Ruthenium(II)-Catalyzed Regio- and Stereoselective C–H Allylation of Indoles with Allyl Alcohols

A ruthenium-catalyzed C–H allylation of indoles with allyl alcohols via β-hydroxide elimination is reported. Without external oxidants and expensive additives, this reaction features mild reaction conditions, compatibility with various functional groups, and good to excellent regioselectivity and stereoselectivity

A Versatile Method to Determine the Cellular Bioavailability of Small-Molecule Inhibitors

The determination of the cellular bioavailability of small-molecule inhibitors is a critical step for interpreting cell-based data and guiding inhibitor optimization. Herein, a HPLC-MS based protocol was developed to determine inhibitor cellular bioavailability. This generalizable protocol allows determination of the accurate intracellular concentrations and characterization of various properties of inhibitors including the extra- and intracellular stability, the dose- and time-dependence of the intracellular concentrations, the cell permeability, and the nonspecific binding with the cell culture plates, the extracellular matrices, and the cell membrane. The inhibitors of the protein–protein interactions, bromodomains, and the β-catenin/B-cell lymphoma 9 (BCL9) interaction were used to examine the protocol, and the cellular bioavailability of the inhibitors in cancer cells was determined. High nonspecific binding and low cellular uptake were observed for two bromodomain inhibitors. The two β-catenin/BCL9 inhibitors had low nonspecific binding but different cellular uptake. These inhibitors exhibited different stability kinetics in cells

High-Throughput Selectivity Assays for Small-Molecule Inhibitors of β‑Catenin/T-Cell Factor Protein–Protein Interactions

Two homogeneous high-throughput assays, AlphaScreen and fluorescence polarization, were established to quantify inhibitor selectivity between different protein–protein complexes. As a first case study, they have been successfully applied to the key protein–protein interactions in the downstream sites of the canonical Wnt signaling pathway. The aberrant formation of the β-catenin/T-cell factor (Tcf) complex is the major driving force for many cancers and fibroses. Crystallographic and biochemical studies reveal that the binding modes of Tcf, E-cadherin, and adenomatous polyposis coli (APC) to β-catenin are identical and mutually exclusive. In the present study, two highly sensitive and robust assays were established to quantitatively evaluate inhibitor selectivity between β-catenin/Tcf, β-catenin/E-cadherin, and β-catenin/APC interactions. A pilot screen demonstrated the feasibility of the assays and yielded four hits for the disruption of β-catenin/Tcf interactions. A potent and dual-selective β-catenin/Tcf inhibitor was identified

Discovery of Selective Small-Molecule Inhibitors for the β‑Catenin/T-Cell Factor Protein–Protein Interaction through the Optimization of the Acyl Hydrazone Moiety

Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the β-catenin/Tcf protein–protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor optimization resulted in chemically stable derivatives that disrupt the β-catenin/Tcf PPI. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure–activity relationship studies. This series of inhibitors with a new scaffold exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. One derivative of this series suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells. This compound represents a solid starting point for the development of potent and selective β-catenin/Tcf inhibitors

Structure-Based Optimization of Small-Molecule Inhibitors for the β‑Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

Structure-based optimization was conducted to improve the potency, selectivity, and cell-based activities of β-catenin/B-cell lymphoma 9 (BCL9) inhibitors based on the 4′-fluoro-<i>N</i>-phenyl-[1,1′-biphenyl]-3-carboxamide scaffold, which was designed to mimic the side chains of the hydrophobic α-helical hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7. Compound <b>29</b> was found to disrupt the β-catenin/BCL9 protein–protein interaction (PPI) with a <i>K</i>_i of 0.47 μM and >1900-fold selectivity for β-catenin/BCL9 over β-catenin/E-cadherin PPIs. The proposed binding mode of new inhibitors was consistent with the results of site-directed mutagenesis and structure–activity relationship studies. Cell-based studies indicated that <b>29</b> disrupted the β-catenin/BCL9 interaction without affecting the β-catenin/E-cadherin interaction, selectively suppressed transactivation of Wnt/β-catenin signaling, downregulated expression of Wnt target genes, and inhibited viability of Wnt/β-catenin-dependent cancer cells in dose-dependent manners. A comparison of the biochemical and cell-based assay results offered the directions for future inhibitor optimization

