Probing the Protein Kinases' Cysteinome by Covalent Fragments.

Protein kinases are important drug targets, yet specific inhibitors have been developed for only a fraction of the more than 500 human kinases. A major challenge in designing inhibitors for highly related kinases is selectivity. Unlike their non-covalent counterparts, covalent inhibitors offer the advantage of selectively targeting structurally similar kinases by modifying specific protein side chains, particularly non-conserved cysteines.

The C-terminal PHDVC5HCH tandem domain of NSD2 is a combinatorial reader of unmodified H3K4 and tri-methylated H3K27 that regulates transcription of cell adhesion genes in multiple myeloma.

Histone methyltransferase NSD2 (MMSET) overexpression in multiple myeloma (MM) patients plays an important role in the development of this disease subtype. Through the expansion of transcriptional activating H3K36me2 and the suppression of repressive H3K27me3 marks, NSD2 activates an aberrant set of genes that contribute to myeloma growth, adhesive and invasive activities. NSD2 transcriptional activity also depends on its non-catalytic domains, which facilitate its recruitment to chromatin through histone binding.

Critical assessment of LC3/GABARAP ligands used for degrader development and ligandability of LC3/GABARAP binding pockets.

Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome.

Thieno[3,2-b]pyridine: Attractive scaffold for highly selective inhibitors of underexplored protein kinases with variable binding mode.

Protein kinases are key regulators of numerous biological processes and aberrant kinase activity can cause various diseases, particularly cancer. Herein, we report the identification of new series of highly selective kinase inhibitors based on the thieno[3,2-b]pyridine scaffold. The weak interaction of the thieno[3,2-b]pyridine core with the kinase hinge region allows for profoundly different binding modes all of which maintain high kinome-wide selectivity, as illustrated by the isomers MU1464 and MU1668.

Arachidonic Acid Directly Activates the Human DP2 Receptor.

Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis, and other atopic diseases, with an alarming prevalence in relevant parts of the Western world. A bulk of evidence points out the important role of the DP2 receptor in these inflammation processes. A screening of different polyunsaturated fatty acids at a fluorescence resonance energy transfer-based DP2 receptor conformation sensor expressed in human embryonic kidney (HEK) cells revealed an agonistic effect of the prostaglandin (PG)-D precursor arachidonic acid on DP2 receptor activity of about 80% of the effect induced by PGD In a combination of experiments at the conformation sensor and using a bioluminescence resonance energy transfer-based G protein activation sensor expressed together with DP2 receptor wild type in HEK cells, we found that arachidonic acid acts as a direct activator of the DP2 receptor, but not the DP1 receptor, in a concentration range considered physiologically relevant.

RUNX1 interacts with lncRNA SMANTIS to regulate monocytic cell functions.

Monocytes, the circulating macrophage precursors, contribute to diseases like atherosclerosis and asthma. Long non-coding RNAs (lncRNAs) have been shown to modulate the phenotype and inflammatory capacity of monocytes. We previously discovered the lncRNA SMANTIS, which contributes to cellular phenotype expression by controlling BRG1 in mesenchymal cells.

Inhibition of bromodomain and extra-terminal proteins targets constitutively active NFκB and STAT signaling in lymphoma and influences the expression of the antiapoptotic proteins BCL2A1 and c-MYC.

The antiapoptotic protein BCL2A1 is highly, but very heterogeneously expressed in Diffuse Large B-cell Lymphoma (DLBCL). Particularly in the context of resistance to current therapies, BCL2A1 appears to play an important role in protecting cancer cells from the induction of cell death. Reducing BCL2A1 levels may have therapeutic potential, however, no specific inhibitor is currently available.

Substantial Differences in Proanthocyanidin Contents among Ginkgo biloba Leaf Extracts in Herbal Medicinal Products Obtained from the German Market.

Pharmacologic activity of proanthocyanidins in leaf extract has recently been reported. The objective of the present study was to screen proanthocyanidin contents in herbal medicinal products containing extracts. A recently published HPLC method for quantification of proanthocyanidins in leaf extract EGb 761 was adopted to also be suitable for finished herbal medicinal products.

A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7.

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs).

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

Objective: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC).

Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway.

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors.

Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells.

The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown.

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, and to humans.

Synthesis and Biological Evaluation of 2-Substituted Quinazolin-4(3)-Ones with Antiproliferative Activities.

Sixteen new 2-substituted quinazolines were synthesized using a straightforward methodology starting from 2-methoxybezoic acid or 3-methoxy-2-naphthoic acid. The anti-proliferative activity of the target compounds was evaluated against nine cancer cell lines. Additionally, all the compounds were screened for their potency and selectivity against a panel of 109 kinases and four bromodomains, using Differential Scanning Fluorimetry (DSF).

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

Background: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury.

Methods: Native endothelial cells were isolated from young (20±3.

Quality Control of Chemogenomic Library Using LC-MS.

In chemical biology, using compounds with incorrect identity or insufficient purity can lead to misleading biological activity data. Chemical quality control for confirmation of purity and compound identity is thus central to chemogenomics. We have established a medium-throughput LC-MS-based semi-automated quality control (QC) workflow with a minimal requirement for materials suitable for chemogenomics and other small molecule libraries.

Deciphering the mitophagy receptor network identifies a crucial role for OPTN (optineurin) in acute myeloid leukemia.

The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXC motif, a target for redox modifications.

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge.

Design and Synthesis of Pyrazole-Based Macrocyclic Kinase Inhibitors Targeting BMPR2.

Bone morphogenetic protein (BMP) signaling is mediated by transmembrane protein kinases that form heterotetramers consisting of type-I and type-II receptors. Upon BMP binding, the constitutively active type-II receptors activate specific type-I receptors by transphosphorylation, resulting in the phosphorylation of SMAD effector proteins. Drug discovery in the receptor tyrosine kinase-like (TKL) family has largely focused on type-I receptors, with few inhibitors that have been published targeting type-II receptors.

Led-Seq: ligation-enhanced double-end sequence-based structure analysis of RNA.

Structural analysis of RNA is an important and versatile tool to investigate the function of this type of molecules in the cell as well as in vitro. Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals.

Design, synthesis and characterisation of a novel type II B-RAF paradox breaker inhibitor.

The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (B-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization.

Toward effective Atg8-based ATTECs: Approaches and perspectives.

Induction of Atg8-family protein (LC3/GABARAP proteins in human) interactions with target proteins of interest by proximity-inducing small molecules offers the possibility for novel targeted protein degradation approaches. However, despite intensive screening campaigns during the last 5 years, no potent ligands for LC3/GABARAPs have been developed, rendering this approach largely unexplored and unsuitable for therapeutic exploitation. In this Viewpoint, we analyze the reported attempts identifying LC3/GABARAP inhibitors and provide our own point of view why no potent inhibitors have been found.