Correction: Zwergel et al. Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. Cancers 2020, 12, 447.

In the original publication [...].

Chemoproteomic Profiling Maps Zinc-Dependent Cysteine Reactivity.

As a vital micronutrient, zinc is integral to the structure, function, and signaling networks of diverse proteins. Dysregulated zinc levels, due to either excess intake or deficiency, are associated with a spectrum of health disorders. In this context, understanding zinc-regulated biological processes at the molecular level holds significant relevance to public health and clinical practice. Identifying and characterizing zinc-regulated proteins in their diverse proteoforms, however, remain a difficult task in advancing zinc biology. Herein, we address this challenge by developing a quantitative chemical proteomics platform that globally profiles the reactivities of proteinaceous cysteines upon cellular zinc depletion. Exploiting a protein-conjugated resin for the selective removal of Zn2+ from culture media, we identify an array of zinc-sensitive cysteines on proteins with diverse functions based on their increased reactivity upon zinc depletion. Notably, we find that zinc regulates the enzymatic activities, post-translational modifications, and subcellular distributions of selected target proteins such as peroxiredoxin 6 (PRDX6), platelet-activating factor acetylhydrolase IB subunit alpha1 (PAFAH1B3), and phosphoglycerate kinase (PGK1).

Hemisynthetic alkaloids derived from trilobine are antimalarials with sustained activity in multidrug-resistant Plasmodium falciparum.

Malaria eradication requires the development of new drugs to combat drug-resistant parasites. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus that are active against Plasmodium falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC50 ranging from 35 to 88 nM). Chemical optimization led to compound 125 with significantly improved preclinical properties. 125 delays the onset of parasitemia in Plasmodium berghei infected mice and inhibits P. falciparum transmission stages in vitro (culture assays), and in vivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P. falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, by chemical pull-down strategy, we characterized the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that are not targeted by the actual antimalarial drugs or implicated in drug-resistance mechanisms.

Clinical Antiviral Drug Arbidol Inhibits Infection by SARS-CoV-2 and Variants through Direct Binding to the Spike Protein.

Arbidol (ARB) is a broad-spectrum antiviral drug approved in Russia and China for the treatment of influenza. ARB was tested in patients as a drug candidate for the treatment at the early onset of COVID-19 caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite promising clinical results and multiple ongoing trials, preclinical data are lacking and the molecular mechanism of action of ARB against SARS-CoV-2 remains unknown. Here, we demonstrate that ARB binds to the spike viral fusion glycoprotein of the SARS-CoV-2 Wuhan strain as well as its more virulent variants from the United Kingdom (strain B.1.1.7) and South Africa (strain B.1.351). We pinpoint the ARB binding site on the S protein to the S2 membrane fusion domain and use an infection assay with Moloney murine leukemia virus (MLV) pseudoviruses (PVs) pseudotyped with the S proteins of the Wuhan strain and the new variants to show that this interaction is sufficient for the viral cell entry inhibition by ARB. Finally, our experiments reveal that the ARB interaction leads to a significant destabilization and eventual lysosomal degradation of the S protein in cells. Collectively, our results identify ARB as the first clinically approved small molecule drug binder of the SARS-CoV-2 S protein and place ARB among the more promising drug candidates for COVID-19.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Chemoproteomics-Enabled De Novo Discovery of Photoswitchable Carboxylesterase Inhibitors for Optically Controlled Drug Metabolism.

Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.

Combined Omics Approach Identifies Gambogic Acid and Related Xanthones as Covalent Inhibitors of the Serine Palmitoyltransferase Complex.

In this study, we identify the natural product gambogic acid as well as structurally related synthetic xanthones as first-in-class covalent inhibitors of the de novo sphingolipid biosynthesis. We apply chemoproteomics to determine that gambogic acid binds to the regulatory small subunit B of the serine palmitoyltransferase complex (SPTSSB). We then test structurally related synthetic xanthones to identify 18 as an equally potent but more selective binder of SPTSSB and show that 18 reduces sphingolipid levels in situ and in vivo. Finally, using various biological methods, we demonstrate that 18 induces cellular responses characteristic for diminished sphingosine-1-phosphate (S1P) signaling. This study demonstrates that SPTSSB may become a viable therapeutic target in various diseases with pathological S1P signaling. Furthermore, we believe that our compound will become a valuable tool for studying the sphingolipid metabolism and serve as a blueprint for the development of a new generation of sphingolipid biosynthesis inhibitors.

Bisubstrate-Type Chemical Probes Identify GRP94 as a Potential Target of Cytosine-Containing Adenosine Analogs.

We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type chemical probes and a chemical-biology methodology, this work opens the way to the development of a new family of GRP94 inhibitors that could potentially be of therapeutic interest.

Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation.

DNA methyltransferases (DNMTs) play a relevant role in epigenetic control of cancer cell survival and proliferation. Since only two DNMT inhibitors (azacitidine and decitabine) have been approved to date for the treatment of hematological malignancies, the development of novel potent and specific inhibitors is urgent. Here we describe the design, synthesis, and biological evaluation of a new series of compounds acting at the same time as DNMTs (mainly DNMT3A) inhibitors and degraders. Tested against leukemic and solid cancer cell lines, 2a-c and 4a-c (the last only for leukemias) displayed up to submicromolar antiproliferative activities. In HCT116 cells, such compounds induced EGFP gene expression in a promoter demethylation assay, confirming their demethylating activity in cells. In the same cell line, 2b and 4c chosen as representative samples induced DNMT1 and -3A protein degradation, suggesting for these compounds a double mechanism of DNMT3A inhibition and DNMT protein degradation.

Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells.

The implication of DNA methylation in cancer is today clearly established. Despite that nucleoside analogues are currently used for leukaemia treatment, their low stability in physiological conditions and their lack of selectivity arise the need for the identification of non-nucleoside DNA methyltransferase inhibitors. Here, we describe the synthesis and pharmacological characterisation of a novel class of DNA methyltransferase inhibitors: the 3-halo-3-nitroflavanones. We showed that 3-bromo-3-nitroflavanones 3b and 4a have a micromolar DNMT inhibition and an increased potency in a cell reporter model. Interestingly they are significantly more stable than the reference compounds and induce a low cytotoxicity, supporting them as new candidates for the development of non-cytotoxic cell-reprogramming epi-drugs for anticancer treatment.

Identification of novel quinazoline derivatives as potent antiplasmodial agents.

Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs.

Hijacking DNA methyltransferase transition state analogues to produce chemical scaffolds for PRMT inhibitors.

DNA, RNA and histone methylation is implicated in various human diseases such as cancer or viral infections, playing a major role in cell process regulation, especially in modulation of gene expression. Here we developed a convergent synthetic pathway starting from a protected bromomethylcytosine derivative to synthesize transition state analogues of the DNA methyltransferases. This approach led to seven 5-methylcytosine-adenosine compounds that were, surprisingly, inactive against hDNMT1, hDNMT3Acat, TRDMT1 and other RNA human and viral methyltransferases. Interestingly, compound 4 and its derivative 2 showed an inhibitory activity against PRMT4 in the micromolar range. Crystal structures showed that compound 4 binds to the PRMT4 active site, displacing strongly the S-adenosyl-l-methionine cofactor, occupying its binding site, and interacting with the arginine substrate site through the cytosine moiety that probes the space filled by a substrate peptide methylation intermediate. Furthermore, the binding of the compounds induces important structural switches. These findings open new routes for the conception of new potent PRMT4 inhibitors based on the 5-methylcytosine-adenosine scaffold.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Rational Design of Bisubstrate-Type Analogues as Inhibitors of DNA Methyltransferases in Cancer Cells.

Aberrant DNA hypermethylation of promoter of tumor suppressor genes is commonly observed in cancer, and its inhibition by small molecules is promising for their reactivation. Here we designed bisubstrate analogues-based inhibitors, by mimicking each substrate, the S-adenosyl-l-methionine and the deoxycytidine, and linking them together. This approach resulted in quinazoline-quinoline derivatives as potent inhibitors of DNMT3A and DNMT1, some showing certain isoform selectivity. We highlighted the importance of (i) the nature and rigidity of the linker between the two moieties for inhibition, as (ii) the presence of the nitrogen on the quinoline group, and (iii) of a hydrophobic group on the quinazoline. The most potent inhibitors induced demethylation of CDKN2A promoter in colon carcinoma HCT116 cells and its reactivation after 7 days of treatment. Furthermore, in a leukemia cell model system, we found a correlation between demethylation of the promoter induced by the treatment, chromatin opening at the promoter, and the reactivation of a reporter gene.

DNA methyltransferase inhibitors in cancer: From pharmacology to translational studies.

DNA methylation is a mammalian epigenetic mark that participates to define where and when genes are expressed, both in normal cells and in the context of diseases. Like other epigenetic marks, it is reversible and can be modulated by chemical agents. Because it plays an important role in cancer by silencing certain genes, such as tumour suppressor genes, it is a promising therapeutic target. Two compounds are already approved to treat haematological cancers, and many efforts have been carried out to discover new molecules that inhibit DNA methyltransferases, the enzymes responsible for DNA methylation. Here, we analyse the molecular mechanisms and cellular pharmacology of these inhibitors, pointing out the necessity for new pharmacological models and paradigms. The parameters of pharmacological responses need to be redefined: the aim is cellular reprogramming rather than general cytotoxicity. Thus, "epigenetic" rather than cytotoxic dosages are defined. Another issue is the delay of the response: cellular reprogramming can take several generations to produce observable phenotypes. Is this compatible with laboratory scale experiments? Finally, it is important to consider the specificity for cancer cells compared to normal cells and the appearance of resistance. We also discuss different techniques that are used and the selection of pharmacological models.

Compounds for use in the treatment of mycobacterial infections: a patent evaluation (WO2014049107A1).

Tuberculosis is one of the main causes of mortality with 1.5 million deaths a year worldwide. The growing emergence of multi- and extremely resistant strains highlights the urgent need of novel antibiotic strategies. Ethionamide, interfering with the mycobacterial membrane biosynthesis, is used in second-line treatment. This molecule is a prodrug, which requires activation by EthA. The patent described in this evaluation (WO2014049107A1) claimed a new family of molecules and their use as antibiotic treatment against mycobacteria such as Mycobacterium tuberculosis, M. leprae and atypical mycobacteria, either as a single active agent or in combination with antibiotics activable by EthA pathway.