Combining 1,3-Ditriazolylbenzene and Quinoline to Discover a New G-Quadruplex-Interactive Small Molecule Active against Cancer Stem-Like Cells.

Quadruplex nucleic acids are promising targets for cancer therapy. In this study we used a fragment-based approach to create new flexible G-quadruplex (G4) DNA-interactive small molecules with good calculated oral drug-like properties, based on quinoline and triazole heterocycles. G4 melting temperature and polymerase chain reaction (PCR)-stop assays showed that two of these compounds are selective G4 ligands, as they were able to induce and stabilize G4s in a dose- and DNA sequence-dependent manner. Molecular docking studies have suggested plausible quadruplex binding to both the G-quartet and groove, with the quinoline module playing the major role. Compounds were screened for cytotoxicity against four cancer cell lines, where 4,4'-(4,4'-(1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(1-methylquinolin-1-ium) (1 d) showed the greater activity. Importantly, dose-response curves show that 1 d is cytotoxic in the human colon cancer HT-29 cell line enriched in cancer stem-like cells, a subpopulation of cells implicated in chemoresistance. Overall, this study identified a new small molecule as a promising lead for the development of drugs targeting G4 in cancer stem cells.

Cryptolepine and quindoline: understanding their photophysics.

Quindoline (QUIND, indolo[3,2-b]quinoline) and cryptolepine (CRYPT, 5-methyl-10H-indolo[3,2-b]quinoline) together with their corresponding derivatives have been studied for decades due to their important biological activity against diseases like malaria. The biological activity of drugs is routinely investigated using fluorescence based methods. However, recent reports show that the photophysics of CRYPT and its analogues is not yet understood. Herein, the photophysics of CRYPT and QUIND is studied in aqueous solutions at different pH values and in both protic and aprotic solvents of different polarities. CRYPT and QUIND are shown to exist in different prototropic forms depending on pH and solvent polarity. CRYPT is found to be more sensitive to the solvent nature. Both compounds are shown to have two-photon stimulated emission. Their two-photon absorption (TPA) cross-sections were measured in the 710-960 nm range. The TPA cross-section is relatively low but allows for the observation of both compounds in HEK 293 T cells, where CRYPT is found mostly in the nucleus and QUIND accumulates in the cytoplasm.

Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA.

A new family of 99mTc(I)- tricarbonyl complexes and 125I-heteroaromatic compounds bearing an acridine orange (AO) DNA targeting unit was evaluated for Auger therapy. Characterization of the DNA interaction, performed with the non-radioactive Re and 127I congeners, confirmed that all compounds act as DNA intercalators. Both classes of compounds induce double strand breaks (DSB) in plasmid DNA but the extent of DNA damage is strongly dependent on the linker between the Auger emitter (99mTc or 125I) and the AO moiety. The in vitro evaluation was complemented with molecular docking studies and Monte Carlo simulations of the energy deposited at the nanometric scale, which corroborated the experimental data. Two of the tested compounds, 125I-C5 and 99mTc-C3, place the corresponding radionuclide at similar distances to DNA and produce comparable DSB yields in plasmid and cellular DNA. These results provide the first evidence that 99mTc can induce DNA damage with similar efficiency to that of 125I, when both are positioned at comparable distances to the double helix. Furthermore, the high nuclear retention of 99mTc-C3 in tumoral cells suggests that 99mTc-labelled AO derivatives are more promising for the design of Auger-emitting radiopharmaceuticals than the 125I-labelled congeners.

Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives.

BACKGROUND: A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. METHODOLOGY/PRINCIPAL FINDINGS: IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 µM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). CONCLUSIONS: Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer.

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

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 p21(KRAS) 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.

Indolo[3,2-c]quinoline G-quadruplex stabilizers: a structural analysis of binding to the human telomeric G-quadruplex.

