Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity.

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/β-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling.

Cyanopyrrolidine Inhibitors of Ubiquitin Specific Protease 7 Mediate Desulfhydration of the Active-Site Cysteine.

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine.

Lead Optimization Yields High Affinity Frizzled 7-Targeting Peptides That Modulate Toxin B Pathogenicity in Epithelial Cells.

Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors.

A structural mechanism for directing corepressor-selective inverse agonism of PPARγ.

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity. X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism.

Defining a conformational ensemble that directs activation of PPARγ.

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Here, we present a nuclear receptor co-regulator-binding surface structural ensemble in solution, viewed through the lens of fluorine-19 (F) nuclear magnetic resonance (NMR) and molecular simulations, and the response of this ensemble to ligands, co-regulator peptides and heterodimerization.

Direct Comparison of 4 Very Early Rule-Out Strategies for Acute Myocardial Infarction Using High-Sensitivity Cardiac Troponin I.

Background: Four strategies for very early rule-out of acute myocardial infarction using high-sensitivity cardiac troponin I (hs-cTnI) have been identified. It remains unclear which strategy is most attractive for clinical application.

Methods: We prospectively enrolled unselected patients presenting to the emergency department with symptoms suggestive of acute myocardial infarction.

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor.

GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPARγ). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPARγ ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPARγ LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists.

Probing the Complex Binding Modes of the PPARγ Partial Agonist 2-Chloro-N-(3-chloro-4-((5-chlorobenzo[d]thiazol-2-yl)thio)phenyl)-4-(trifluoromethyl)benzenesulfonamide (T2384) to Orthosteric and Allosteric Sites with NMR Spectroscopy.

In a previous study, a cocrystal structure of PPARγ bound to 2-chloro-N-(3-chloro-4-((5-chlorobenzo[d]thiazol-2-yl)thio)phenyl)-4-(trifluoromethyl)benzenesulfonamide (1, T2384) revealed two orthosteric pocket binding modes attributed to a concentration-dependent biochemical activity profile. However, 1 also bound an alternate/allosteric site that could alternatively account for the profile. Here, we show ligand aggregation afflicts the activity profile of 1 in biochemical assays.

Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation.

Protein arginine phosphorylation is a recently discovered modification that affects multiple cellular pathways in Gram-positive bacteria. In particular, the phosphorylation of arginine residues by McsB is critical for regulating the cellular stress response. Given that the highly efficient protein arginine phosphatase YwlE prevents arginine phosphorylation under non-stress conditions, we hypothesized that this enzyme negatively regulates arginine phosphorylation and acts as a sensor of cell stress.

Identification of a Binding Site for Unsaturated Fatty Acids in the Orphan Nuclear Receptor Nurr1.

Nurr1/NR4A2 is an orphan nuclear receptor, and currently there are no known natural ligands that bind Nurr1. A recent metabolomics study identified unsaturated fatty acids, including arachidonic acid and docosahexaenoic acid (DHA), that interact with the ligand-binding domain (LBD) of a related orphan receptor, Nur77/NR4A1. However, the binding location and whether these ligands bind other NR4A receptors were not defined.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization.

Protein Arginine Methylation and Citrullination in Epigenetic Regulation.

The post-translational modification of arginine residues represents a key mechanism for the epigenetic control of gene expression. Aberrant levels of histone arginine modifications have been linked to the development of several diseases including cancer. In recent years, great progress has been made in understanding the physiological role of individual arginine modifications and their effects on chromatin function.

Synthesis and Use of a Phosphonate Amidine to Generate an Anti-Phosphoarginine-Specific Antibody.

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody.

Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design.

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation.

Protein arginine methyltransferase 5 catalyzes substrate dimethylation in a distributive fashion.

Protein arginine methyltransferase 5 (PRMT5) is a histone-modifying enzyme whose activity is aberrantly upregulated in various cancers and thereby contributes to a progrowth phenotype. Indeed, knockdown of PRMT5 leads to growth arrest and apoptosis, suggesting that inhibitors targeting this enzyme may have therapeutic utility in oncology. To aid the development of inhibitors targeting PRMT5, we initiated mechanistic studies geared to understand how PRMT5 selectively catalyzes the symmetric dimethylation of its substrates.

Chasing Phosphoarginine Proteins: Development of a Selective Enrichment Method Using a Phosphatase Trap.

Arginine phosphorylation is an emerging post-translational protein modification implicated in the bacterial stress response. Although early reports suggested that arginine phosphorylation also occurs in higher eukaryotes, its overall prevalence was never studied using modern mass spectrometry methods, owing to technical difficulties arising from the acid lability of phosphoarginine. As shown recently, the McsB and YwlE proteins from Bacillus subtilis function as a highly specific protein arginine kinase and phosphatase couple, shaping the phosphoarginine proteome.

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine.

Targeting the arginine phosphatase YwlE with a catalytic redox-based inhibitor.

Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) family, was shown to efficiently target phosphoarginine.

Structural basis for recognizing phosphoarginine and evolving residue-specific protein phosphatases in gram-positive bacteria.

Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a protein modification in bacteria prompted us to analyze the molecular basis of targeting phospho-arginine. In this work, we characterize an annotated tyrosine phosphatase, YwlE, that counteracts the protein arginine kinase McsB.

Chemical and biological methods to detect post-translational modifications of arginine.

Post-translational modifications (PTMs) of protein embedded arginines are increasingly being recognized as playing an important role in both prokaryotic and eukaryotic biology, and it is now clear that these PTMs modulate a number of cellular processes including DNA binding, gene transcription, protein-protein interactions, immune system activation, and proteolysis. There are currently four known enzymatic PTMs of arginine (i.e.

McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR.

All living organisms face a variety of environmental stresses that cause the misfolding and aggregation of proteins. To eliminate damaged proteins, cells developed highly efficient stress response and protein quality control systems. We performed a biochemical and structural analysis of the bacterial CtsR/McsB stress response.