3 research outputs found
Characterization of the Artemisinin Binding Site for Translationally Controlled Tumor Protein (TCTP) by Bioorthogonal Click Chemistry
Despite
the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent
studies, the alkylation mechanism and accurate binding site of artemisinin–protein
interaction have remained elusive. Here, we report the chemical-probe-based
enrichment of the artemisinin-binding peptide and characterization
of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment
within the N-terminal region of TCTP was enriched and found to be
alkylated by an artemisinin-derived probe. MS2 fragments showed that
artemisinin could alkylate multiple amino acids from Phe12 to Tyr22
of TCTP, which was supported by labeling experiments upon site-directed
mutagenesis and computational modeling studies. Taken together, the
“capture-and-release” strategy affords consolidated
advantages previously unavailable in artemisinin–protein binding
site studies, and our results deepened the understanding of the mechanism
of protein alkylation via heme-activated artemisinin
Site-Selective Protein Immobilization by Covalent Modification of GST Fusion Proteins
The immobilization of functional
proteins onto solid supports using
affinity tags is an attractive approach in recent development of protein
microarray technologies. Among the commonly used fusion protein tags,
glutathione <i>S</i>-transferase (GST) proteins have been
indispensable tools for protein–protein interaction studies
and have extensive applications in recombinant protein purification
and reversible protein immobilization. Here, by utilizing pyrimidine-based
small-molecule probes with a sulfonyl fluoride reactive group, we
report a novel and general approach for site-selective immobilization
of Schistosoma japonicum GST (<i>sj</i>GST) fusion proteins through irreversible and specific
covalent modification of the tyrosine-111 residue of the <i>sj</i>GST tag. As demonstrated by <i>sj</i>GST-tagged eGFP and <i>sj</i>GST-tagged kinase activity assays, this immobilization
approach offers the advantages of high immobilization efficiency and
excellent retention of protein structure and activity
Discovery of a Series of 2,5-Diaminopyrimidine Covalent Irreversible Inhibitors of Bruton’s Tyrosine Kinase with in Vivo Antitumor Activity
Bruton’s tyrosine kinase (Btk)
is an attractive drug target for treating several B-cell lineage cancers.
Ibrutinib is a first-in-class covalent irreversible Btk inhibitor
and has demonstrated impressive effects in multiple clinical trials.
Herein, we present a series of novel 2,5-diaminopyrimidine covalent
irreversible inhibitors of Btk. Compared with ibrutinib, these inhibitors
exhibited a different selectivity profile for the analyzed kinases
as well as a dual-action mode of inhibition of both Btk activation
and catalytic activity, which counteracts a negative regulation loop
for Btk. Two compounds from this series, <b>31</b> and <b>38</b>, showed potent antiproliferative activities toward multiple
B-cell lymphoma cell lines, including germinal center B-cell-like
diffuse large B cell lymphoma (GCB-DLBCL) cells. In addition, compound <b>31</b> significantly prevented tumor growth in a mouse xenograft
model