A library of 5-methylindolo[3,2-c]quinolones (IQc) with various substitution patterns of alkyldiamine side chains were evaluated for G-quadruplex (G4) binding mode and efficiency. Fluorescence resonance energy transfer melting assays showed that IQcs with a positive charge in the heteroaromatic nucleus and two weakly basic side chains are potent and selective human telomeric (HT) and gene promoter G4 stabilizers. Spectroscopic studies with HT G4 as a model showed that an IQc stabilizing complex involves the binding of two IQc molecules (2,9-bis{[3-(diethylamino)propyl]amino}-5-methyl-11H-indolo[3,2-c]quinolin-5-ium chloride, 3 d) per G4 unit, in two non-independent but equivalent binding sites. Molecular dynamics studies suggest that end-stacking of 3 d induces a conformational rearrangement in the G4 structure, driving the binding of a second 3 d ligand to a G4 groove. Modeling studies also suggest that 3 d, with two three-carbon side chains, has the appropriate geometry to participate in direct or water-mediated hydrogen bonding to the phosphate backbone and/or G4 loops, assisted by the terminal nitrogen atoms of the side chains. Additionally, antiproliferative studies showed that IQc compounds 2 d (2-{[3-(diethylamino)propyl]amino}-5-methyl-11H-indolo[3,2-c]quinolin-5-ium chloride) and 3 d are 7- to 12-fold more selective for human malignant cell lines than for nonmalignant fibroblasts.

N10,N11-di-alkylamine indolo[3,2-b]quinolines as hemozoin inhibitors: design, synthesis and antiplasmodial activity.

We recently reported that potent N10,O11-bis-alkylamine indolo[3,2-b]quinoline antimalarials act as hemozoin (Hz) growth inhibitors. To improve access and binding to the target we have now designed novel N10,N11-di-alkylamine bioisosteres. 3-Chloro derivatives (10a-f) showed selectivity for malaria parasite compared to human cells, high activity against Plasmodium falciparum chloroquine (CQ)-resistant strain W2 (IC50s between 20 and 158nM), good correlation with ß-hematin inhibition and improved vacuolar accumulation ratios, thus suggesting inhibition of Hz growth as one possible mechanism of action for these compounds. Moreover, our studies show that Hz is a valid target for the development of new antimalarials able to overcome CQ resistance.

Bis-alkylamine Indolo[3,2-b]quinolines as hemozoin ligands: implications for antimalarial cytostatic and cytocidal activities.

To get insight into the relevance of targeting hemozoin (Hz) crystals, two isomeric series, N5,N10-bis-alkylamine (2a-k) and N10,O11-bis-alkylamine (3a-k) indolo[3,2-b]quinolines, were evaluated for their in vitro activity against chloroquine (CQ)-resistant and sensitive strains of Plasmodium falciparum. In general, compounds of series 3 were more active than isomers 2, with IC50/LD50 ranging from 25/233 nM (3i) to 1.3 (3a)/10.7 (3b) µM. SAR analyses showed that lipophilicity and chlorine substitution at C3 increased both cytostatic and cytocidal activities. Both series bound to hematin monomer, inhibited ß-hematin formation in vitro, delayed intraerythrocytic parasite development with apparent inhibition of Hz biocrystallization, and showed higher cytocidal activity against schizonts. In addition, cytostatic and cytocidal activities of series 3, but not those of isomers 2, correlated with calculated vacuole accumulation ratios, suggesting different capacities of 2 and 3 to bind to the Hz crystal face {001} exposed on the vacuole aqueous medium and different mechanisms of cytocidal potency.

Synthesis, G-quadruplex stabilisation, docking studies, and effect on cancer cells of indolo[3,2-b]quinolines with one, two, or three basic side chains.

G-quadruplex (G4) DNA structures in telomeres and oncogenic promoter regions are potential targets for cancer therapy, and G4 ligands have been shown to modulate telomerase activity and oncogene transcription. Herein we report the synthesis and G4 thermal stabilisation effects, determined by FRET melting assays, of 20 indolo[3,2-b]quinolines mono-, di-, and trisubstituted with basic side chains. Molecular modelling studies were also performed in an attempt to rationalise the ligands' binding poses with G4. Overall, the results suggest that ligand binding and G4 DNA thermal stabilisation increase with an N5-methyl or a 7-carboxylate group and propylamine side chains, whereas selectivity between G4 and duplex DNA appears to be modulated by the number and relative position of basic side chains. From all the indoloquinoline derivatives studied, the novel trisubstituted compounds 3 d and 4 d, bearing a 7-(aminoalkyl)carboxylate side chain, stand out as the most promising compounds; they show high G4 thermal stabilisation (ΔTm values between 17 and 8 °C) with an inter-G4 ΔTm trend of Hsp90A>KRas21R≈F21T>c-Kit2, 10-fold selectivity for G4 over duplex DNA, and 100-fold selectivity for the HCT116 cancer cell line (IC50 and IC90: <10 µM) over primary rat hepatocytes. Compounds 3 d and 4 d also decreased protein expression levels of Hsp90 and KRas in HCT116 cancer cells.

Antitrypanosomal and cysteine protease inhibitory activities of alkyldiamine cryptolepine derivatives.

