Study of phenoxy radical couplings using the enzymatic secretome of Botrytis cinerea.

Phenoxy radical coupling reactions are widely used in nature for the synthesis of complex molecules such as lignin. Their use in the laboratory has great potential for the production of high value compounds from the polyphenol family. While the enzymes responsible for the generation of the radicals are well known, the behavior of the latter is still enigmatic and difficult to control in a reaction flask. Previous work in our laboratory using the enzymatic secretome of B. cinerea containing laccases has shown that incubation of stilbenes leads to dimers, while incubation of phenylpropanoids leads to dimers as well as larger coupling products. Building on these previous studies, this paper investigates the role of different structural features in phenoxy radical couplings. We first demonstrate that the presence of an exocyclic conjugated double bond plays a role in the generation of efficient reactions. In addition, we show that the formation of phenylpropanoid trimers and tetramers can proceed via a decarboxylation reaction that regenerates this reactive moiety. Lastly, this study investigates the reactivity of other phenolic compounds: stilbene dimers, a dihydro-stilbene, a 4-O-methyl-stilbene and a simple phenol with the enzymatic secretome of B. cinerea. The observed efficient dimerization reactions consistently correlate with the presence of a para-phenol conjugated to an exocyclic double bond. The absence of this structural feature leads to variable results, with some compounds showing low conversion or no reaction at all. This research has allowed the development of a controlled method for the synthesis of specific dimers and tetramers of phenylpropanoid derivatives and novel stilbene derivatives, as well as an understanding of features that can promote efficient radical coupling reactions.

Diapocynin treatment induces functional and structural improvements in an advanced disease state in the mdx5Cv mice.

Duchenne muscular dystrophy (DMD) is the most common muscular disorder affecting children. It affects nearly 1 male birth over 5000. Oxidative stress is a pervasive feature in the pathogenesis of DMD. Recent work shows that the main generators of ROS are NADPH oxidases (NOX), suggesting that they are an early and promising target in DMD. In addition, skeletal muscles of mdx mice, a murine model of DMD, overexpress NOXes. We investigated the impact of diapocynin, a dimer of the NOX inhibitor apocynin, on the chronic disease phase of mdx5Cv mice. Treatment of these mice with diapocynin from 7 to 10 months of age resulted in decreased hypertrophy of several muscles, prevented force loss induced by tetanic and eccentric contractions, improved muscle and respiratory functions, decreased fibrosis of the diaphragm and positively regulated the expression of disease modifiers. These encouraging results ensure the potential role of diapocynin in future treatment strategies.

Effects of a diverse prebiotic fibre supplement on HbA1c, insulin sensitivity and inflammatory biomarkers in pre-diabetes: a pilot placebo-controlled randomised clinical trial.

Prebiotic fibre represents a promising and efficacious treatment to manage pre-diabetes, acting via complementary pathways involving the gut microbiome and viscosity-related properties. In this study, we evaluated the effect of using a diverse prebiotic fibre supplement on glycaemic, lipid and inflammatory biomarkers in patients with pre-diabetes. Sixty-six patients diagnosed with pre-diabetes (yet not receiving glucose-lowering medications) were randomised into treatment (thirty-three) and placebo (thirty-three) interventions. Participants in the treatment arm consumed 20 g/d of a diverse prebiotic fibre supplement, and participants in the placebo arm consumed 2 g/d of cellulose for 24 weeks. A total of fifty-one and forty-eight participants completed the week 16 and week 24 visits, respectively. The intervention was well tolerated, with a high average adherence rate across groups. Our results extend upon previous work, showing a significant change in glycated haemoglobin (HbA1c) in the treatment group but only in participants with lower baseline HbA1c levels (< 6 % HbA1c) (P = 0·05; treatment -0·17 ± 0·27 v. placebo 0·07 ± 0·29, mean ± sd). Within the whole cohort, we showed significant improvements in insulin sensitivity (P = 0·03; treatment 1·62 ± 5·79 v. placebo -0·77 ± 2·11) and C-reactive protein (P FWE = 0·03; treatment -2·02 ± 6·42 v. placebo 0·94 ± 2·28) in the treatment group compared with the placebo. Together, our results support the use of a diverse prebiotic fibre supplement for physiologically relevant biomarkers in pre-diabetes.

A critical view on autoantibodies in lupus nephritis: Concrete knowledge based on evidence.

