5 research outputs found
Production of crystallizable human chymase from a Bacillus subtilis system
AbstractA Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3–0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF-treated chymase were optimized. The first is C2 with a=47.94 Å, b=85.23 Å, c=174.18 Å, β=96.74°, and diffracts to at least 2.1 Å, while the second is P212121, with cell dimensions a=43.93 Å, b=58.16 Å, and c=86.09 Å, and a diffraction limit of approximately 1.9 Å. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit
Battling Btk Mutants With Noncovalent Inhibitors That Overcome Cys481 and Thr474 Mutations
The
Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib has
shown impressive clinical efficacy in a range of B-cell malignancies.
However, acquired resistance has emerged, and second generation therapies
are now being sought. Ibrutinib is a covalent, irreversible inhibitor
that modifies Cys481 in the ATP binding site of Btk and renders the
enzyme inactive, thereby blocking B-cell receptor signal transduction.
Not surprisingly, Cys481 is the most commonly mutated Btk residue
in cases of acquired resistance to ibrutinib. Mutations at other sites,
including Thr474, a gatekeeper residue, have also been detected. Herein,
we describe noncovalent Btk inhibitors that differ from covalent inhibitors
like ibrutinib in that they do not interact with Cys481, they potently
inhibit the ibrutinib-resistant Btk C481S mutant <i>in vitro</i> and in cells, and they are exquisitely selective for Btk. Noncovalent
inhibitors such as GNE-431 also show excellent potency against the
C481R, T474I, and T474M mutants. X-ray crystallographic analysis of
Btk provides insight into the unique mode of binding of these inhibitors
that explains their high selectivity for Btk and their retained activity
against mutant forms of Btk. This class of noncovalent Btk inhibitors
may provide a treatment option to patients, especially those who have
acquired resistance to ibrutinib by mutation of Cys481 or Thr474