iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

New Hybrid Compounds Incorporating Natural Products as Multifunctional Agents against Alzheimer's Disease.

A series of new hybrid derivatives 1a-c, 2a-c, 3a-c, 4a-c, 5a-c, inspired by nature, were synthesized and studied as multifunctional agents for the treatment of Alzheimer's disease (AD). These compounds were designed to merge together the trifluoromethyl benzyloxyaminic bioactive moiety, previously identified, with different acids available in nature. The ability of the synthesized compounds to chelate biometals, such as Cu2+, Zn2+ and Fe2+, was studied by UV-Vis spectrometer, and through a preliminary screening their antioxidant activity was evaluated by DPPH. Then, selected compounds were tested by in vitro ABTS free radical method and ex vivo rat brain TBARS assay. Compounds 2a-c, combining the strongest antioxidant and biometal chelators activities, were studied for their ability to contrast Aß1-40 fibrillization process. Finally, starting from the promising profile obtained for compound 2a, we evaluated if it could be able to induce a positive cross-interaction between transthyretin (TTR) and Aß in presence and in absence of Cu2+.

Identification of N-Acyl Hydrazones as New Non-Zinc-Binding MMP-13 Inhibitors by Structure-Based Virtual Screening Studies and Chemical Optimization.

Matrix metalloproteinase 13 plays a central role in osteoarthritis (OA), as its overexpression induces an excessive breakdown of collagen that results in an imbalance between collagen synthesis and degradation in the joint, leading to progressive articular cartilage degradation. Therefore, MMP-13 has been proposed as a key therapeutic target for OA. Here we have developed a virtual screening workflow aimed at identifying selective non-zinc-binding MMP-13 inhibitors by targeting the deep S1' pocket of MMP-13. Three ligands were found to inhibit MMP-13 in the µM range, and one of these showed selectivity over other MMPs. A structure-based analysis guided the chemical optimization of the hit compound, leading to the obtaining of a new N-acyl hydrazone-based derivative with improved inhibitory activity and selectivity for the target enzyme.

Pomegranate: A Source of Multifunctional Bioactive Compounds Potentially Beneficial in Alzheimer's Disease.

Pomegranate fruit (PF) is a fruit rich in nutraceuticals. Nonedible parts of the fruit, especially peels, contain high amounts of bioactive components that have been largely used in traditional medicine, such as the Chinese, Unani, and Ayurvedic ones, for treating several diseases. Polyphenols such as anthocyanins, tannins, flavonoids, phenolic acids, and lignans are the major bioactive molecules present in PF. Therefore, PF is considered a source of natural multifunctional agents that exert simultaneously antioxidant, anti-inflammatory, antitumor, antidiabetic, cardiovascular, and neuroprotective activities. Recently, several studies have reported that the nutraceuticals contained in PF (seed, peel, and juice) have a potential beneficial role in Alzheimer's disease (AD). Research suggests that the neuroprotective effect of PF is mostly due to its potent antioxidant and anti-inflammatory activities which contribute to attenuate the neuroinflammation associated with AD. Despite the numerous works conducted on PF, to date the mechanism by which PF acts in combatting AD is not completely known. Here, we summarize all the recent findings (in vitro and in vivo studies) related to the positive effects that PF and its bioactive components can have in the neurodegeneration processes occurring during AD. Moreover, considering the high biotransformation characteristics of the nutraceuticals present in PF, we propose to consider the chemical structure of its active metabolites as a source of inspiration to design new molecules with the same beneficial effects but less prone to be affected by the metabolic degradation process.

Bacterial Zinc Metalloenzyme Inhibitors: Recent Advances and Future Perspectives.

Human deaths caused by Gram-negative bacteria keep rising due to the multidrug resistance (MDR) phenomenon. Therefore, it is a priority to develop novel antibiotics with different mechanisms of action. Several bacterial zinc metalloenzymes are becoming attractive targets since they do not show any similarities with the human endogenous zinc-metalloproteinases. In the last decades, there has been an increasing interest from both industry and academia in developing new inhibitors against those enzymes involved in lipid A biosynthesis, and bacteria nutrition and sporulation, e.g., UDP-[3-O-(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), thermolysin (TLN), and pseudolysin (PLN). Nevertheless, targeting these bacterial enzymes is harder than expected and the lack of good clinical candidates suggests that more effort is needed. This review gives an overview of bacterial zinc metalloenzyme inhibitors that have been synthesized so far, highlighting the structural features essential for inhibitory activity and the structure-activity relationships. Our discussion may stimulate and help further studies on bacterial zinc metalloenzyme inhibitors as possible novel antibacterial drugs.

