The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif

High-Confidence Protein–Ligand Complex Modeling by NMR-Guided Docking Enables Early Hit Optimization

Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein–ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein–ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein–ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders

High-Confidence Protein–Ligand Complex Modeling by NMR-Guided Docking Enables Early Hit Optimization

Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein–ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein–ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein–ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders

High-Confidence Protein–Ligand Complex Modeling by NMR-Guided Docking Enables Early Hit Optimization

Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein–ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein–ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein–ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders

Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit

Abstract Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11–13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED

<div><p>Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.</p></div

Synthesis, Binding Mode, and Antihyperglycemic Activity of Potent and Selective (5-Imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine Inhibitors of Glycogen Synthase Kinase 3

In an effort to identify new antidiabetic agents, we have discovered a novel family of (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)­[2-(2-pyridylamino)­ethyl]­amine analogues which are inhibitors of human glycogen synthase kinase 3 (GSK3). We developed efficient synthetic routes to explore a wide variety of substitution patterns and convergently access a diverse array of analogues. Compound <b>1</b> (CHIR-911, CT-99021, or CHIR-73911) emerged from an exploration of heterocycles at the C-5 position, phenyl groups at C-4, and a variety of differently substituted linker and aminopyridine moieties attached at the C-2 position. These compounds exhibited GSK3 IC₅₀s in the low nanomolar range and excellent selectivity. They activate glycogen synthase in insulin receptor-expressing CHO-IR cells and primary rat hepatocytes. Evaluation of lead compounds <b>1</b> and <b>2</b> (CHIR-611 or CT-98014) in rodent models of type 2 diabetes revealed that single oral doses lowered hyperglycemia within 60 min, enhanced insulin-stimulated glucose transport, and improved glucose disposal without increasing insulin levels