Christoph Arkona

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo.

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure‐based rational design. Seventy pyridazolon‐4‐ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS‐493 (4‐{(2Z)‐2‐[1,3‐bis(4‐nitrophenyl)‐5‐oxo‐1,5‐dihydro‐4H‐pyrazol‐4‐yliden]hydrazino}benzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71±15 nM in the enzyme assay and was 29‐ and 45‐fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)‐stimulated epithelial–mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non‐small‐cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes

Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes Are aliphatic alkynes truly bioorthogonal? In an attempt to prepare clickable ubiquitin derivatives bearing a C-terminal propargyl amide, two groups have now independently discovered propargylamides to be irreversible inhibitors of cysteine proteases. The unexpected findings demonstrate the unexpected reactivity of alkynes in protein-templated reactions and introduce a novel class of activity-based protein probes. . Angewandte Highlights

Synthesis and functional characterization of tridegin and its analogues: inhibitors and substrates of factor XIIIa.

Tridegin, a 66‐mer peptide isolated from the leech Haementeria ghilianii, is a potent inhibitor of the coagulation factor XIIIa. This paper describes the chemical synthesis of tridegin by two different strategies—solid‐phase assembly and native chemical ligation—both followed by oxidation in solution phase. Tridegin and truncated analogues were examined for their activity and revealed a particular importance of the C‐terminal region of the parent peptide. Based on these studies a minimal sequence required for factor XIIIa inhibition could be identified. Our data revealed that the glutamine residue at position 52 (Q52) of tridegin most likely binds to the active site of factor XIIIa and was therefore suggested to react with the enzyme. The function of the N‐terminal region is also discussed, as the isolated C‐terminal segment of tridegin lost its inhibitory activity rapidly in the presence of factor XIIIa, whereas this was not the case for the full‐length inhibitor.

Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme–inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.

Protein‐Templated Fragment Ligations—From Molecular Recognition to Drug Discovery

Abstract Protein‐templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein‐templated fragment ligations are chemical reactions between small molecules (“fragments”) utilizing a proteins surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small‐molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein‐templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed.

Proteintemplat‐gesteuerte Fragmentligationen – von der molekularen Erkennung zur Wirkstofffindung

Proteintemplat‐gesteuerte Fragmentligationen sind ein neuartiges Konzept zur Unterstützung der Wirkstofffindung und können dazu beitragen, die Wirksamkeit von Proteinliganden zu verbessern. Es handelt sich dabei um chemische Reaktionen zwischen niedermolekularen Verbindungen (“Fragmenten”), die die Oberfläche eines Proteins als Reaktionsgefäß verwenden, um die Bildung eines Proteinliganden mit erhöhter Bindungsaffinität zu katalysieren. Die Methode nutzt die molekulare Erkennung kleiner reaktiver Fragmente durch die Proteine sowohl zur Assemblierung der Liganden als auch zur Identifizierung bioaktiver Fragmentkombinationen. Chemische Synthese und Bioassay werden dabei in einem Schritt vereint. Dieser Aufsatz diskutiert die biophysikalischen Grundlagen der reversiblen und irreversiblen Fragmentligationen und gibt einen Überblick über die Methoden, mit denen die durch das Proteintemplat gebildeten Ligationsprodukte detektiert werden können. Der chemische Reaktionsraum und aktuelle Anwendungen wie auch die Bedeutung dieses Konzeptes für die Wirkstofffindung werden erörtert.

High yield expression of catalytically active USP18 (UBP43) using a Trigger Factor fusion system.

BackgroundCovalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens.ResultsHigh-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone.ConclusionThe results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.

Phenylthiomethyl Ketone-Based Fragments Show Selective and Irreversible Inhibition of Enteroviral 3C Proteases

Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.