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.
Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide.
Thec atalytic hydrophosphination reaction is one of the most sustainablec hemical transformations today.H ere,t he palladium-catalyzed asymmetric P À Ha ddition of diarylphosphines with N-enoylbenzotriazoles and analogues is described. Chiral phosphinep roductsa re obtained in 100%a tom economy and without cumbersome protection-deprotection manipulations.T he obtained productsc an be subsequently transformedt ob ear various functionalities,i ncluding phosphino-carboxylic esters which play critical roles in catalysisa nd as synthetic aids.A nti-tumour activities of the corresponding gold-phosphine complexes have been explored, contributing to existing chemotherapeuticr esearchi n cancer treatment.
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.
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