Single-molecule views of MutS on mismatched DNA

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.     

Cooperative dynamics of a DNA polymerase replicating complex

Engineered DNA polymerases continue to be the workhorses of many applications in biotechnology, medicine and nanotechnology. However, the dynamic interplay between the enzyme and the DNA remains unclear. In this study, we performed an extensive replica exchange with flexible tempering (REFT) molecular dynamics simulation of the ternary replicating complex of the archaeal family B DNA polymerase from the thermophile Thermococcus gorgonarius, right before the chemical step. The convoluted dynamics of the enzyme are reducible to rigid-body motions of six subdomains. Upon binding to the enzyme, the DNA double helix conformation changes from a twisted state to a partially untwisted state. The twisted state displays strong bending motion, whereby the DNA oscillates between a straight and a bent conformation. The dynamics of double-stranded DNA are strongly correlated with rotations of the thumb toward the palm, which suggests an assisting role of the enzyme during DNA translocation. In the complex, the primer–template duplex displays increased preference for the B-DNA conformation at the n ? 2 and n ? 3 dinucleotide steps. Interactions at the primer 3′ end indicate that Thr541 and Asp540 are the acceptors of the first proton transfer in the chemical step, whereas in the translocation step both residues hold the primer 3′ terminus in the vicinity of the priming site, which is crucial for high processivity.     

Discovery of ZAP70 inhibitors by high-throughput docking into a conformation of its kinase domain generated by molecular dynamics

Very few selective inhibitors of the zeta-chain associated protein kinase 70 kDa (ZAP70) have been reported despite its importance in autoimmune diseases. Here, to induce a fit of the so-called gatekeeper residue (Met414) and hydrophobic pocket next to it, a potent Janus kinase 2 (JAK2) inhibitor was first docked into the ATP binding site of ZAP70 by structural alignment of the kinase domains. The resulting model of the complex between ZAP70 and the JAK2 inhibitor was then relaxed by an explicit solvent molecular dynamics simulation with restraints on the backbone atoms. High-throughput docking into the induced-fit conformation of ZAP70 generated by molecular dynamics has revealed 10 low-micromolar inhibitors which correspond to six distinct chemotypes. One of these ZAP70 inhibitors has an IC₅₀ of 110 nM for JAK2.     

3D-QSAR (CoMFA,CoMSIA), molecular docking and molecular dynamics simulations study of 6-aryl-5-cyano-pyrimidine derivatives to explore the structure requirements of LSD1 inhibitors

Recently, Histone Lysine Specific Demethylase 1 (LSD1) was regarded as a promising anticancer target for the novel drug discovery. And several small molecules as LSD1 inhibitors in different structures have been reported. In this work, we carried out a molecular modeling study on the 6-aryl-5-cyano-pyrimidine fragment LSD1 inhibitors using three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics simulations. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to generate 3D-QSAR models. The results show that the best CoMFA model has q² = 0.802, r²_ncv = 0.979, and the best CoMSIA model has q² = 0.799, r²_ncv = 0.982. The electrostatic, hydrophobic and H-bond donor fields play important roles in the models. Molecular docking studies predict the binding mode and the interactions between the ligand and the receptor protein. Molecular dynamics simulations results reveal that the complex of the ligand and the receptor protein are stable at 300 K. All the results can provide us more useful information for our further drug design.     

Discovery of novel DNA methyltransferase 3A inhibitors via structure-based virtual screening and biological assays

DNA methyltransferases are involved in diverse biological processes and abnormal methylation patterns play essential roles in cancer initiation and progression. DNA methyltransferase 3A (DNMT3A) acting as a de novo DNA methyltransferase, has gained widespread attention especially in haematological diseases. To date, large numbers of DNMTs inhibitors have been discovered, however, the small molecular inhibitors targeting DNMT3A are still in its infancy. In this study, structure-based virtual screening in combination with biological assays was performed to discovery potent novel DNMT3A inhibitors. Compound 40 and 40_3 displayed comparable in vitro inhibitory activity against DNMT3A with IC₅₀ values of 46.5 μM and 41 μM, respectively. Further binding mode analysis suggested these molecules inhibit DNMT3A activity through binding the S-adenosyl-l-methionine (SAM) pocket. Overall, 40 and 40_3 may serve as novel scaffolds for further optimization and small molecular probes for investigating DNMT3A function.     

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (K_i = 0.29 ± 0.07 μM, ΔT_m = 8.5 °C). This compound’s effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.     

Structural and diffusion properties of formamide/water mixture interacting with TiO2 surface

The photoreaction and adsorption properties on surfaces, thermal decomposition, chemical transformation, and other properties of the formamide molecule are widely used to understand the origins of the formation of biological molecules (nucleosides, amino acids, DNA, monolayers, etc.) needed for life. The titanium oxide (TiO₂) surface can act both as a template on which the accumulation of adsorbed molecules like formamide occurs through the concentration effect, and as a catalytic material that lowers the activation energy needed for the formation of intermediate products. In this paper, a formamide–water solution interacting with TiO₂ (anatase) surface is simulated using the molecular dynamics method. The structural, diffusion and density properties of formamide–water mixture on TiO₂ are established for a wide temperature range from T = 250 K up to T = 400 K.     

Effects of hydroxysafflor yellow A on ALDH1: Inhibition kinetics and molecular dynamics simulation

Hydroxysafflor yellow A (HSYA) is a potent natural antioxidant that displays important neuroprotective activity. Inhibition of acetaldehyde dehydrogenase 1 (ALDH1) has attracted the attention of researchers due to its overexpression in several types of cancers. We studied the effects of HSYA on ALDH1 by evaluating the inhibitory kinetics based on its antioxidant properties and performing computational simulation integrating methods. HSYA reversibly inhibited human recombinant ALDH1 via non-competitive inhibition (K_i = 0.267 ± 0.024 mM). We also investigated the tertiary structural changes via measuring intrinsic and ANS-binding fluorescence. The results indicated that the inactivation induced by HSYA was associated with structural changes. To obtain further information, we simulated the 3D structure of ALDH1 and conducted computational docking simulations as well as molecular dynamics simulations. The results indicated that 4 rings of HSYA interact with several residues near the ALDH1 active site. Our study provides insight into the inhibition of ALDH1 accompanied by structural changes. Based on its ALDH1-inhibiting effect and its potential as a natural antioxidant, HSYA is a potential agent for treating ALDH1-associated cancers.     

Molecular modeling studies of atorvastatin analogues as HMGR inhibitors using 3D-QSAR,molecular docking and molecular dynamics simulations

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is generally regarded as targets for the treatment of hypercholesterolemia. HMGR inhibitors (more commonly known as statins) are discovered as plasma cholesterol lowering molecules. In this work, 120 atorvastatin analogues were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulation. The results show that the best CoMFA (comparative molecular field analysis) model has q² = 0.558 and r² = 0.977, and the best CoMSIA (comparative molecular similarity indices analysis) model has q² = 0.582 and r² = 0.919. Molecular docking and MD simulation explored the binding relationship of the ligand and the receptor protein. The calculation results indicated that the hydrophobic and electrostatic fields play key roles in QSAR model. After MD simulation, we found four vital residues (Lys735, Arg590, Asp690 and Asn686) and three hydrophobic regions in HMGR binding site. The calculation results show that atorvastatin analogues obtained by introduction of F atoms or gem-difluoro groups could obviously improve the inhibitory activity. The new HMGR inhibitor analogues design in this Letter had been submitted which is being currently synthesized by our laboratories.     

Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis

We studied the inhibitory effect of gastrodin on tyrosinase using inhibition kinetics and computational simulation. Gastrodin reversibly inhibited tyrosinase in a mixed-type manner with K_i = 123.8 ± 20.2 mM. Time-interval kinetics revealed the inhibition to be a first-order process with mono- and bi-phasic components. Using AutoDock Vina, we calculated a binding energy of ?6.3 kcal/mol for gastrodin and tyrosinase, and we performed a molecular dynamics simulation of the tyrosinase–gastrodin interaction. The simulation results suggested that gastrodin interacts primarily with histidine residues in the active site. A 10-ns molecular dynamics simulation showed that one copper ion in the tyrosinase active site was responsible for the interaction with gastrodin. Our study provides insight into the inhibition of tyrosinase by the hydroxyl groups of gastrodin. A combination of inhibition kinetics and computational calculations may help to confirm the inhibitory action of gastrodin on tyrosinase and define the mechanisms of inhibition.     

