Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug distribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and specificity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, nanomolar concentrations, allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understanding of target engagement.

Two decades after their discovery, stapled peptide methodologies have evolved to a point where they can be used with confidence to generate therapeutic leads. Research groups across the world are testing innovative methodologies for their design, with dozens of publications released every month. A number of stapled peptide drug candidates have recently entered clinical trials. In this review, we provide an overview of successful methods for their construction, highlight trends in the deposited crystal structures of stapled peptide complexed to their targets and discuss properties that contribute towards improved pharmacological profiles.

Small heteroaryl‐diyne (Het‐DY) tags with distinct vibrational frequencies, and physiologically relevant cLog P were designed for multiplexed bioorthogonal Raman imaging. Pd−Cu catalyzed coupling, combined with the use of Lei ligand, was shown to improve overall yields of the desired heterocoupled Het‐DY tags, minimizing the production of homocoupled side‐products. Spectral data were in agreement with the trends predicted by DFT calculations and systematic introduction of electron‐ rich/poor rings stretched the frequency limit of aryl‐capped diynes (2209–2243 cm−1). The improved Log P of these Het‐DY tags was evident from their diffuse distribution in cellular uptake studies and functionalizing tags with organelle markers allowed the acquisition of location‐specific biological images. LC–MS‐ and NMR‐based assays showed that some heteroaryl‐capped internal alkynes are potential nucleophile traps with structure‐dependent reactivity. These biocompatible Het‐DY tags, equipped with covalent reactivity, open up new avenues for Raman bioorthogonal imaging.

Ponatinib is a clinically approved tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. In this study stimulated Raman scattering (SRS) microscopy was used to allow label-free imaging of intracellular ponatinib with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. SRS microscopy represents a powerful imaging tool for visualizing label-free drug molecules within cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing the intracellular concentrations of drug molecules to be accurately determined. Intracellular visualization of ponatinib was achieved at biologically relevant, nanomolar concentrations for the first time using SRS. It was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not re-sensitise cells to ponatinib, confirming BCR-ABL independent resistance mechanism in this CML cell model. In summary, we have visualized intracellular ponatinib localization for the first time using SRS, demonstrating that acquired drug resistance can influence drug uptake and localisation in CML, which in turn has an effect on target engagement. Citation Format: Kristel Sepp, Martin Lee, Marie T. Bluntzer, G. Vignir Helgason, Alison N. Hulme, Valerie G. Brunton. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5267.