14 research outputs found
Humanization and Characterization of an Anti-Human TNF-α Murine Monoclonal Antibody
A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG1 was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG1 possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 µg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use
Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1)
Mutations in leucine-rich repeat
kinase 2 (LRRK2), such as G2019S,
are associated with an increased risk of developing Parkinson’s
disease. Surrogates for the LRRK2 kinase domain based on checkpoint
kinase 1 (CHK1) mutants were designed, expressed in insect cells infected
with baculovirus, purified, and crystallized. X-ray structures of
the surrogates complexed with known LRRK2 inhibitors rationalized
compound potency and selectivity. The CHK1 10-point mutant was preferred,
following assessment of surrogate binding affinity with LRRK2 inhibitors.
Fragment hit-derived arylpyrrolo[2,3-<i>b</i>]pyridine
LRRK2 inhibitors underwent structure-guided optimization using this
crystallographic surrogate. LRRK2-pSer935 HEK293 IC<sub>50</sub> data
for <b>22</b> were consistent with binding to Ala2016 in LRRK2
(equivalent to Ala147 in CHK1 10-point mutant structure). Compound <b>22</b> was shown to be potent, moderately selective, orally available,
and brain-penetrant in wild-type mice, and confirmation of target
engagement was demonstrated, with LRRK2-pSer935 IC<sub>50</sub> values
for <b>22</b> in mouse brain and kidney being 1.3 and 5 nM,
respectively
Crystal structure of the M-fragment of a-catenin: implications for modulation of cell adhesion
The cytoskeletal protein α-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of α-catenin (residues 377–633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of α-catenin and the tail region of vinculin. These results suggest that α-catenin is composed of repeating antiparallel helical domains. The region of α-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of α-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell–cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule