhibitor was 34.33 ±0.18) nM.
WP2: Combining PRETI with imaging.
Task 5: Establishment and validation of cellular assay for studying RET trafficking.
SH-SY5Y cells with a CRISPR generated RET knockout were virally transduced to add back RET51 with a C-terminal mCherry tag. Both RET wildtype and kinase dead (K758M) cell lines were generated. Control experiments to confirm normal cell growth, activation of RET51 (by GDNF) and proper expression of RET51-mCherry (Figure 4) had been carried out. Real-time fluorescence confocal microscopy will be implemented to compare the behaviour of the RET trafficking with or without BLU-667.
Task 6: Monitor RET trafficking in real time using light as an external control element.
This part of the project could not be finished within the 24 moths project time. However, the imaging experiments, set to take place in late autumn or early winter, will be expertly conducted by another postdoctoral fellow within the Grøtli Lab. See the final periodic report for details. In brief, this cellular assay will be used to determine: (a) Localisation of the RET enzyme within the cell. (b) How does the intra cellular RET distribution depend on the dose of inhibitor? (c) Does the RET distribution vary depending on when the RET enzyme is inhibited?
