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KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2- c ]quinolines

View Article: PubMed Central - PubMed

ABSTRACT

KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5′-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 < IC50 < 4.80 μM), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy.

No MeSH data available.


Related in: MedlinePlus

FRET stabilization temperatures (ΔTm) of KRAS21R G4 and hairpin ds-DNA (T-loop) at 0.2 μM, in K-cacodylate buffer (pH = 7.4, 60 mM K+), stabilized by IQc derivatives and TMPyP4 at 1 μM.a) SD ≤ 0.2°C.
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f2: FRET stabilization temperatures (ΔTm) of KRAS21R G4 and hairpin ds-DNA (T-loop) at 0.2 μM, in K-cacodylate buffer (pH = 7.4, 60 mM K+), stabilized by IQc derivatives and TMPyP4 at 1 μM.a) SD ≤ 0.2°C.

Mentions: Fluorescence resonance energy transfer (FRET) studies with DNA sequences labelled at the 5′ and 3′ ends, were conducted to evaluate the G4 DNA stabilizing properties of IQcs (at 0.1 to 5 μM ligand concentrations). The G4-forming sequence KRAS21R11, together with T-loop, a 26-mer hairpin double-stranded DNA (ds-DNA) sequence were used in the first screening assay. Reproducible IQc dose-dependent melting curves (Sup. Inf.; Figure S3) and stabilization temperature (ΔTm) values were obtained (Figure 2 and Table S5). Alkyldiamine mono-substituted IQc compounds 2b–g gave moderate stabilization of the KRAS21R G4 structure, increasing Tm between 10 and 15°C; the di-substituted alkyldiamine derivatives 3f–j showed similar behaviour whereas some (3d, e) produced greater stabilization with ΔTm values varying between 12 and 22°C. IQcs lacking alkyldiamine side chains (1, 2a and 3c) showed reduced or negligible ability to stabilize the KRAS21R G4. Interestingly, those derivatives with a bulky benzyl N5-substituent (3c and 3j) showed decreased KRAS21R G4 stabilization when compared with their mono-substituted counterparts 2a and 3e. Moreover, all IQcs showed apparent lower binding affinity to ds-DNA (1.5 < ΔTm < 9.1°C for 2b–g and 1.6 < ΔTm < 6.5°C for 3d–j), which suggests selectivity of compounds to G4 compared to ds-DNA.


KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2- c ]quinolines
FRET stabilization temperatures (ΔTm) of KRAS21R G4 and hairpin ds-DNA (T-loop) at 0.2 μM, in K-cacodylate buffer (pH = 7.4, 60 mM K+), stabilized by IQc derivatives and TMPyP4 at 1 μM.a) SD ≤ 0.2°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382548&req=5

f2: FRET stabilization temperatures (ΔTm) of KRAS21R G4 and hairpin ds-DNA (T-loop) at 0.2 μM, in K-cacodylate buffer (pH = 7.4, 60 mM K+), stabilized by IQc derivatives and TMPyP4 at 1 μM.a) SD ≤ 0.2°C.
Mentions: Fluorescence resonance energy transfer (FRET) studies with DNA sequences labelled at the 5′ and 3′ ends, were conducted to evaluate the G4 DNA stabilizing properties of IQcs (at 0.1 to 5 μM ligand concentrations). The G4-forming sequence KRAS21R11, together with T-loop, a 26-mer hairpin double-stranded DNA (ds-DNA) sequence were used in the first screening assay. Reproducible IQc dose-dependent melting curves (Sup. Inf.; Figure S3) and stabilization temperature (ΔTm) values were obtained (Figure 2 and Table S5). Alkyldiamine mono-substituted IQc compounds 2b–g gave moderate stabilization of the KRAS21R G4 structure, increasing Tm between 10 and 15°C; the di-substituted alkyldiamine derivatives 3f–j showed similar behaviour whereas some (3d, e) produced greater stabilization with ΔTm values varying between 12 and 22°C. IQcs lacking alkyldiamine side chains (1, 2a and 3c) showed reduced or negligible ability to stabilize the KRAS21R G4. Interestingly, those derivatives with a bulky benzyl N5-substituent (3c and 3j) showed decreased KRAS21R G4 stabilization when compared with their mono-substituted counterparts 2a and 3e. Moreover, all IQcs showed apparent lower binding affinity to ds-DNA (1.5 < ΔTm < 9.1°C for 2b–g and 1.6 < ΔTm < 6.5°C for 3d–j), which suggests selectivity of compounds to G4 compared to ds-DNA.

View Article: PubMed Central - PubMed

ABSTRACT

KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5&prime;-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 &lt; IC50 &lt; 4.80 &mu;M), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy.

No MeSH data available.


Related in: MedlinePlus