Discovery of a potent
NUDT7 inhibitor by fragment merging. (A)
Chemical structures of similar hit compounds that exhibited 68% (PCM-0102716),
88% (PCM-0102512), and 100% (PCM-0102558, PCM-0102298, PCM-0102951,
and PCM-0102938) labeling of NUDT7 in the primary screen. Compound
NUDT7-REV-1 is a noncovalent fragment (purple) that was identified
as a NUDT7 binder in a crystallography soaking screen (see (E)). NUDT7-COV-1
(blue) is a merged compound based on PCM-0102716 (magenta) and NUDT7-REV-1.
(B) The six hits identified in the primary screen stabilize NUDT7
by 4.5–8.1 °C in a Tm shift
assay. (C) Labeling percentage of compounds PCM-0102558, PCM-0102951,
PCM-0102298, PCM-0102716, PCM-0102512, and PCM-0102938 at 5–200
μM, 24 h, 4 °C. (D) Cocrystal structures of NUDT7 with
compounds PCM-0102951, PCM-0102558, and PCM-0102716. (E) Overlay of
the crystal structures of NUDT7 with compound PCM-0102716 (pink) and
with the noncovalent fragment NUDT7-REV-1 (purple). (F) The cocrystal
structure of NUDT7 with the merged compound NUDT7-COV-1 adopts the
exact same binding mode as the two separate fragments. (G) Enzymatic
inhibition of NUDT7 by NUDT7-COV-1 and NUDT7-REV-1. The data shown
include results with and without a 30 min protein preincubation in
the presence of the compounds. (H) Intracellular target engagement
is demonstrated by thermal stabilization of FLAG-NUDT7 by NUDT7-COV-1
in intact HEK293 cells. After transfection, cells were treated with
20 μM NUDT7-COV-1 or DMSO for 30 min before being heated to
the indicated temperatures.