In-Cell Quantification of Drugs by Magic-Angle Spinning Dynamic Nuclear Polarization NMR

Abstract

The determination of intracellular drug concentrations can provide a better understanding of the drug function and efficacy. Ideally, this should be performed nondestructively, with no modification of either the drug or the target, and with the capability to detect low amounts of the molecule of interest, in many cases in the μM to nM range (pmol to fmol per million cells). Unfortunately, it is currently challenging to have an experimental technique that provides direct quantitative measurements of intracellular drug concentrations that simultaneously satisfies these requirements. Here, we show that magic-angle spinning dynamic nuclear polarization (MAS DNP) can be used to fulfill these requirements. We apply a quantitative 15 N MAS DNP approach in combination with 15 N labeling to quantify the intracellular amount of the drug [ 15 N]CHIR-98014, an activator of the Wingless and Int-1 signaling pathway, determining intracellular drug amounts in the range of tens to hundreds of picomoles per million cells. This is, to our knowledge, the first time that MAS DNP has been used to successfully estimate intracellular drug amounts.

Highly bioresistant, hydrophilic and rigidly linked trityl-nitroxide biradicals for cellular high-field dynamic nuclear polarization

“…[4][5][6] Coupling dynamic nuclear polarization (DNP 7,8 ) with ssNMR overcomes this sensitivity limitation by transferring large electron spin polarization to NMR active nuclei. [9][10][11] As a result, DNP-ssNMR has been utilized to study crude cell lysates 12,13 , globular 10,14 , and membrane [15][16][17] proteins in their native settings as well as quantify drug concentrations directly within mammalian cells 18 .…”

“…9–11 As a result, DNP-ssNMR has been utilized to study crude cell lysates, 12,13 globular, 10,14 and membrane 15–17 proteins in their native settings as well as quantify drug concentrations directly within mammalian cells. 18…”

“…[9][10][11] As a result, DNP-ssNMR has been utilized to study crude cell lysates, 12,13 globular, 10,14 and membrane [15][16][17] proteins in their native settings as well as quantify drug concentrations directly within mammalian cells. 18 DNP-supported ssNMR experiments rely on tailored polarizing agents (PAs) that enhance the sensitivity of ssNMR by one to two orders of magnitude. However, development of PAs suitable for cellular DNP is still confronted with several challenges: (I) the unfavorable magnetic eld dependence of many PAs which greatly reduces their DNP enhancements at high magnetic elds; [19][20][21] (II) signicantly lower DNP enhancements for biological macromolecules as compared to those for small molecules; 10,12,22 (III) fast bioreduction of the currently available PAs in cellular environments.…”

The conformational ensemble of an intrinsically disordered protein explains peak shapes under DNP conditions

“…[15,17] The increase in experimental sensitivity can be sufficient to enable detection of molecules present at low micromolar concentrations, even in complex biological mixtures. [9,18] However, low temperatures often have an unfavorable effect on NMR line shapes due to inhomogeneous line broadening from the freezing out of molecular motion. [19][20][21][22] At room temperature, regions of intrinsic disorder sample many conformations on the fast exchange time scale which results in a single peak in the NMR spectrum at the population weighted average chemical shift value.…”

“…15,17 The increase in experimental sensitivity can be sufficient to enable detection of molecules present at low micromolar concentrations, even in complex biological mixtures. 9,18…”