Structure-Based Optimization of Small-Molecule Inhibitors for the β‑Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction

Structure-based optimization was conducted to improve the potency, selectivity, and cell-based activities of β-catenin/B-cell lymphoma 9 (BCL9) inhibitors based on the 4′-fluoro-<i>N</i>-phenyl-[1,1′-biphenyl]-3-carboxamide scaffold, which was designed to mimic the side chains of the hydrophobic α-helical hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7. Compound <b>29</b> was found to disrupt the β-catenin/BCL9 protein–protein interaction (PPI) with a <i>K</i>_i of 0.47 μM and >1900-fold selectivity for β-catenin/BCL9 over β-catenin/E-cadherin PPIs. The proposed binding mode of new inhibitors was consistent with the results of site-directed mutagenesis and structure–activity relationship studies. Cell-based studies indicated that <b>29</b> disrupted the β-catenin/BCL9 interaction without affecting the β-catenin/E-cadherin interaction, selectively suppressed transactivation of Wnt/β-catenin signaling, downregulated expression of Wnt target genes, and inhibited viability of Wnt/β-catenin-dependent cancer cells in dose-dependent manners. A comparison of the biochemical and cell-based assay results offered the directions for future inhibitor optimization

Targeting the Tcf4 G¹³ANDE¹⁷ Binding Site To Selectively Disrupt β‑Catenin/T-Cell Factor Protein–Protein Interactions

Selective disruption of protein–protein interactions by small molecules is important for probing the structure and dynamic aspects of cellular network. It can also provide new therapeutic targets. β-Catenin of the canonical Wnt signaling pathway uses the same positively charged groove to bind with T-cell factor (Tcf), cadherin, and adenomatous polysis coli (APC). The extravagant formation of β-catenin/Tcf interactions drives the initiation and progression of many cancers and fibroses, while β-catenin/cadherin and β-catenin/APC interactions are essential for cell–cell adhesion and β-catenin degradation. In this study, a selective binding site that can differentiate β-catenin/Tcf, β-catenin/cadherin, and β-catenin/APC interactions was identified by alanine scanning and biochemical assays. A new peptidomimetic strategy that incorporates SiteMap and multiple-copy simultaneous search was used to design selective small-molecule inhibitors for β-catenin/Tcf interactions. A potent inhibitor was discovered to bind with β-catenin and completely disrupt β-catenin/Tcf interactions. It also exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC interactions in both biochemical and cell-based assays. This study provides a proof of concept for designing selective inhibitors for β-catenin/Tcf interactions

Rational Design of Selective Small-Molecule Inhibitors for β‑Catenin/B-Cell Lymphoma 9 Protein–Protein Interactions

Selective inhibition of α-helix-mediated protein–protein interactions (PPIs) with small organic molecules provides great potential for the discovery of chemical probes and therapeutic agents. Protein Data Bank data mining using the HippDB database indicated that (1) the side chains of hydrophobic projecting hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7 of an α-helix had few orientations when interacting with the second protein and (2) the hot spot pockets of PPI complexes had different sizes, shapes, and chemical groups when interacting with the same hydrophobic projecting hot spots of α-helix. On the basis of these observations, a small organic molecule, 4′-fluoro-<i>N</i>-phenyl-[1,1′-biphenyl]-3-carboxamide, was designed as a generic scaffold that itself directly mimics the binding mode of the side chains of hydrophobic projecting hot spots at positions <i>i</i>, <i>i</i> + 3, and <i>i</i> + 7 of an α-helix. Convenient decoration of this generic scaffold led to the selective disruption of α-helix-mediated PPIs. A series of small-molecule inhibitors selective for β-catenin/B-cell lymphoma 9 (BCL9) over β-catenin/cadherin PPIs was designed and synthesized. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure–activity relationship studies. This new class of inhibitors can selectively disrupt β-catenin/BCL9 over β-catenin/cadherin PPIs, suppress the transactivation of canonical Wnt signaling, downregulate the expression of Wnt target genes, and inhibit the growth of Wnt/β-catenin-dependent cancer cells

The Mobility of a Conserved Tyrosine Residue Controls Isoform-Dependent Enzyme–Inhibitor Interactions in Nitric Oxide Synthases

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS–inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity