posted on 2023-01-05, 15:01authored byAarti Kawatkar, Roger A. Clark, Lorna Hopcroft, Debora Ann Roaquin, Ronald Tomlinson, Andrea M. Zuhl, Gillian M. Lamont, Jason G. Kettle, Susan E. Critchlow, M. Paola Castaldi, Frederick W. Goldberg, Andrew X. Zhang
Lactic
acid transport is a key process maintaining glycolytic
flux
in tumors. Inhibition of this process will result in glycolytic shutdown,
impacting on cell growth and survival and thus has been pursued as
a therapeutic approach for cancers. Using a cell-based screen in a
MCT4-dependent cell line, we identified and optimized compounds for
their ability to inhibit the efflux of intracellular lactic acid with
good physical and pharmacokinetic properties. To deconvolute the mechanism
of lactic acid efflux inhibition, we have developed three assays to
measure cellular target engagement. Specifically, we synthesized a
biologically active photoaffinity probe (IC50 < 10 nM),
and using this probe, we demonstrated selective engagement of MCT4
of our parent molecule through a combination of confocal microscopy
and in-cell chemoproteomics. As an orthogonal assay, the cellular
thermal shift assay (CETSA) confirmed binding to MCT4 in the cellular
system. Comparisons of lactic acid efflux potencies in cells with
differential expression of MCT family members further confirmed that
the optimized compounds inhibit the efflux of lactic acid through
the inhibition of MCT4. Taken together, these data demonstrate the
power of orthogonal chemical biology methods to determine cellular
target engagement, particularly for proteins not readily amenable
to traditional biophysical methods.