Deposition of autoantibodies in glomeruli is a key factor in the development of lupus nephritis (LN). For a long time, anti-dsDNA and anti-C1q antibodies were thought to be the main cause of the kidney damage. However, recent studies have shown that the list of autoantibidies that have renal tropism and deposit in the kidney in LN is increasing and the link between anti-dsDNA and renal pathology is weak due to potential confounders. Aspecific bindings of dsDNA with cationic antibodies and of anti-dsDNA with several renal antigens such as actinin, laminin, entactin, and annexinA2 raised doubts about the specific target of these antibodies in the kidney. Moreover, the isotype of anti-dsDNA in SLE and LN has never received adequate interest until the recent observation that IgG2 are preponderant over IgG1, IgG3 and IgG4. Based on the above background, recent studies investigated the involvement of anti-dsDNA IgG2 and of other antibodies in LN. It was concluded that circulating anti-dsDNA IgG2 levels do not distinguish between LN versus non-renal SLE, and, in patients with LN, their levels do not change over time. Circulating levels of other antibodies such as anti-ENO1 and anti-H2 IgG2 were, instead, higher in LN vs non-renal SLE at the time of diagnosis and decreased following therapies. Finally, new classes of renal antibodies that potentially modify the anti-inflammatory response in the kidney are emerging as new co-actors in the pathogenetic scenario. They have been defined as 'second wave antibodies' for the link with detoxifying mechanisms limiting the oxidative stress in glomeruli that are classically stimulated in a second phase of inflammation. These findings have important clinical implications that may modify the laboratory approach to LN. Serum levels of anti-ENO1 and anti-H2 IgG2 should be measured in the follow up of patients for designing the length of therapies and identify those patients who respond to treatments. Anti-SOD2 could help to monitor and potentiate the anti-inflammatory response in the kidney.

Enhancing Oral Delivery of Biologics: A Non-Competitive and Cross-Reactive Anti-Leptin Receptor Nanofitin Demonstrates a Gut-Crossing Capacity in an Ex Vivo Porcine Intestinal Model.

Biotherapeutics exhibit high efficacy in targeted therapy, but their oral delivery is impeded by the harsh conditions of the gastrointestinal (GI) tract and limited intestinal absorption. This article presents a strategy to overcome the challenges of poor intestinal permeability by using a protein shuttle that specifically binds to an intestinal target, the leptin receptor (LepR), and exploiting its capacity to perform a receptor-mediated transport. Our proof-of-concept study focuses on the characterization and transport of robust affinity proteins, known as Nanofitins, across an ex vivo porcine intestinal model. We describe the potential to deliver biologically active molecules across the mucosa by fusing them with the Nanofitin 1-F08 targeting the LepR. This particular Nanofitin was selected for its absence of competition with leptin, its cross-reactivity with LepR from human, mouse, and pig hosts, and its shuttle capability associated with its ability to induce a receptor-mediated transport. This study paves the way for future in vivo demonstration of a safe and efficient oral-to-systemic delivery of targeted therapies.

Generation of potent antibacterial compounds through enzymatic and chemical modifications of the trans-δ-viniferin scaffold.

Stilbene dimers are well-known for their diverse biological activities. In particular, previous studies have demonstrated the high antibacterial potential of a series of trans-δ-viniferin-related compounds against gram-positive bacteria such as Staphylococcus aureus. The trans-δ-viniferin scaffold has multiple chemical functions and can therefore be modified in various ways to generate derivatives. Here we report the synthesis of 40 derivatives obtained by light isomerization, O-methylation, halogenation and dimerization of other stilbene monomers. The antibacterial activities of all generated trans-δ-viniferin derivatives were evaluated against S. aureus and information on their structure-activity relationships (SAR) was obtained using a linear regression model. Our results show how several parameters, such as the O-methylation pattern and the presence of halogen atoms at specific positions, can determine the antibacterial activity. Taken together, these results can serve as a starting point for further SAR investigations.

Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model.

