Factors governing the assembly of cationic phospholipid-DNA complexes.

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength

Characterizing inhibitors of human AP endonuclease 1

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme

APE1 chemical shift perturbations induced by compound 6 (MLS000552981).

(a) 15N-TROSY spectra for APE1 (0.10 mM) in the absence (black) or presence (red) of compound 6 (0.30 mM). (b) Bar chart of chemical shift perturbations (Δδ) for backbone 1H, 15N resonances (combined) versus amino acid residue. (TIF)</p

APE1 chemical shift perturbations induced by 5-fluoroindole-2-carboxylic acid (2).

(a) 15N-TROSY spectra for APE1 (0.15 mM) in the absence (black) or presence (red) of 2 (1 mM). (b) Bar chart of chemical shift perturbations (Δδ) for backbone 1H, 15N resonances (combined) versus amino acid residue. Dashed lines are shown at Δδ values of 0.015 and 0.030. Residues exhibiting Δδ ≥0.015 are labeled in both figures. (TIF)</p

NMR perturbations for Mg²⁺-APE1 as induced by compound 6 (MLS000552981).

NMR experiments were performed using 0.30 mM compound 6 and 0.10 mM APE1 in the presence of MgCl2 at a concentration of (a) 1.0 mM or (b) 0.25 mM. (TIF)</p

NMR CSPs induced by the indole compounds mapped to a crystal structure of APE1 bound to nicked abasic DNA (enzyme-product complex; PDB ID: 5DFF).

In the upper row (a, c, e, g), APE1 is shown in surface format, DNA in cartoon format, and the Mg2+ cofactor is shown as a small green sphere, which, together with the nicked site of the DNA, locates the active site. Residues for which the compound induces a CSP (Δδ ≥0.015 ppm) have a sphere centered at the backbone N and colored according to CSP magnitude (log Δδ), with red and blue representing high and low values, respectively (red, Δδ = 0.6; blue, Δδ = 0.001). In the lower row (b, d, f, h), the same APE1 structure is shown in nontransparent surface format and in a different orientation; residues are colored according to CSP magnitude, with cyan for the largest CSPs (Δδ ≥ 0.030 ppm) and magenta for moderate CSPs (0.015 ppm ≤ Δδ f shows compound 3 bound in the remote pocket, as determined by our new crystal structure (Fig 3).</p