The rat sarcoma (RAS) oncogene
was discovered as a genetic element of the Harvey and Kirsten rat
sarcoma (KRAS) viruses capable of immortalizing mammalian cells.
1−3 Subsequently, RAS protooncogenes were identified in normal cells.
Mutated RAS oncogenes (i.e., HRAS, NRAS, and KRAS) are found in 30%
of all human cancers. KRAS mutations are found in >90% of pancreatic
cancers, 50% of colon cancers, and 25% of lung adenocarcinomas.
4 RAS proteins are members of a family of guanine-nucleotide-binding
proteins with intrinsic GTPase activity that are often found associated
with the plasma membrane as a consequence of posttranslational modifications.
In the GTP-bound form, RAS proteins are active and serve as nucleotide
switches converting extracellular cues (e.g., from growth factors)
into intracellular signals. Missense mutations within the RAS gene
result in oncoproteins with reduced GTPase and therefore overactive
in signaling. Tumor cells harboring RAS mutations are often dependent
upon RAS protein constitutive activity for survival—a defining
feature of “oncogene addiction”. Consequently, KRAS
is an exceptional albeit challenging target for targeted cancer therapies.
Direct targeting of KRAS function with small molecules has not yet
proven achievable; therefore, alternative approaches of selectively
targeting KRAS-dependent tumors have been explored. One of these involves
the targeting of a nononcogene codependency evident exclusively in
the context of KRAS dependency.
5,6In 2009, RNAi
experiments revealed that lowering mRNA levels of
a transcript encoding the serine/threonine kinase STK33 was selectively
toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule
inhibitors of STK33 might selectively target KRAS-dependent cancers.
7 Although its function in normal biology is not
fully understood, STK33 was found to be critical for the survival
of KRAS-dependent hematopoietic cancer cell lines (acute myeloid leukemia,
multiple myeloma, and T cell acute lymphoblastic leukemia) and epithelial
cancer cell lines (colon, breast, pancreatic, and lung cancer). Using
mutations in the ATP-binding loop, the kinase activity of STK33 was
inferred to be required for the survival of KRAS-dependent cancer
cell lines. These results raised the possibility that small-molecule
inhibitors of STK33 may lead to the selective killing of KRAS cancers.
After we initiated our studies, small-molecule inhibitors of STK33
were published by two groups (
1 and
2, Figure ).
8 Both
of these compounds show excellent in vitro inhibition of STK33 but
do not show selective killing of KRAS-dependent cancer cells. Kinase
profiling experiments revealed that compounds
1 and
2 also inhibited a number of other kinases in addition to
STK33. For example,
1 is 2-fold selective for Aurora-B
(AurB) versus STK33, and
2 is only ~5-fold selective
for STK33 versus both AurB and PKA. This lack of selectivity could
render difficult the correlation between the STK33 activity and the
observed phenotype and thus could make it difficult to understand
the role of STK33 in KRAS-dependent and -independent cell lines.
A small molecule that demonstrates exclusive selectivity for STK33
would be ideal, but this may not be achievable. Hence, a collection
of small molecules that demonstrates different kinase selectivities
could be useful for interpreting results from genomic cancer cell-line
profiling. On the basis of the kinase profiling results of
1 and
2, we chose PKA and AurB as counter screens in
an effort to develop a selective STK33 inhibitor that would have a
significantly different kinase profile. In addition, PKA and STK33
belong to a structurally related class of protein kinases.
10 Hence, PKA inhibition serves as a measure of
selectivity against kinases closely related to STK33. AurB falls into
the atypical structural class and hence was expected to report on
broader kinase selectivity.
Our studies began with a high-throughput screening
to find STK33
inhibitors. Screening of 321811 compounds from the MLSMR library in
1536-well plate format led to the identification of 13 hits with submicromolar
IC50 values for STK33. On the basis of synthetic tractability
and selectivity against PKA and AurB, we selected quinoxalinone 3 for further development (Figure ).
Quinoxalinone
3 exhibited selectivities
against PKA
and AurB greater than 35-fold and was considered to be a good starting
point for SAR study. The synthetic strategy for accessing analogues
of
3 is described in Scheme
1.
Benzene-1,2-diamine derivatives
4 were first condensed
with isatin derivatives
5 to deliver intermediate compounds
6. These intermediates were then used as common building blocks
to prepare a series of amide analogues
7a–
t (through amide bond coupling), amine analogues
8a–
e (through reductive amination reactions), and
sulfonamide analogues
9a,
b (through coupling
reactions with sulfonyl chlorides). SAR on the northern portion of
3 is presented in Tables
1 and
2.
| Table 1SAR at the Northern Position: Amidesa |
| Table 2SAR at the Northern Position: Amines
and Sulfonamidesa |
To replace the thiophene ring present in the hit compound,
a large
number of amide analogues were synthesized (Table
1). Replacing the thiophene ring with a phenyl group (
7b) led to a slight increase in activity. Using deactivated
thiophene such as 5-chlorothiophene (
7c) or thiazole
(
7d) led to a slight decrease in activity. Replacement
of the thiophene ring by a 2-pyridyl (
7e) led to a 4-fold
increase in activity, but the compound was found to be chemically
unstable as a DMSO solution (LCMS follow-up over a few days showed
decomposition). The 3-pyridyl analogue (
7f) was more
stable, but it was less potent than the hit compound. The 4-pyridyl
analogue was also synthesized but could not be obtained in sufficient
purity, probably because of its chemical instability. Substituted
benzoic acid derivatives were then used in the amide bond coupling
reaction. The 4-fluorophenyl analogue (
7g) led to a 2-fold
increase in potency. However, similar to what was observed with the
2-pyridyl analogue, the 4-fluorophenyl analogue was found to be unstable.
Introduction of a chloro
(
7h) or methoxy
substituent (
7i) led to decreases in activity, independent
of their position on the phenyl ring. Using aliphatic groups in place
of the thiophene ring led to inactive compounds; the cyclohexyl analogue
(
7j) is shown as a representative.
Amine and sulfonamide
analogues were then investigated using reductive
amination and coupling reactions with sulfonyl chloride, respectively
(Table
2). In most cases, the compounds were
found to be inactive against STK33. Remarkably, in the amine series,
the thiophene (
8a), phenyl (
8b), 2-pyridyl
analogue (
8c), and 4-fluorophenyl analogues (
8d) were completely inactive against STK33, showing that the carbonyl
group present in the corresponding amide analogues is critical for
activity.
Next, the influence of substituents on the southern
phenyl ring
was investigated (Table
3) and was limited
to symmetric diamines to avoid regioselectivity issues during the
condensation reaction with isatin. The use of 4,5-dichloro- (
7k), 4,5-difluoro- (
7l), or 4,5-dimethyl substituents
(
7m) led to a decrease in potency and selectivity versus
PKA.
| Table 3SAR at the Southern Phenyl Ring |
Next, we investigated the SAR on the eastern phenyl
ring (Table
4). In this case, the synthetic
strategy relied on
amidation of diversely substituted isatins with thiophene-2-carbonyl
chloride, followed by a condensation of the obtained intermediate
with benzene-1,2-diamine (see the
Supporting Information). Introduction of a 4-chloro substituent (
7n) led to
a 2-fold increase in activity. Introduction of a 4-fluoro
- substituent (
7o) led to a slight decrease in potency.
The use of a 4-methoxy (
7p) or a 4-trifluoromethoxy substituent
(
7q) led to an increase in potency. The use of a bulkier,
electron-donating 4-isopropyl substituent (
7r, ML281)
provided a 20-fold increase in activity against STK33. With an IC
50 of 14 nM, ML281 showed greater than 700-fold selectivity
over PKA. Introducing electron-withdrawing groups at the 5-position
(
7s and
7t) also led to an increase in activity,
and the 5-fluoro analogue
7t showed an IC
50 value against STK33 of 78 nM.
| Table 4SAR on the Eastern Phenyl Ring |
ML281 showed a solubility of 5.8 μM in PBS,
high plasma protein
binding (99.6% in human and 99.9% in mouse), and variable plasma stability
(80.3% in human and 10.0% in mouse).
Throughout this study,
AurB was also used as a counter screen,
and the results for selected quinoxalinone analogs are presented in
Table
5. The most potent STK33 inhibitors were
found to be mostly inactive against AurB. ML281 showed a 550-fold
selectivity over AurB and greater than 700-fold selectivity over PKA.
We also resynthesized
1 (see the
Supporting Information) and compared it with ML281 and
2 (Table
5). Compound
1 shows an IC
50 of 7 nM against STK33 and 28- and 0.4-fold
selectivities over PKA and AurB, respectively. Similarly, the fasudil
analogue
2 shows an IC
50 of 11 nM against
STK33 and 5-fold selectivities over PKA and AurB. Hence, ML281 does
not inhibit kinases that are strongly inhibited by
1 and
2 and will constitute a valuable complement tool to better
correlate STK33 activity to phenotype in cells.
| Table 5Selectivity versus AurB for Selected
Analogues |
ML281 was then profiled against a panel of 83 kinases
chosen for
diversity and toxicity (Figure ). ML281 was
found to be extremely selective and inhibits only two kinases other
than STK33 in the panel tested at a level of 25% or more: FLT3 (a
proto-oncogene) and KDR (VEGF R2 associated with vascularization).
Full data for the 83 kinases tested are provided in the
Supporting Information.
ML281 was finally profiled in cellular assays to
test the hypothesis
of STK33 synthetic lethality in KRAS-dependent cell lines. Figure shows the relative viability of two KRAS-dependent
(NOMO-1 and SKM-1) and two KRAS-independent (THP-1 and U937) AML-derived
cell lines. ML281 does not appear to significantly alter viability
of any of the tested cell lines at concentrations of up to 10 μM.
This experiment has been repeated with more than 20 KRAS-dependent
and KRAS-independent cell lines, and no significant correlation between
KRAS dependency and cell viability was found (see the
Supporting Information). Whether ML281 is not inhibiting STK33
in cells (due to off-target effects or high plasma protein binding)
or whether STK33 inhibition is not synthetic lethal to KRAS-dependent
cell line remains to be determined. In this context, biomarkers of
STK33 activity should greatly help the cancer research community to
answer that question.
The small-molecule probe ML281 is a nanomolar inhibitor
of STK33
and is in a novel chemical class as compared to the other recently
reported inhibitors. Although the potency of ML281 for in vitro STK33
inhibition is not significantly improved as compared to 1 and 2, it exhibits greater selectivity against PKA
and AurB (as well as excellent selectivity when profiled against 83
protein kinases) and provides a new chemical class and selectivity
profile of STK33 inhibitors. This new probe may be useful to elucidate
the cellular functions of STK33. Compounds 1 and 2 have proven unsuccessful in selectively killing KRAS-dependent
cancer cell lines. Our own results with ML281 shed more light on the
biology of STK33 and further suggest that the synthetic lethality
with KRAS oncogenes cannot be recapitulated with selective STK33 small-molecule
inhibitors. However, the development of STK33 biomarkers is strongly
desired to understand the role of STK33 in cells and to correlate
inhibitors activity to cell fate better. ML281 has been registered
with NIH Molecular Libraries Program and is available upon request.
