posted on 2022-04-06, 17:45authored byAndrea Bertarello, Pierrick Berruyer, Markus Artelsmair, Charles S. Elmore, Sepideh Heydarkhan-Hagvall, Markus Schade, Elisabetta Chiarparin, Staffan Schantz, Lyndon Emsley
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 15N MAS DNP approach in combination with 15N labeling to
quantify the intracellular amount of the drug [15N]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.