MoA Studies of the TEAD P-Site Binding Ligand MSC-4106 and Its Optimization to TEAD1-Selective Amide M3686.

Taking the structural information into account, we were able to tune the TEAD selectivity for a specific chemotype. However, different TEAD selectivity profiles did not affect the compound potency or efficacy in the NCI-H226 viability assay. Amides based on or analogues showed improved viability efficacy compared with the corresponding acids.

The HSP90/R2TP Quaternary Chaperone Scaffolds Assembly of the TSC Complex.

The R2TP chaperone is composed of the RUVBL1/RUVBL2 AAA+ ATPases and two adapter proteins, RPAP3 and PIH1D1. Together with HSP90, it functions in the assembly of macromolecular complexes that are often involved in cell proliferation. Here, proteomic experiments using the isolated PIH domain reveals additional R2TP partners, including the Tuberous Sclerosis Complex (TSC) and many transcriptional complexes.

Identification of novel candidate predisposing genes in familial nonmedullary thyroid carcinoma implicating DNA damage repair pathways.

The genetic basis of nonsyndromic familial nonmedullary thyroid carcinoma (FNMTC) is still poorly understood, as the susceptibility genes identified so far only account for a small percentage of the genetic burden. Recently, germline mutations in DNA repair-related genes have been reported in cases with thyroid cancer. In order to clarify the genetic basis of FNMTC, 94 genes involved in hereditary cancer predisposition, including DNA repair genes, were analyzed in 48 probands from FNMTC families, through targeted next-generation sequencing (NGS).

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale.

CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations.

Deciphering cellular and molecular determinants of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries.

Optimization of TEAD P-Site Binding Fragment Hit into In Vivo Active Lead .

The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable.

Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitors.

The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation.

Development of Dl1.72, a Novel Anti-DLL1 Antibody with Anti-Tumor Efficacy against Estrogen Receptor-Positive Breast Cancer.

The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER) subtypes.

Development of antibodies against the notch ligand Delta-Like-1 by phage display with activity against breast cancer cells.

Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER breast cancer cells is reported.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics.

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis.

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58.

Screening Pyridine Derivatives against Human Hydrogen Sulfide-synthesizing Enzymes by Orthogonal Methods.

Biosynthesis of hydrogen sulfide (HS), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased HS production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human HS-synthesizing enzymes may therefore prove beneficial for pharmacological applications.

X-ray structure of full-length human RuvB-Like 2 - mechanistic insights into coupling between ATP binding and mechanical action.

RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology.

Insights into the Structures of Superoxide Reductases from the Symbionts Ignicoccus hospitalis and Nanoarchaeum equitans.

Superoxide reductases (SORs) are enzymes that detoxify the superoxide anion through its reduction to hydrogen peroxide and exist in both prokaryotes and eukaryotes. The substrate is transformed at an iron catalytic center, pentacoordinated in the ferrous state by four histidines and one cysteine. SORs have a highly conserved motif, (E)(K)HxP-, in which the glutamate is associated with a redox-driven structural change, completing the octahedral coordination of the iron in the ferric state, whereas the lysine may be responsible for stabilization and donation of a proton to catalytic intermediates.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers.

Production and characterization of a novel Delta-like 1 functional unit as a tool for Notch pathway activation and generation of a specific antibody.

Notch signalling is an evolutionary conserved cell-to-cell communication pathway crucial for development and tissue homeostasis. Abnormal Notch signalling by mutations or deregulated expression of its receptors and/or ligands can lead to cancer making it a potential therapeutic target. Delta-like1 (DLL1) is a ligand of the Notch pathway implicated in different types of cancer, including breast cancer.

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine -Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine -synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (HS). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic mutations is not yet fully understood.

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR.

Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.

Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species.