Redox-Based Inactivation of Cysteine Cathepsins by Compounds Containing the 4-Aminophenol Moiety

BACKGROUND: Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming. METHODS AND FINDINGS: In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a k(inact)/K(I) of 36.7 ± 13.6 M(-1) s(-1) using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with k(inact)/K(I) values of 406.4 ± 10.8 and 36.5 ± 1.3 M(-1) s(-1). No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS) by 4-aminophenols in various redox environments was determined by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H(2)O(2) is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid. CONCLUSIONS: In this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases

Synthesis and penicillin‐binding protein inhibitory assessment of dipeptidic 4‐phenyl‐β‐lactams from α‐amino acid‐derived imines

Monocyclic beta-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-beta-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically

Virtual Screening and Biochemical Testing of Borocycles as Immunoproteasome Inhibitors

Inhibition of the immunoproteasome (iCP) offers new opportunities in the treatment of cancer, autoimmune disorders and neurodegenerative diseases. Inspired by the success of boronic acids as proteasome inhibitors we have complied a virtual library of commercially available 5- and 6-membered borocycles and performed a structure based virtual screening against the chymotrypsin-like (β5i) subunit of the iCP. The top scored docking poses were visually inspected to select compounds for experimental testing. Six compounds with 5-membered ring and another six compounds with 6-membered ring were subjected to biochemical tests. All compounds exhibited detectable inhibitory activity at 100 µM concentration and these are the first reported cyclic boronic acid inhibitors of the iCP. Structural variations including the ring size and the substitution of the borocyles and the substitution pattern of the attached aromatic ring resulted in no major variation of the inhibitory activity. We propose that the evaluation of larger cycling boronic acid libraries is needed to fully elucidate the potential of these structures

Design and synthesis of amino-substituted N-arylpiperidinyl-based inhibitors of the (immuno)proteasome

The constitutive proteasome and the immunoproteasome represent validated targets for pharmacological intervention in the context of various diseases, such as cancer, inflammation, and autoimmune diseases. The development of novel chemical scaffolds of non-peptidic nature, capable of inhibiting different catalytically active subunits of both isoforms, is a viable approach against these diseases. Such compounds are also useful as leads for the development of biochemical probes that enable the studies of the roles of both isoforms in various biological contexts. Here, we present a ligand-based computational design of (immuno)proteasome inhibitors, which resulted in the amino-substituted N-arylpiperidine-based compounds that can inhibit different subunits of the (immuno)proteasome in the low micromolar range. The compounds represent a useful starting point for further structure-activity relationship studies that will, hopefully, lead to non-peptidic compounds that could be used in pharmacological and biochemical studies of both proteasomes

New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

Redox active or thiol reactive?

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action

Tunable heteroaromatic nitriles for selective bioorthogonal click reaction with cysteine

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science

Discovery of novel small-molecule compounds with selective cytotoxicity for Burkittʼs lymphoma cells using 3D ligand-based virtual screening

We describe a ligand-based approach towards compounds with more specific targeting for Burkittʼs lymphoma. Using three-dimensional ligand-based similarity searches and a previously described hit compound, we have identified six compounds that are chemically different but with similar spatial conformations. Biological evaluation revealed that one compound has better growth inhibition and improved selectivity towards Burkittʼs lymphoma cells than the query compound. However, initial mechanism-of-action studies show a different target profile in comparison with the previous hit compound, which does not involve the inhibition of the proteasome or the NFκB pathway. The data from this study provide a solid basis for further efforts in the search for selective agents against Burkittʼs lymphoma

Simulation- and AI-directed optimization of 4,6-substituted 1,3,5-triazin-2(1H)-ones as inhibitors of human DNA topoisomerase IIα

The 4,6-substituted-1,3,5-triazin-2(1H)-ones are promising inhibitors of human DNA topoisomerase IIα. To further develop this chemical class targeting the enzyme´s ATP binding site, the triazin-2(1H)-one substitution position 6 was optimized. Inspired by binding of preclinical substituted 9H-purine derivative, bicyclic substituents were incorporated at position 6 and the utility of this modification was validated by a combination of molecular simulations, dynamic pharmacophores, and free energy calculations. Considering also predictions of Deepfrag, a software developed for structure-based lead optimization based on deep learning, compounds with both bicyclic and monocyclic substitutions were synthesized and investigated for their inhibitory activity. The SAR data showed that the bicyclic substituted compounds exhibited good inhibition of topo IIα, comparable to their mono-substituted counterparts. Further evaluation on a panel of human protein kinases showed selectivity for the inhibition of topo IIα. Mechanistic studies indicated that the compounds acted predominantly as catalytic inhibitors, with some exhibiting topo IIα poison effects at higher concentrations. Integration of STD NMR experiments and molecular simulations, provided insights into the binding model and highlighted the importance of the Asn120 interaction and hydrophobic interactions with substituents at positions 4 and 6. In addition, NCI-60 screening demonstrated cytotoxicity of the compounds with bicyclic substituents and identified sensitive human cancer cell lines, underlining the translational relevance of our findings for further preclinical development of this class of compounds. The study highlights the synergy between simulation and AI-based approaches in efficiently guiding molecular design for drug optimization, which has implications for further preclinical development of this class of compounds