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Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

ABSTRACT: The ability to label active caspase-3 represents a useful pharmacodynamic strategy to determine the efficacy of anti-tumour drugs. Activity-based probes (ABPs) provide a method for the labelling of activated caspases and the recent development of hybrid combinatorial substrate libraries (HyCoSuL) has allowed for the generation of highly selective ABPs to discriminately label these proteases. Here using this approach, a novel caspase-3 selective ABP (CS1) has been developed and validated in apoptotic cells to selectively bind caspase-3 over the closely related caspase-7. However, a critical bottleneck for ABPs is their cell penetrance and therefore this cell-impermeable CS1 probe was subsequently formulated into PLGA-based nanoparticles (CS1-NPs). We demonstrate the ability of these particles to be taken up by the cells and facilitate intracellular delivery of the ABP to effectively label caspase 3 in response to apoptotic stimuli. This work forms the foundation of a novel approach for the labelling of caspase 3 and may have downstream utility to measure real time apoptosis in tumours and other organs.

SUBMITTER: Cogo F

PROVIDER: S-EPMC7331821 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

Cogo Francesco F Poreba Marcin M Rut Wioletta W Groborz Katarzyna K Smyth Peter P Johnston Michael C MC Williams Richard R Longley Daniel B DB Burden Roberta E RE Salvesen Guy S GS Drag Marcin M Scott Christopher J CJ

Nanoscale 20190101 2

The ability to label active caspase-3 represents a useful pharmacodynamic strategy to determine the efficacy of anti-tumour drugs. Activity-based probes (ABPs) provide a method for the labelling of activated caspases and the recent development of hybrid combinatorial substrate libraries (HyCoSuL) has allowed for the generation of highly selective ABPs to discriminately label these proteases. Here using this approach, a novel caspase-3 selective ABP (CS1) has been developed and validated in apopt ...[more]

PMID: 30566168

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Project description:BackgroundThe cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging.ResultsA library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far.ConclusionsIn conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.

Dataset Information

Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

Publications

Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

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