Adjusting the molecular size, the valency and the pharmacokinetics of drug conjugates are as many leverages to improve their therapeutic window, notably by affecting tumor penetration, renal clearance, and short systemic exposure. In that regard, small tumor-targeting ligands are gaining attention. In this study, we demonstrate the benefits of the small Nanofitin alternative scaffolds (7 kDa) as selective tumor-targeting modules for the generation of drug conjugates, focusing on Nanofitins B10 and D8 directed against the EGFR. Owing to their small size and monovalent format, the two Nanofitins displayed a fast and deep tumor penetration in EGFR-positive A431 xenografts in BALB/c nude mice after intravenous administration, yielding to a targeting of respectively 67.9% ± 14.1 and 98.9% ± 0.7 of the tumor cells as demonstrated by IHC. Conjugation with the monomethyl auristatin E toxin provided homogeneous Nanofitin-drug conjugates, with an overall yield of ≥97%, for in vivo assessment in a curative xenograft model using bioluminescent, EGFR-positive, A431 cells in BALB/c nude mice. Internalization was found critical for efficient release of the toxin. Hence, the intravenous administration of the D8-based construct showed significant antitumor effect in vivo as determined by monitoring tumor volumes and bioluminescence levels over 2 months.

Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1.

Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway - a developmental pathway with many implications in health and disease - yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.

Challenges and Opportunities in the Oral Delivery of Recombinant Biologics.

Recombinant biological molecules are at the cutting-edge of biomedical research thanks to the significant progress made in biotechnology and a better understanding of subcellular processes implicated in several diseases. Given their ability to induce a potent response, these molecules are becoming the drugs of choice for multiple pathologies. However, unlike conventional drugs which are mostly ingested, the majority of biologics are currently administered parenterally. Therefore, to improve their limited bioavailability when delivered orally, the scientific community has devoted tremendous efforts to develop accurate cell- and tissue-based models that allow for the determination of their capacity to cross the intestinal mucosa. Furthermore, several promising approaches have been imagined to enhance the intestinal permeability and stability of recombinant biological molecules. This review summarizes the main physiological barriers to the oral delivery of biologics. Several preclinical in vitro and ex vivo models currently used to assess permeability are also presented. Finally, the multiple strategies explored to address the challenges of administering biotherapeutics orally are described.

Identification of Natural Products Inhibiting SARS-CoV-2 by Targeting Viral Proteases: A Combined in Silico and in Vitro Approach.

In this study, an integrated in silico-in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (Mpro) and papain-like protease (PLpro), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 Mpro by more than 60% at a concentration of 20 µM, and four of them with high potency (IC50 < 10 µM). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels-Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC50 values of 4.6, 8.0, and 7.6 µM and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 Mpro proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive 1H relaxation dispersion NMR spectroscopy.

In silico investigation of cytochrome bc1 molecular inhibition mechanism against Trypanosoma cruzi.

Chagas' disease is a neglected tropical disease caused by the kinetoplastid protozoan Trypanosoma cruzi. The only therapies are the nitroheterocyclic chemicals nifurtimox and benznidazole that cause various adverse effects. The need to create safe and effective medications to improve medical care remains critical. The lack of verified T. cruzi therapeutic targets hinders medication research for Chagas' disease. In this respect, cytochrome bc1 has been identified as a promising therapeutic target candidate for antibacterial medicines of medical and agricultural interest. Cytochrome bc1 belongs to the mitochondrial electron transport chain and transfers electrons from ubiquinol to cytochrome c1 by the action of two catalytic sites named Qi and Qo. The two binding sites are highly selective, and specific inhibitors exist for each site. Recent studies identified the Qi site of the cytochrome bc1 as a promising drug target against T. cruzi. However, a lack of knowledge of the drug mechanism of action unfortunately hinders the development of new therapies. In this context, knowing the cause of binding site selectivity and the mechanism of action of inhibitors and substrates is crucial for drug discovery and optimization processes. In this paper, we provide a detailed computational investigation of the Qi site of T. cruzi cytochrome b to shed light on the molecular mechanism of action of known inhibitors and substrates. Our study emphasizes the action of inhibitors at the Qi site on a highly unstructured portion of cytochrome b that could be related to the biological function of the electron transport chain complex.

CEMIP (HYBID, KIAA1199): structure, function and expression in health and disease.

CEMIP (cell migration-inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions.

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells.

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 µM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.

Discovery of anti-Formin-like 1 protein (FMNL1) antibodies in membranous nephropathy and other glomerular diseases.

Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.

Correction to "Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase".

[This corrects the article DOI: 10.1021/acsomega.2c00507.].

Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase.

The anaplastic lymphoma kinase (ALK) is abnormally expressed and hyperactivated in a number of tumors and represents an ideal therapeutic target. Despite excellent clinical responses to ALK inhibition, drug resistance still represents an issue and novel compounds that overcome drug-resistant mutants are needed. We designed, synthesized, and evaluated a large series of azacarbazole inhibitors. Several lead compounds endowed with submicromolar potency were identified. Compound 149 showed selective inhibition of native and mutant drug-refractory ALK kinase in vitro as well as in a Ba/F3 model and in human ALK+ lymphoma cells. The three-dimensional (3D) structure of a 149:ALK-KD cocrystal is reported, showing extensive interaction through the hinge region and the catalytic lysine 1150.

