Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins.

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

Protein-Templated Ugi Reactions versus In-Situ Ligation Screening: Two Roads to the Identification of SARS-CoV-2 Main Protease Inhibitors.

Protein-templated fragment ligation was established as a method for the rapid identification of high affinity ligands, and multicomponent reactions (MCR) such as the Ugi four-component reaction (Ugi 4CR) have been efficient in the synthesis of drug candidates. Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein-templated Ugi 4CR quantitatively using a fluorescence-based enzyme assay, HPLC-QTOF mass spectrometry (MS), and native protein MS with SARS-CoV-2 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three-component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non-templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in-situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS-CoV-2 protease inhibitors.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-ß-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-ß-lactamases (MBL) such as New Delhi metallo-ß-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential ß-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine.

SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8 µM at a molecular weight of 211 g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease.

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments.

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.

Nanoparticular Inhibitors of Flavivirus Proteases from Zika, West Nile and Dengue Virus Are Cell-Permeable Antivirals.

Viral proteases have been established as drug targets in several viral diseases including human immunodeficiency virus and hepatitis C virus infections due to the essential role of these enzymes in virus replication. In contrast, no antiviral therapy is available to date against flaviviral infections including those by Zika virus (ZIKV), West Nile virus (WNV), or dengue virus (DENV). Numerous potent inhibitors of flaviviral proteases have been reported; however, a huge gap remains between the in vitro and intracellular activities, possibly due to low cellular uptake of the charged compounds. Here, we present an alternative, nanoparticular approach to antivirals. Conjugation of peptidomimetic inhibitors and cell-penetrating peptides to dextran yielded chemically defined nanoparticles that were potent inhibitors of flaviviral proteases. Peptide-dextran conjugates inhibited viral replication and infection in cells at nontoxic, low micromolar or even nanomolar concentrations. Thus, nanoparticular antivirals might be alternative starting points for the development of broad-spectrum antiflaviviral drugs.

Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions.

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.

Catching a Moving Target: Comparative Modeling of Flaviviral NS2B-NS3 Reveals Small Molecule Zika Protease Inhibitors.

The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs.

The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation.

Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.

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.

Protein-Templated Fragment Ligations-From Molecular Recognition to Drug Discovery.

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 protein's 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.

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.

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.

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

BACKGROUND: Covalent 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. RESULTS: High-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. CONCLUSION: The 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.

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.

Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.