Discovery of Novel Quinoline-Based Proteasome Inhibitors for Human African Trypanosomiasis (HAT).

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Effect of experimental temperature on the permeation of model diffusants across porcine buccal mucosa.

The influence of experimental temperature on the permeability of model diffusants across porcine buccal mucosa was investigated in vitro. The permeability increased significantly as the experimental temperature was increased in increments of approximately 7°C. It was observed that the apparent permeability and temperature were related by an exponential relationship that conformed to the Arrhenius equation. Diffusants with higher lipophilicities--buspirone and bupivacaine--had lower activation energies for diffusion when compared to hydrophilic diffusants--antipyrine and caffeine. The activation energy for diffusion of the model diffusants decreased linearly with increasing distribution coefficients across porcine buccal mucosa. The results suggested that the buccal mucosa acts as a stronger barrier to the diffusion of hydrophilic diffusants than the lipophilic ones. The log-linear relationship between permeability and temperature indicates that temperature should be carefully controlled in diffusion experiments. These results also point to the possibility of developing heat-generating buccal delivery devices, especially for hydrophobic diffusants.

Porcine buccal mucosa as in vitro model: effect of biological and experimental variables.

Porcine buccal mucosa has been used as an in vitro model to assess the potential of delivering a molecule via the transbuccal route. However, permeation studies across porcine buccal mucosa show high variability due to various experimental and biological factors. The variability associated with the use of different mucosal regions, tissue storage conditions and tissue processing methods on drug permeation was investigated in this study. The permeability of model diffusants was significantly higher in the region behind the lip when compared to the cheek region because the latter has a thicker epithelium. Porcine buccal mucosa retained its integrity in Kreb's bicarbonate ringer solution at 4 degrees C for 24 h while many other storage conditions resulted in loss of epithelial integrity. Separation of the epithelium from underlying connective tissue either surgically or by heat treatment resulted in an epithelial thickness of approximately 150 microm. Separation of epithelium from the underlying connective tissue by heat treatment did not adversely affect its permeability and integrity characteristics. Investigation of these important biological and experimental variables provides guidance for conducting in vitro transbuccal permeation studies.

Porcine buccal mucosa as an in vitro model: relative contribution of epithelium and connective tissue as permeability barriers.

Porcine buccal mucosa has been extensively used as an in vitro model to study the permeability of various diffusants and to assess their potential to be delivered through the buccal route. The relative contribution of each component of the buccal mucosa on drug permeability was assessed in this study. The permeability of model diffusants decreased significantly with an increase in the mucosal thickness. A bilayer membrane model was developed to delineate the relative contribution to the barrier function offered by the epithelium and the connective tissue region. The decrease in permeability with mucosal thickness was attributed to the increase in the thickness of connective tissue. However, the epithelium acted as a primary barrier to permeation of all diffusants studied at a mucosal thickness up to 500 microm. In addition, the epithelium exhibited higher resistance to the permeation of hydrophilic diffusants than to lipophilic diffusants. With an increase in buccal mucosal membrane thickness, the lag time for the diffusants increased. Based on the analysis of permeation data, the buccal membrane, as a composite of epithelium and connective tissue, is considered as a heterogeneous permeation barrier. A mucosal tissue thickness of about 500 microm is recommended for in vitro transbuccal permeation studies since the epithelium remained the major permeability barrier for all diffusants at this thickness.