Chronic liver diseases: From development to novel pharmacological therapies: IUPHAR Review 37.

Chronic liver diseases comprise a broad spectrum of burdensome diseases that still lack effective pharmacological therapies. Our research group focuses on fibrosis, which is a major precursor of liver cirrhosis. Fibrosis consists in a progressive disturbance of liver sinusoidal architecture characterised by connective tissue deposition as a reparative response to tissue injury. Multifactorial events and several types of cells participate in fibrosis initiation and progression, and the process still needs to be completely understood. The development of experimental models of liver fibrosis alongside the identification of critical factors progressing fibrosis to cirrhosis will facilitate the development of more effective therapeutic approaches for such condition. This review provides an overlook of the main process leading to hepatic fibrosis and therapeutic approaches that have emerged from a deep knowledge of the molecular regulation of fibrogenesis in the liver. LINKED ARTICLES: This article is part of a themed issue on Translational Advances in Fibrosis as a Therapeutic Target. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.22/issuetoc.

Traditional Knowledge Evolution over Half of a Century: Local Herbal Resources and Their Changes in the Upper Susa Valley of Northwest Italy.

Susa Valley, located in the Italian Western Alps, has served as a meeting point for cultural, spiritual, and commercial exchange for a long period of history. The valley's role as one of the main connecting routes between south and southwestern Europe resulted in its acquisition of a rich traditional ecological knowledge. However, like other Italian mountainous valleys, this valley has suffered from abandonment and depopulation in the past 50 years. Our study aims to investigate the current ethnobotanical medicinal knowledge in the valley and to compare our findings with a study conducted over 50 years ago in the same area. In 2018, we conducted 30 in-depth semi-structured interviews on medicinal plants and food-medicines used in the Susa Valley. We documented 36 species, of which 21 species were used for medical purposes and 15 species were used as food-medicine. The comparison with the previous study on medicinal herbs conducted in 1970 in the valley demonstrated a significant decrease in both the knowledge and use of medicinal plants, which could be attributed to socioeconomic, cultural, and possibly environmental changes that occurred in the past half-century. Our study highlights several promising species for future use as nutraceuticals, food, and medicinal products, such as Taraxacum officinale, Urtica dioica, and Artemisia genipi.

Loss of activating transcription factor 3 prevents KRAS-mediated pancreatic cancer.

The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3-/-) combined with acinar-specific induction of oncogenic KRAS (Ptf1acreERT/+KrasG12D/+). Atf3-/- mice with (termed APK) and without KRASG12D were exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis, Atf3-/- mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia (PanIN) formation and PDAC in Ptf1acreERT/+KrasG12D/+ mice. In response to injury, KRASG12D bypassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. Compared to Ptf1acreERT/+KrasG12D/+ mice, APK mice exhibited a significant decrease in pancreatic and total body weight, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.

NUPR1 interacts with eIF2α and is required for resolution of the ER stress response in pancreatic tissue.

Nuclear protein 1 (NUPR1) is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1's well-established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely debated. In this study, we uncover a new role for NUPR1, participating in the unfolded protein response (UPR) and the integrated stress response. Biochemical results and ultrastructural morphological observations revealed alterations in the UPR of acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein synthesis after stress induction. Bioinformatic analysis of NUPR1-interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays confirmed the interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Furthermore, our data suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower poststress protein synthesis recovery when compared to wild-type. Combined, these data propose a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK branch activity and resolution through a unique interaction with eIF2α.

