Rachel R. Kroe

NUTS and BOLTS: applications of fluorescence-detected sedimentation.

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors, absorbance and interference, impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes, allowing the characterization of very dilute solutions of macromolecules, applications we call normal use tracer sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high-affinity interactions. Second, the AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call biological on-line tracer sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieus such as cell lysates and serum. Examples that embody features of both NUTS and BOLTS applications are presented along with our observations on these applications.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.

Structure-based design and subsequent optimization of 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) inhibitors of p38 MAP kinase

A computer-aided drug design strategy leads to the identification of a new class of p38 inhibitors based on the 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) scaffold. The tolyl triazole amides provided a potent platform amenable to optimization. Further exploration leads to compounds with greater than 100-fold improvement in binding affinity to p38. Derivatives prepared to alter the physicochemical properties produced inhibitors with IC(50)s in human whole blood as low as 83 nM.

Discovery and characterization of the N-phenyl-N′-naphthylurea class of p38 kinase inhibitors

An effort aimed at exploring structural diversity in the N-pyrazole-N-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.