KRASG12C has emerged as a promising target
in the treatment
of solid tumors. Covalent inhibitors targeting the mutant cysteine-12
residue have been shown to disrupt signaling by this long-“undruggable”
target; however clinically viable inhibitors have yet to be identified.
Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99)
we identified in KRASG12C to identify inhibitors suitable
for clinical development. Structure-based design efforts leading to
the identification of a novel quinazolinone scaffold are described,
along with optimization efforts that overcame a configurational stability
issue arising from restricted rotation about an axially chiral biaryl
bond. Biopharmaceutical optimization of the resulting leads culminated
in the identification of AMG 510, a highly potent, selective, and
well-tolerated KRASG12C inhibitor currently in phase I
clinical trials (NCT03600883).
Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimer's disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC(50) values ranging from 0.99 +/- 0.04 to 25.3 +/- 1.3 microM, whereas CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC(50) values > 60 microM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (K(i) = 11.0 +/- 1.3 microM), whereas inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 +/- 1.1 and 7.8 +/- 0.9 microM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC(50) 5.5 +/- 0.7 microM, K(i) = 5.4 +/- 0.3 microM) with less effect on other enzymes evaluated (IC(50) > 29 microM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC(50) = 8.8 +/- 1.2 microM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC(50) > 50 microM). Based on these data and expected tissue concentrations of inhibitors, we predict that a p.o. administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa.
Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between (1)H and (13)C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion.
A comprehensive understanding of
structure–reactivity relationships
is critical to the design and optimization of cysteine-targeted covalent
inhibitors. Herein, we report glutathione (GSH) reaction rates for N-phenyl acrylamides with varied substitutions at the α-
and β-positions of the acrylamide moiety. We find that the GSH
reaction rates can generally be understood in terms of the electron
donating or withdrawing ability of the substituent. When installed
at the β-position, aminomethyl substituents with amine pK
a’s > 7 accelerate, while those with
pK
a’s < 7 slow the rate of GSH
addition at pH 7.4, relative to a hydrogen substituent. Although a
computational model was able to only approximately capture experimental
reactivity trends, our calculations do not support a frequently invoked
mechanism of concerted amine/thiol proton transfer and C–S
bond formation and instead suggest that protonated aminomethyl functions
as an electron-withdrawing group to reduce the barrier for thiolate
addition to the acrylamide.
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