Identification of non-ATP-competitive α-carboline inhibitors of the anaplastic lymphoma kinase.

The Anaplastic Lymphoma Kinase (ALK) is a therapeutic target for personalized medicine in selected cancers. Despite excellent clinical responses to ALK inhibitors, most patients develop drug resistance and relapse. New compounds with alternative binding modes are needed to overcome resistant mutants. Here we describe a medicinal chemistry effort to the design and development of novel ALK inhibitors based on a 4,6-substituted α-carboline scaffold. Active compounds were able to inhibit the gatekeeper L1196M mutant, in several cases better than the wild-type enzyme. Compound 43 showed potent non-ATP-competitive inhibition of wild-type and mutant ALK, including G1202R, in biochemical and cellular assays, as well as in xenograft mouse models.

Chemoenzymatic Synthesis of Original Stilbene Dimers Possessing Wnt Inhibition Activity in Triple-Negative Breast Cancer Cells Using the Enzymatic Secretome of Botrytis cinerea Pers.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancer and triple-negative breast cancer (TNBC) in particular depend upon Wnt pathway overactivation. Despite this importance, no Wnt pathway-targeting drugs are currently available, which necessitates novel approaches to search for therapeutically relevant compounds targeting this oncogenic pathway. Stilbene analogs represent an under-explored field of therapeutic natural products research. In the present work, a library of complex stilbene derivatives was obtained through biotransformation of a mixture of resveratrol and pterostilbene using the enzymatic secretome of Botrytis cinerea. To improve the chemodiversity, the reactions were performed using i-PrOH, n-BuOH, i-BuOH, EtOH, or MeOH as cosolvents. Using this strategy, a series of 73 unusual derivatives was generated distributed among 6 scaffolds; 55 derivatives represent novel compounds. The structure of each compound isolated was determined by nuclear magnetic resonance and high-resolution mass spectrometry. The inhibitory activity of the isolated compounds against the oncogenic Wnt pathway was comprehensively quantified and correlated with their capacity to inhibit the growth of the cancer cells, leading to insights into structure-activity relationships of the derivatives. Finally, we have dissected mechanistic details of the stilbene derivatives activity within the pathway.

Laboratory Selection of Trypanosomatid Pathogens for Drug Resistance.

The selection of parasites for drug resistance in the laboratory is an approach frequently used to investigate the mode of drug action, estimate the risk of emergence of drug resistance, or develop molecular markers for drug resistance. Here, we focused on the How rather than the Why of laboratory selection, discussing different experimental set-ups based on research examples with Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The trypanosomatids are particularly well-suited to illustrate different strategies of selecting for drug resistance, since it was with African trypanosomes that Paul Ehrlich performed such an experiment for the first time, more than a century ago. While breakthroughs in reverse genetics and genome editing have greatly facilitated the identification and validation of candidate resistance mutations in the trypanosomatids, the forward selection of drug-resistant mutants still relies on standard in vivo models and in vitro culture systems. Critical questions are: is selection for drug resistance performed in vivo or in vitro? With the mammalian or with the insect stages of the parasites? Under steady pressure or by sudden shock? Is a mutagen used? While there is no bona fide best approach, we think that a methodical consideration of these questions provides a helpful framework for selection of parasites for drug resistance in the laboratory.

Linkers as Game-changers in PROTAC Technology: Emphasizing General Trends in PROTAC Pharmacokinetics for their Rational Design.

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that act as degraders. They selectively remove disease-associated proteins by hijacking the Ubiquitin-Proteasome System (UPS). Chemically, they consist of three parts: an E3 ligase ligand, a target of interest (TOI) ligand, and a linker, which connects the two moieties. The rapid expansion of PROTAC Technology as an innovative therapeutic modality in cancer fostered the drug discovery effort to optimize their physicochemical properties. Due to their large size, their features are far from the traditional 'drug-like' properties. This short review highlights some of the structural modifications in the linker component to optimize the PROTAC Drug Metabolism and Pharmacokinetics (DMPK) profile. In particular, we discussed aspects related to solubility, cell permeability, active transporters efflux and, metabolic stability.