2 research outputs found
Synthesis and Enzymatic Studies of Bisubstrate Analogues for Farnesyl Diphosphate Synthase
Farnesyl
diphosphate synthase catalyzes the sequential chain elongation
reactions between isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP) to form geranyl diphosphate (GPP) and between
IPP and GPP to give farnesyl diphosphate (FPP). Bisubstrate analogues
containing the allylic and homoallylic substrates were synthesized
by joining fragments for IPP and the allylic diphosphates with a C–C
bond between the methyl group at C3 in IPP and the <i>Z</i>-methyl group at C3 in DMAPP (<b>3-OPP</b>) and GPP (<b>4-OPP</b>), respectively. These constructs placed substantial
limits on the conformational space available to the analogues relative
to the two substrates. The key features of the synthesis of bisubstrate
analogues <b>3-OPP</b> and <b>4-OPP</b> are a regioselective <i>C</i>-alkylation of the dianion of 3-methyl-3-buten-1-ol (<b>5</b>), a <i>Z</i>-selective cuprate addition of alkyl
groups to an α,β-alkynyl ester intermediate, and differential
activation of allylic and homoallylic alcohols in the analogues, followed
by a simultaneous displacement of the leaving groups with trisÂ(tetra-<i>n</i>-butylammonium) hydrogen diphosphate to give the corresponding
bisdiphosphate analogues. The bisubstrate analogues were substrates
for FPP synthase, giving novel seven-membered ring analogues of GPP
and FPP. The catalytic efficiencies for cyclization of <b>3-OPP</b> and <b>4-OPP</b> were similar to those for chain elongation
with IPP and DMAPP
Dual Allosteric Inhibition of SHP2 Phosphatase
SHP2 is a cytoplasmic protein tyrosine
phosphatase encoded by the <i>PTPN11</i> gene and is involved
in cell proliferation, differentiation, and survival. Recently, we
reported an allosteric mechanism of inhibition that stabilizes the
auto-inhibited conformation of SHP2. SHP099 (<b>1</b>) was identified
and characterized as a moderately potent, orally bioavailable, allosteric
small molecule inhibitor, which binds to a tunnel-like pocket formed
by the confluence of three domains of SHP2. In this report, we describe
further screening strategies that enabled the identification of a
second, distinct small molecule allosteric site. SHP244 (<b>2</b>) was identified as a weak inhibitor of SHP2 with modest thermal
stabilization of the enzyme. X-ray crystallography revealed that <b>2</b> binds and stabilizes the inactive, closed conformation of
SHP2, at a distinct, previously unexplored binding siteî—¸a cleft
formed at the interface of the <i>N</i>-terminal SH2 and
PTP domains. Derivatization of <b>2</b> using structure-based
design resulted in an increase in SHP2 thermal stabilization, biochemical
inhibition, and subsequent MAPK pathway modulation. Downregulation
of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520
cancer cells. Remarkably, simultaneous occupation of both allosteric
sites by <b>1</b> and <b>2</b> was possible, as characterized
by cooperative biochemical inhibition experiments and X-ray crystallography.
Combining an allosteric site 1 inhibitor with an allosteric site 2
inhibitor led to enhanced pharmacological pathway inhibition in cells.
This work illustrates a rare example of dual allosteric targeted protein
inhibition, demonstrates screening methodology and tactics to identify
allosteric inhibitors, and enables further interrogation of SHP2 in
cancer and related pathologies