Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR

Thermodynamic and structural studies are commonly utilized to optimize small molecules for specific DNA interactions, and, thus, a significant amount of binding data is available. However, the dynamic processes that are involved in minor groove complex formation and maintenance are not fully understood. To help define the processes involved, we have conducted 1D and 2D NMR in conjunction with biosensor-SPR experiments with a variety of compounds and symmetric, as well as asymmetric, AT tract DNA sequences. Surprisingly, the NMR data clearly show exchange between equivalent binding sites for strongly binding compounds like netropsin and DB921 (<i>K</i>_a > 10⁸ M^–1) that does not involve dissociation off the DNA. A quantitative analysis of the data revealed that these bound exchange rates are indeed much faster than the macroscopic dissociation rates which were independently determined by biosensor-SPR. Additionally, we could show the existence of at least two 1:1 compound DNA complexes at the same site for the interaction of these compounds with an asymmetric DNA sequence. To explain this behavior we introduced a model in which the ligand is rapidly flipping between two orientations while in close association with the DNA. The ligand reorientation will contribute favorably to the binding entropy. As the potential of minor groove binders to form more than a single complex with asymmetric, as well as symmetric, duplexes is widely unknown, the consequences for binding thermodynamics and compound design are discussed

Pharmacokinetics of DB868 and DB829 in uninfected vervet monkeys after the final (10^th) oral dose of DB868.

<p>Key: C_{4 h}, concentration at 4 h; C_{24 h}, concentration at 24 h; AUC_last, area under the curve from time zero to the last measurable concentration; AUC_0-∞, area under the curve from time zero to infinite time; t_½, terminal elimination half-life; BLQ, below limit of quantitation; NC, not calculable.</p

Metabolism of the prodrug DB868 in monkey liver microsomes.

<p>HPLC/UV chromatograms (A) and concentration-time profiles (B) of the prodrug DB868, intermediate metabolites, and active compound (DB829) following incubation of DB868 with male vervet monkey liver microsomes were shown. Incubation mixtures (1 mL at pH 7.4) contained 10 µM DB868, 0.5 mg/mL monkey liver microsomes, and 1 mM NADPH. Aliquots were removed at 1, 5, 15, 30 and 120 min, and analyzed for DB868, three intermediate metabolites (M1, M2, M3), and DB829 by HPLC/UV. Metabolite M4 was not quantified due to the lack of a synthetic standard. Symbols and error bars represent means and SDs of triplicate incubations.</p

Plasma concentration-time profiles of DB829 following administration of the prodrug DB868 to infected vervet monkeys.

<p>The monkeys confirmed to have first stage HAT were administered DB868 orally, beginning at 7 days post-infection, at 20 mg/kg/day for 5 days, 10 mg/kg/day for 7 days, or 3 mg/kg/day for 7 days. The inset graph shows the extended DB829 profiles up to 150 days post-last DB868 dose. * denotes the time (4 days post-last drug dose) that monkey 686 was euthanized due to clinical morbidity (peritoneal abscesses).</p

Efficacy of oral DB868 and intramuscular pentamidine against first stage <i>T. b. rhodesiense</i> infection in vervet monkeys.

<p>Key: P = parasitaemia; EoT = end of treatment; Neg = negative; Pos = positive; WD = withdrawn.</p

Changes in plasma biomarkers of liver injury in uninfected vervet monkeys administered DB868.

<p>DB868 was administered orally at 10 mg/kg/day (n = 4) or 30 mg/kg/day (n = 4) for 10 days, day −9 to day 0 post-last drug dose. Symbols and error bars represent means and SEs, respectively, of (<b>A</b>) alanine aminotransferase (ALT), (<b>B</b>) aspartate aminotransferase (AST), and (<b>C</b>) total and direct bilirubin (T. BIL and D. BIL, respectively) levels.</p

Changes in mean parasitaemia values of vervet monkeys treated with DB868.

<p>Starting at 7 days post-infection with <i>T. b. rhodesiense</i> KETRI 2537, monkeys confirmed to have first stage HAT were treated with either DB868 orally or pentamidine intramuscularly. DB868 at 20 mg/kg/day for 5 days (n = 2); DB868 at 10 mg/kg/day for 7 days (n = 2); DB868 at 3 mg/kg/day for 7 days (n = 2); pentamidine at 4 mg/kg/day for 7 days (n = 3). Symbols and error bars represent means and interindividual differences (range), respectively.</p

Changes in plasma biomarkers of kidney injury in uninfected vervet monkeys administered DB868.

<p>DB868 was administered orally at 10 mg/kg/day (n = 4) or 30 mg/kg/day (n = 4) for 10 days, day −9 to day 0 post-last drug dose. Symbols and error bars represent means and SEs, respectively, of (<b>A</b>) creatinine and (<b>B</b>) urea.</p

Structures of the prodrug (DB868) and active compound (DB829).

<p>Structures of the prodrug (DB868) and active compound (DB829).</p

Changes in body weight and haematological parameters in uninfected vervet monkeys administered DB868.

<p>The monkeys (n = 4) were administered DB868 orally at 30 mg/kg/day for 10 days, day −9 to day 0 post-last drug dose. Symbols and error bars represent means and SEs, respectively, of body weight, red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT).</p