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC(50) of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.

1H NMR spectroscopic identification of protonable sites in cryptolepines with C-11 substituents containing two amino functionalities.

Knowledge of protonable sites and acid dissociation constants of cryptolepine derivatives having C-11 substituents containing two amino functionalities is of great importance to the understanding of the mechanism of their antimalarial action, which may contribute to their further development as drug candidates. In this work, we applied (1)H NMR titration to investigate the acid-base characteristics of these polyprotic compounds in the pH range 3-13. We identified three acid-base equilibria with most acid dissociation constants (pK(a)*) being greater than 10.5, which prevented us from using the potentiometric method. Overall, (1)H NMR titration was sensitive and suitable for the determination of pK(a) values for these drug leads.

Incorporation of basic side chains into cryptolepine scaffold: structure-antimalarial activity relationships and mechanistic studies.

The synthesis of cryptolepine derivatives containing basic side-chains at the C-11 position and their evaluations for antiplasmodial and cytotoxicity properties are reported. Propyl, butyl, and cycloalkyl diamine side chains significantly increased activity against chloroquine-resistant Plasmodium falciparum strains while reducing cytotoxicity when compared with the parent compound. Localization studies inside parasite blood stages by fluorescence microscopy showed that these derivatives accumulate inside the nucleus, indicating that the incorporation of a basic side chain is not sufficient enough to promote selective accumulation in the acidic digestive vacuole of the parasite. Most of the compounds within this series showed the ability to bind to a double-stranded DNA duplex as well to monomeric hematin, suggesting that these are possible targets associated with the observed antimalarial activity. Overall, these novel cryptolepine analogues with substantially improved antiplasmodial activity and selectivity index provide a promising starting point for development of potent and highly selective agents against drug-resistant malaria parasites.

C-11 diamino cryptolepine derivatives NSC748392, NSC748393, and NSC748394: anticancer profile and G-quadruplex stabilization.

G-Quadruplex DNA ligands are promising novel anticancer agents with potentially fewer side effects and greater selectivity than standard anticancer drugs. However, the design of G-quadruplex ligands remains challenging since known chemical features increasing selectivity have often compromised drugability. Three C-11 diamino cryptolepine derivatives, with significant chemical differences between the side chains, low cytotoxicity to mammalian non-tumor cells (Vero cells) and drug-like properties, were selected for anticancer drug screening in the NCI Developmental Therapeutics Program. The three compounds showed good in vitro anticancer profiles with GI(50) averages at sub-micromolar concentrations (0.32-0.78 µM), cytostatic effects (TGI) at micromolar concentrations (1.3-6.9 µM) and moderate cytotoxic effects to cancer cells (LC(50)) also at micromolar concentrations (4.7-33 µM), but only the compound with a linear alkylamine side chain (NSC748393) showed a good score in the in vivo anticancer Hollow Fiber assay. compare analysis of growth inhibition profile of NSC748393 suggested a multi-target mechanism. G-Quadruplex DNA binding affinity and selectivity studies by FRET-melting assays showed that NSC748392 and NSC478393, with aliphatic amine side chains, are good G-quadruplex ligands but not selective, whereas a C-11 aromatic side chain, as in NSC748394, increases selectivity although with decreasing binding affinity. Overall, NSC748393 can be considered a lead molecule for the design of effective but more selective anticancer drugs targeting telomeric G-quadruplexes.

Bis-alkylamine quindolone derivatives as new antimalarial leads.

Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave N,O-, N,N- and O-alkylated products containing one or two basic side-chains. The compounds were evaluated for antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum W2 strain and for cytotoxicity in HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones into moderate or highly active and selective antimalarial compounds. The most active and selective compound, 5c, showed an IC(50)=51 nM for P. falciparum and a selectivity ratio of 98.

Cryptolepine analogues containing basic aminoalkyl side-chains at C-11: synthesis, antiplasmodial activity, and cytotoxicity.

A series of cryptolepine derivatives has been synthesized through the incorporation of short basic side-chains in the C-11 position of the 10H-indolo[3,2-b]quinoline scaffold. Their antiplasmodial activity was evaluated in vitro against the chloroquine resistant Plasmodium falciparum W2 strain, showing IC(50) values between 22 and 184 nM, while their cytotoxicity was assessed using HUVEC cells, revealing three compounds with a selectivity ratio higher than 10. The most selective of these derivatives, 4d, with a selectivity ratio of 46, was also the least cytotoxic of the series.