Towards understanding the unbound state of drug compounds: Implications for the intramolecular reorganization energy upon binding

There has been an explosion of structural information for pharmaceutical compounds bound to biological targets, but the conformations and dynamics of compounds free in solution are poorly characterized, if at all. Yet, knowledge of the unbound state is essential to understand the fundamentals of molecular recognition, including the much debated conformational intramolecular reorganization energy of a compound upon binding (ΔE_Reorg). Also, dependable observation of the unbound compounds is important for ligand-based drug discovery, e.g. with pharmacophore modelling. Here, these questions are addressed with long (⩾0.5 μs) state-of-the-art molecular dynamics (MD) simulations of 26 compounds (including 7 approved drugs) unbound in explicit solvent. These compounds were selected to be chemically diverse, with a range of flexibility, and good quality bioactive X-ray structures. The MD-simulated free compounds are compared to their bioactive structure and conformers generated with ad hoc sampling in vacuo or with implicit generalized Born (GB) aqueous solvation models. The GB conformational models clearly depart from those obtained in explicit solvent, and suffer from conformational collapse almost as severe as in vacuo. Thus, the global energy minima in vacuo or with GB are not suitable representations of the unbound state, which can instead be extensively sampled by MD simulations. Many, but not all, MD-simulated compounds displayed some structural similarity to their bioactive structure, supporting the notion of conformational pre-organization for binding. The ligand–protein complexes were also simulated in explicit solvent, to estimate ΔE_Reorg as an enthalpic difference ΔH_Reorg between the intramolecular energies in the bound and unbound states. This fresh approach yielded ΔH_Reorg values ⩽ 6 kcal/mol for 18 out of 26 compounds. For three particularly polar compounds 15 ⩽ ΔH_Reorg ⩽ 20 kcal/mol, supporting the notion that ΔH_Reorg can be substantial. Those large ΔH_Reorg values correspond to a redistribution of electrostatic interactions upon binding. Overall, the study illustrates how MD simulations offer a promising avenue to characterize the unbound state of medicinal compounds.     

Towards alpha-glucosidase folding induced by trifluoroethanol: Kinetics and computational prediction

Alpha-glucosidase (EC 3.2.1.20) is an enzyme, which is related with diabetes mellitus type 2 clinically, and is also generally used to convert starch to fermentable sugars in the industry. Therefore, study on this enzyme structures and functions is important. In this study, we investigated structural changes in the alpha-glucosidase during trifluoroethanol (TFE)-induced unfolding. The activity of alpha-glucosidase was significantly inactivated by TFE in a dose-dependent manner. The inactivation was composed of two-phases. TFE inhibited alpha-glucosidase in a parabolic mixed-type reaction (K_i = 0.72 ± 0.08 M). TFE directly induced the unfolding and hydrophobic exposure of alpha-glucosidase. We also simulated the docking between alpha-glucosidase and TFE, as well as molecular dynamics. The computational simulations suggested that several residues, such as ASP68, TYR71, VAL108, HIS111, PHE177, ASP214, THR215, GLU276, HIS348, ASP349, and ARG439, interact with TFE. The molecular dynamics simulation confirmed the binding mechanisms, between the alpha-glucosidase and TFE, and suggested that TFE inhibits the glucose binding site. Our study provides insights into the alpha-glucosidase folding behaviors, and cosolvent binding under a 3D structural simulation.     

Structure-related protein tyrosine phosphatase 1B inhibition by naringenin derivatives

Naturally occurring flavonoids co-exist as glycoside conjugates, which dominate aglycones in their content. To unveil the structure-activity relationship of a naturally occurring flavonoid, we investigated the effects of the glycosylation of naringenin on the inhibition of enzyme systems related to diabetes (protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase) and on glucose uptake in the insulin-resistant state. Among the tested naringenin derivatives, prunin, a single-glucose-containing flavanone glycoside, potently inhibited PTP1B with an IC₅₀ value of 17.5 ± 2.6 µM. Naringenin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC₅₀: 5.4 ± 0.30 µM). In addition, prunin significantly enhanced glucose uptake in a dose-dependent manner in insulin-resistant HepG2 cells. Regarding the inhibition of α-glucosidase, naringenin exhibited more potent inhibitory activity (IC₅₀: 10.6 ± 0.49 µM) than its glycosylated forms and the reference inhibitor, acarbose (IC₅₀: 178.0 ± 0.27 µM). Among the glycosides, only prunin (IC₅₀: 106.5 ± 4.1 µM) was more potent than the positive control. A molecular docking study revealed that prunin had lower binding energy and higher binding affinity than glycosides with higher numbers of H-bonds, suggesting that prunin is the best fit to the PTP1B active site cavity. Therefore, in addition to the number of H-bonds present, possible factors affecting the protein binding and PTP1B inhibition of flavanones include their fit to the active site, hydrogen-bonding affinity, Van der Waals interactions, H-bond distance, and H-bond stability. Furthermore, this study clearly depicted the association of the intensity of bioactivity with the arrangement and characterization of the sugar moiety on the flavonoid skeleton.     

DNA-binding affinity and anticancer activity of β-carboline–chalcone conjugates as potential DNA intercalators: Molecular modelling and synthesis

A new series of DNA-interactive β-carboline–chalcone conjugates have been synthesized and evaluated for their in vitro cytotoxicity and DNA-binding affinity. It has been observed that most of these new hybrids have shown potent cytotoxic activities on A-549 (lung adenocarcinoma) cell lines with IC₅₀ values lower than 10 μM. The hybrid 7b is more effective against some of the selected cancer cell lines with IC₅₀ values less than 50 μM. In addition, compounds 7e, 7k, 7p–u has displayed significant elevation in ΔT_m of DNA in comparison to Adriamycin, suggesting significant interaction and remarkable DNA stabilization. The DNA intercalation of these new hybrids has been investigated by fluorescence titration, DNA viscosity measurements, molecular docking as well as molecular dynamics and the results are in agreement with the thermal denaturation studies.     

Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V_un) by the velocity of translocation on ssDNA in nucleotides per second (V_trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s, and the average translocation velocity was 267 ± 15 nt/s. The V_un/V_trans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.     

The Primary and Secondary Translocase Activities within E. coli RecBC Helicase Are Tightly Coupled to ATP Hydrolysis by the RecB Motor

Escherichia coli RecBC, a rapid and processive DNA helicase with only a single ATPase motor (RecB), possesses two distinct single‐stranded DNA (ssDNA) translocase activities that can operate on each strand of an unwound duplex DNA. Using a transient kinetic assay to detect phosphate release, we show that RecBC hydrolyzes the same amount of ATP when translocating along ssDNA using only its primary translocase (0.81 ± 0.05 ATP/nt), only its secondary translocase (1.12 ± 0.06 ATP/nt), or both translocases simultaneously (1.07 ± 0.09 ATP/nt). A mutation within RecB (Y803H) that slows the primary translocation rate of RecBC also slows the secondary translocation rate to the same extent. These results indicate that the ATPase activity of the single RecB motor drives both the primary and secondary RecBC translocases in a tightly coupled reaction. We further show that RecBC also hydrolyzes the same amount of ATP (0.95 ± 0.08 ATP/bp) while processively unwinding duplex DNA, suggesting that the large majority, possibly all, of the ATP hydrolyzed by RecBC during DNA unwinding is used to fuel ssDNA translocation rather than to facilitate base pair melting. A model for DNA unwinding is proposed based on these observations.     

Investigating the binding interactions of galantamine with β-amyloid peptide

The anti-Alzheimer’s agent galantamine is known to possess anti-amyloid properties. However the exact mechanisms are not clear. We studied the binding interactions of galantamine with amyloid peptide dimer (Aβ_1–40) through molecular docking and molecular dynamics simulations. Galantamine’s binding site within the amyloid peptide dimer was identified by docking experiments and the most stable complex was analyzed by molecular dynamics simulation. These studies show that galantamine was interacting with the central region of the amyloid dimer (Lys16–Ala21) and the C-terminal region (Ile31–Val36) with minimum structural drift of Cα atom in those regions. Strikingly, a significant drift was observed at the turn region from Asp23-Gly29 (Cα atom RMSD = 9.2 Å and 11.6 Å at 50 fs and 100 fs respectively). Furthermore, galantamine’s binding mode disrupts the key pi–pi stacking interaction between aromatic rings of Phe19 (chain A) and Phe19 (chain B) and intermolecular hydrogen bonds seen in unbound peptide dimer. Noticeably, the azepine tertiary nitrogen of galantamine was in close proximity to backbone CO of Leu34 (distance <3.5 Å) to stabilize the dimer conformation. In summary, the results indicate that galantamine binding to amyloid peptide dimer leads to a significant conformational change at the turn region (Asp23–Gly29) that disrupts interactions between individual β-strands and promotes a nontoxic conformation of Aβ_1–40 to prevent the formation of neurotoxic oligomers.     

Experimental,computational and chemometrics studies of BSA-vitamin B6 interaction by UV–Vis,FT-IR,fluorescence spectroscopy,molecular dynamics simulation and hard-soft modeling methods

The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV–Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (k_q), dynamic quenching (K_SV) and static quenching (K_LB) at 310 K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster’s non-radiative energy transfer theory and were equal to 41.1% and 2.11 nm.The collected UV–Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5 ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand. Molecular modeling results showed that VB6–BSA complex formed not only on the basis of electrostatic forces, but also on the basis of π–π staking and hydrogen bond. There was an excellent agreement between the experimental and computational results. The results presented in this paper, will offer a reference for detailed and systematic studies on the biological effects and action mechanism of small molecules with proteins.     

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein + chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.