High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)–protein tyrosine kinase interactions

Abstract

The Casitas-B-lineage Lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3-ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that bind over a dozen proteins, including protein tyrosine kinases (PTKs) in a phosphorylation dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and therefore implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However recent studies such as the identification of (a) gain of function c-Cbl mutations in subsets of myeloid cancer and (b) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggests otherwise. Here we report the development of a competitive high throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high throughput screen (HTS) readiness of the assay was demonstrated by screening the Prestwick chemical library ® .

Molecular Pathways: Cbl Proteins in Tumorigenesis and Antitumor Immunity—Opportunities for Cancer Treatment

“…Based on this, Kumar et al . are developing strategies to identify small molecules or peptides that bind the Cbl TKB and block interaction with the RTK (53, 54). The efficacy of such an approach remains to be tested.…”

Peptide Truncation Leads to a Twist and an Unusual Increase in Affinity for Casitas B-Lineage Lymphoma Tyrosine Kinase Binding Domain

“…We recently reported the development and miniaturization of a Cbl(TKB) FP assay for high throughput screening to identify Cbl(TKB) inhibitors. 29 Here, the FP assay was adapted to quantify binding of 12-mer peptide or pentapeptide to Cbl(TKB) and inhibition of binding by various peptides. Peptides 1 and 2 were labeled with a fluorophore at the N-terminus to generate two probes ( 3, Flu-pYTPEP and 4, Flu-TLNSDGpYTPEPA) for the FP assay.…”

“…We, and others, have shown that the phophotyrosine is a key binding component as the non-phosphorylated Tyr-containing peptide does not bind Cbl(TKB). 6, 7, 29 Analysis of the 33G values relative to 1 reveals that each residue modestly contributes to Cbl(TKB) binding (Figure 3A, cyan bars). Interestingly, there is a significant loss of activity in 19 , suggesting that the proline residue at the P+4 position makes substantial contribution to binding.…”

Rôle de l’ubiquitine ligase c-Cbl dans l’ostéogenèse normale et cancéreuse

“…Ces données suggèrent que l'on peut cibler l'interaction entre c-Cbl et les RTK pour favoriser la différenciation ostéoblastique des CSM, ce qui ouvre une nouvelle voie thérapeutique potentielle pour favoriser l'ostéogenèse et la répara-tion osseuse ( Figure 3A). L'utilisation de molécules pharmacologiques capables d'inhiber de façon spécifique l'interaction entre c-Cbl et les RTK, molécules actuellement en développement [26,27], pourrait ainsi permettre de promouvoir la différenciation ostéogénique des cellules souches mésenchymateuses in vivo. E3.…”