Topological Heterogeneity of Protein Kinase C Modulators in Human T-Cells Resolved with In-Cell Dynamic Nuclear...

Area of Science:

• Biophysics
• Chemical Biology
• Molecular Pharmacology

Background:

• Phorbol ester analogs are vital anticancer and HIV latency-reversing agents.
• Their mechanism involves interaction with cellular membranes and protein kinase C (PKC) isoforms.
• Understanding phorbol ester-membrane interactions is crucial for correlating structure with biological activity.

Purpose of the Study:

• To characterize phorbol ester interactions within native cellular membranes using in-cell Dynamic Nuclear Polarization (DNP) NMR.
• To investigate the membrane topology and binding modes of 13C-21,22-phorbol-myristate-acetate (PMA) in T cells.

Main Methods:

• Utilized in-cell Dynamic Nuclear Polarization (DNP) NMR spectroscopy.
• Employed 13C-13C correlation and double quantum filtered NMR techniques.
• Characterized PMA in T cells at therapeutically relevant concentrations.

Main Results:

• Detected 13C-21,22-PMA in T cells down to approximately 60.0 pmol per million cells.
• Identified primary localization of PMA within cellular membranes.
• Observed distinct 13C-21,22-PMA chemical shifts in cells compared to model membranes, indicating unique membrane topologies and shallow binding modes.

Conclusions:

• Established in-cell DNP-NMR as a powerful method for studying drug-membrane interactions in living cells.
• Provided evidence for distinct membrane topologies of PMA, correlating with shallow binding.
• This work lays the foundation for understanding how in-cell structural interactions dictate phorbol ester biological activity.