Evidencing a Pancreatic Ductal Adenocarcinoma Subpopulation Sensitive to the Proteasome Inhibitor Carfilzomib.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a survival rate less than 5%. Multiple chemotherapeutic drugs have been tested to improve patient prognosis; however, the clinical efficacy of these treatments is low. One of the most controversial family of drugs are the proteasome inhibitors, which have displayed promising effects in preclinical studies, but low clinical performance. Here, we unravel a specific transcriptomic signature that discriminates a subgroup of patients sensitive to the proteasome inhibitor carfilzomib. EXPERIMENTAL DESIGN: First, we identified a subpopulation of PDAC-derived primary cells cultures (PDPCC) sensitive to the proteasome inhibitor carfilzomib. Then, we selected a transcriptomic signature that predicts carfilzomib chemosensitivity using independent component analysis on the transcriptome of PDPCC. Finally, we validated the signature in an independent cohort of PDAC biopsy-derived pancreatic organoids. RESULTS: Sensitive phenotype was characterized by a high expression of genes related with a cornified/squamous pathway and a downregulation of epithelial-mesenchymal transition genes. Interestingly, carfilzomib-sensitive transcriptomic profile did not show any association with the proteasome activity but strongly correlates with ATF4 and CHOP expression, which are key markers of the unfolded protein response and critical to trigger the cell death program. Concordantly, sensitive phenotype showed a high level of the de novo RNA and protein synthesis compared with the resistant one and, most important, cell death induced by carfilzomib is dependent of the translational activity. CONCLUSIONS: We demonstrate the existence of a carfilzomib-sensitive PDAC subgroup with a specific transcriptomic phenotype that could explain the biological reason for this responsiveness.

Isoform-selective HDAC1/6/8 inhibitors with an imidazo-ketopiperazine cap containing stereochemical diversity.

A series of hydroxamic acids linked by different lengths to a chiral imidazo-ketopiperazine scaffold were synthesized. The compounds with linker lengths of 6 and 7 carbon atoms were the most potent in histone deacetylase (HDAC) inhibition, and were specific submicromolar inhibitors of the HDAC1, HDAC6 and HDAC8 isoforms. A docking model for the binding mode predicts binding of the hydroxamic acid to the active site zinc cation and additional interactions between the imidazo-ketopiperazine and the enzyme rim. The compounds were micromolar inhibitors of the MV4-11, THP-1 and U937 cancer cell lines. Increased levels of histone H3 and tubulin acetylation support a cellular mechanism of action through HDAC inhibition.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Fluorinated tranylcypromine analogues as inhibitors of lysine-specific demethylase 1 (LSD1, KDM1A).

We report a series of tranylcypromine analogues containing a fluorine in the cyclopropyl ring. A number of compounds with additional m- or p-substitution of the aryl ring were micromolar inhibitors of the LSD1 enzyme. In cellular assays, the compounds inhibited the proliferation of acute myeloid leukemia cell lines. Increased levels of the biomarkers H3K4me2 and CD86 were consistent with LSD1 target engagement.

cis-Cyclopropylamines as mechanism-based inhibitors of monoamine oxidases.

Cyclopropylamines, inhibitors of monoamine oxidases (MAO) and lysine-specific demethylase (LSD1), provide a useful structural scaffold for the design of mechanism-based inhibitors for treatment of depression and cancer. For new compounds with the less common cis relationship and with an alkoxy substituent at the 2-position of the cyclopropyl ring, the apparent affinity determined from docking experiments revealed little difference between the enantiomers. Using the racemate, kinetic parameters for the reversible and irreversible inhibition of MAO were determined. No inhibition of LSD1 was observed. For reversible inhibition, most compounds gave high IC50 values with MAO A, but sub-micromolar values with MAO B. After pre-incubation of the cyclopropylamine with the enzyme, the inhibition was irreversible for both MAO A and MAO B, and the activity was not restored by dilution. Spectral changes during inactivation of MAO A included bleaching at 456 nm and an increased absorbance at 400 nm, consistent with flavin modification. These derivatives are MAO B-selective irreversible inhibitors that do not show inhibition of LSD1. The best inhibitor was cis-N-benzyl-2-methoxycyclopropylamine, with an IC50 of 5 nm for MAO B and 170 nm for MAO A after 30 min pre-incubation. This cis-cyclopropylamine is over 20-fold more effective than tranylcypromine, so may be studied as a lead for selective inhibitors of MAO B that do not inhibit LSD1.

Expanding the druggable space of the LSD1/CoREST epigenetic target: new potential binding regions for drug-like molecules, peptides, protein partners, and chromatin.

Lysine specific demethylase-1 (LSD1/KDM1A) in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD) simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV) software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide) underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.

Protein recognition by short peptide reversible inhibitors of the chromatin-modifying LSD1/CoREST lysine demethylase.

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.

Efficient synthesis and biological evaluation of proximicins A, B and C.

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.