A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression.

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets.

A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography.

Use of fluorescence polarization to monitor MHC-peptide interactions in solution.

We describe here fluorescence polarization-based methods to investigate class I MHC-peptide interactions in solution. Fluorescein-labelled peptides were used to determine MHC/peptide complex association and dissociation constants as well as the equilibrium binding constant (KD). Furthermore, we developed a competition assay for the determination of IC50 values of nonlabelled compounds.

Beta-amino acid scan of a class I major histocompatibility complex-restricted alloreactive T-cell epitope.

An HLA-B27-restricted self-octapeptide known to react with an alloreactive T-cell receptor has been modified by systematic substitution of a beta-amino acid for the natural alpha-amino acid residue, over the whole length of the parent epitope. All modified peptides were shown to bind to recombinant HLA-B*2705 and induce stable major histocompatibility complex-peptide complexes, but with some variation depending on the position of the beta-amino acid on the peptide sequence. Alteration of the natural peptide sequence at the two N-terminal positions (positions 1 and 2) decreases binding affinity and thermodynamic stability of the refolded complex, but all other positions (from position 3 to the C-terminal residue) were insensitive to the beta-amino acid substitution.

Mutation of Cys-67 alters the thermodynamic stability of the human leukocyte antigen HLA0-B*2705.

The B pocket of the class I major histocompatibility complex-encoded protein HLA-B*2705 has recently been suggested to be responsible for the misfolding of this HLA haplotype and thus to induce susceptibility to autoimmune inflammatory diseases. Four mutants of the B*2705 heavy chain were refolded in the presence of three control peptides. The monitoring of the thermal unfolding of the B*2705-peptide complexes by circular dichroism spectroscopy showed that all heterotrimeric mutants were markedly less stable than the corresponding complexes with the wild-type heavy chain.

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations.

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove.

Structure-based design of nonnatural ligands for the HLA-B27 protein.

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy.