The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.
Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an affinity motif and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off-target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context-specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.
Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (K <10 nm), interference with Ras signaling and growth inhibition require 5-20 μm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.
Covalent labeling of amino acids in proteins by reactive small molecules, in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodology is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chemical biology research.
Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6d impairs KRas spatial organization and signaling in cells.H owever, despite potent binding in vitro (K D < 10 nm), interference with Ras signaling and growth inhibition require 5-20 mm compound concentrations.W edemonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6d by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6d with up to 7h ydrogen bonds,r esulting in picomolar affinity.T heir release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6d interaction may impair the growth of tumors driven by oncogenic KRas.
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