AZ628 [3-(Cyano-dimethyl-methyl)-N-[4-methyl-3-(3-methyl-4-oxo-3,4-dihydro-quinazolin-6-ylamino)-phenyl]-benzamide]
is a potent and selective RAF kinase inhibitor with IC50 values
against V600E mutant BRAF and wild type BRAF of 34 nM and 91 nM, respectively
[1]. AZ628 reduces activities of preactivated B-Raf and c-Raf-1 in in vitro kinase assays, with IC50
values of 105 and 29 nM, respectively [2]. Specificity profliling indicates
that AZ628 also inhibits activation of number of tyrosine protein kinases
including VEGFR2, DDR2, Lyn, Flt1, FMS and others. AZ628 inhibits
anchorage-dependent and -independent growth, causes cell cycle arrest, and
induces apoptosis in colon and melanoma cell lines harboring B-RafV600E mutation.
The profile of AZ628 cross-reactivity suggests that similar to sorafenib, AZ628
may be antiangiogenic based on inhibition of VEGFR2 [2].
A genotype-correlated sensitivity to selective
kinase inhibitors by using high-throughput tumor cell line profiling revealed AZ628
(IC50 for WT BRAF 2.14 µM, mutant BRAF 196 nM) has remarkable
sensitivity among melanoma, thyroid, and colorectal cancer cell lines [3]. Biochemical
studies revealed that AZ628 sensitivity is well correlated with its ability to
suppress the downstream pathway component, ERK. Sensitivity to MEK inhibition
has been correlated with BRAF mutations in tumor cell lines. Consistent with
cell line sensitivity findings, researchers observed that, in BRAF mutant
melanoma cells, ERK activation is suppressed by a MEK inhibitor but not by
sorafenib.
The activity of AZ628 is as
follows:
IC50 (wild
type BRAF enzyme assay) = 91 nM
IC50 (V600E mutant BRAF enzyme assay)
= 34 nM
IC50 (preactivated BRAF enzyme assay)
= 105 nM
IC50 (preactivated CRAF enzyme assay)
= 29 nM
Common Name: AZ628
Synonyms: AZ-628; AZ628; AZ 628
IUPAC Name: 3-(Cyano-dimethyl-methyl)-N-[4-methyl-3-(3-methyl-4-oxo-3,4-dihydro-quinazolin-6-ylamino)-phenyl]-benzamide
CAS Number: 878739-06-1
SMILES: CC1=C(C=C(C=C1)NC(=O)C2=CC(=CC=C2)C(C)(C)C#N) NC3=CC4=C(C=C3) N=CN(C4=O)C
Mechanism of Action: Kinase Inhibitor; RAF Kinase Inhibitor
Indication: Various Cancers; Melanomas
Development Stage: Preclinical
Company: AstraZeneca
Activating BRAF kinase mutations arise in about
7% of all human tumors, and pre-clinical studies have validated the RAF-MEK-ERK
signaling cascade as a potentially important therapeutic target in this
setting. Various studies suggests that BRAF inhibitors may prove effective in
the subset of melanomas, colorectal, and thyroid cancers with V600E BRAF mutation,
and at a lower frequency in BRAF mutant brain, gastric, and lung tumors. Activating
somatic mutations in BRAF have been identified in 60% of melanomas, 40% of
thyroid cancers and 20% of colon cancers. The most common BRAF mutation leads
to a substitution of glutamic acid for valine at position 600 (V600E) within
the activation segment of the BRAF kinase domain, which results in elevated
kinase activity and stimulation of downstream MEK-ERK signaling, consequently
promoting tumor cell survival and proliferation.
AZ628 inhibited MAPK signaling and demonstrated
selective antiproliferative activity in cancer cell lines containing
aberrations in key members of MAPK pathway, particularly V600E BRAF mutations. AZ628
potently inhibited PMA-stimulated AP1 reporter activity in 293T cells with an
IC50 value less than 4nM, consistent with MAPK pathway inhibition,
and showed good selectivity over other pathway reporter assays. AZ628 was
further profiled for antiproliferative activity against 70 cancer cell lines
representing a range of tumor types. 24 out of 70 lines were responsive (GI50s
less than 1 µM) to AZ628, with melanoma and colon cell lines being the most
sensitive tumor lineages, whereas the activity range was observed with GI50
values between 0.02 µM to greater than 32 µM. All cell lines tested with a
known V600E or V600G BRAF mutation were responsive to AZ628. Of two melanoma
cell lines with overexpressed wild type BRAF, only the one with elevated pERK
was responsive [1].
AZ628 decreased phospho-ERK (pERK) levels in
Colo205 and A375 cancer cell lines with endogenous V600E BRAF mutations, with EC50 values in the 14-16nM range, and induced a predominant G1 arrest phenotype
in both cell types. pMEK levels were similarly reduced and inhibition of MAPK
signaling was associated with decreased cyclin D1 and increased p27 levels. AZ628
has no effect on pSTAT3 or pPaxillin levels.
To anticipate potential mechanisms of acquired
resistance to RAF inhibitors during the course of treatment, researchers
established drug-resistant clones from a human melanoma-derived cell line
harboring the recurrent V600E activating BRAF mutation. V600E mutant BRAF exhibits
exquisite sensitivity to AZ628. Researchers determined that elevated CRAF
protein levels account for the acquisition of resistance to AZ628 in these
cells, associated with a switch from BRAF to CRAF dependency in tumor cells. They
also found that elevated CRAF protein levels may similarly contribute to
primary insensitivity to RAF inhibition in a subset of BRAF mutant tumor cells.
Interestingly, AZ628-resistant cells demonstrating either primary drug
insensitivity or acquired drug resistance exhibit exquisite sensitivity to the HSP90
inhibitor geldanamycin. Geldanamycin effectively promotes the degradation of
CRAF, thereby revealing a potential therapeutic strategy to overcome resistance
to RAF inhibition in a subset of BRAF-mutant tumors [4].
References:
1. Shen, M.; et. al.
Linking molecular characteristics to the pharmacological response of a panel of
cancer cell lines to the BRAF inhibitor, AZ628. Cancer Res 2007, 67, 5249.
2. Khazak, V.; et. al.
Selective Raf inhibition in cancer therapy. Expert Opin Ther Targets 2007, 11(12),
1587-1609.
3. McDermott, U.;
et. al. Identification of genotype-correlated sensitivity to selective kinase
inhibitors by using high-throughput tumor cell line profiling. Proc Natl Acad
Sci U S A. 2007, 104(50), 19936–19941.
4. Settleman, J.; et. al. Elevated CRAF as a
potential mechanism of acquired resistance to BRAF inhibition in melanoma.
Cancer Res 2008, 68(12), 4853-4861.