Omega-3 PUFAs as a Dietary Supplement in Senile Systemic Amyloidosis.

Eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6), two omega-3 poly-unsaturated fatty acids (PUFAs), are the main components in oil derived from fish and other marine organisms. EPA and DHA are commercially available as dietary supplements and are considered to be very safe and contribute to guaranteeing human health. Studies report that PUFAs have a role in contrasting neurodegenerative processes related to amyloidogenic proteins, such as ß-amyloid for AD, α-synuclein in PD, and transthyretin (TTR) in TTR amyloidosis. In this context, we investigated if EPA and DHA can interact directly with TTR, binding inside the thyroxin-binding pockets (T4BP) that contribute to the tetramer stabilization. The data obtained showed that EPA and DHA can contribute to stabilizing the TTR tetramer through interactions with T4BP.

New isoxazolidinyl-based N-alkylethanolamines as new activators of human brain carbonic anhydrases.

Carbonic anhydrases (CAs) are widespread metalloenzymes which catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and a proton, relevant in many physiological processes. In the last few years, the involvement of CA activation in different metabolic pathways in the human brain addressed the research to the discovery of novel CA activators. Here, a new series of isoxazoline-based amino alcohols as CA activators was investigated. The synthesis and the CA activating effects towards four human CA isoforms expressed in the human brain, that are hCAs I, II, IV and VII, were reported. The best results were obtained for the (methyl)-isoxazoline-amino alcohols 3 and 5 with KA values in the submicromolar range (0.52-0.86 µM) towards hCA VII, and a good selectivity over hCA I. Being hCA VII involved in brain function and metabolism, the newly identified CA activators might be promising hit compounds with potential therapeutic applications in ageing, epilepsy or neurodegeneration.

Identification of a Novel p53 Modulator Endowed with Antitumoural and Antibacterial Activity through a Scaffold Repurposing Approach.

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection.

Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition.

Osteoarthritis (OA) is the most common degenerative joint disease. In 1999, two members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family of metalloproteinases, ADAMTS4 and ADAMTS5, or aggrecanases, were identified as the enzymes responsible for aggrecan degradation in cartilage. The first aggrecanase inhibitors targeted the active site by chelation of the catalytic zinc ion. Due to the generally disappointing performance of zinc-chelating inhibitors in preclinical and clinical studies, inhibition strategies tried to move away from the active-site zinc in order to improve selectivity. Exosite inhibitors bind to proteoglycan-binding residues present on the aggrecanase ancillary domains (called exosites). While exosite inhibitors are generally more selective than zinc-chelating inhibitors, they are still far from fulfilling their potential, partly due to a lack of structural and functional data on aggrecanase exosites. Filling this gap will inform the design of novel potent, selective aggrecanase inhibitors.

Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Development of a fluorogenic ADAMTS-7 substrate.

The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-ß-binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.

Identification of Broad-Spectrum MMP Inhibitors by Virtual Screening.

Matrix metalloproteinases (MMPs) are the family of proteases that are mainly responsible for degrading extracellular matrix (ECM) components. In the skin, the overexpression of MMPs as a result of ultraviolet radiation triggers an imbalance in the ECM turnover in a process called photoaging, which ultimately results in skin wrinkling and premature skin ageing. Therefore, the inhibition of different enzymes of the MMP family at a topical level could have positive implications for photoaging. Considering that the MMP catalytic region is mostly conserved across different enzymes of the MMP family, in this study we aimed to design a virtual screening (VS) workflow to identify broad-spectrum MMP inhibitors that can be used to delay the development of photoaging. Our in silico approach was validated in vitro with 20 VS hits from the Specs library that were not only structurally different from one another but also from known MMP inhibitors. In this bioactivity assay, 18 of the 20 compounds inhibit at least one of the assayed MMPs at 100 µM (with 5 of them showing around 50% inhibition in all the tested MMPs at this concentration). Finally, this VS was used to identify natural products that have the potential to act as broad-spectrum MMP inhibitors and be used as a treatment for photoaging.

Strategies to Target ADAM17 in Disease: From its Discovery to the iRhom Revolution.

For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells.

Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide.

ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of ß-N-acetyl-D-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.

Discovery of Dimeric Arylsulfonamides as Potent ADAM8 Inhibitors.

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration, and angiogenesis. The use of bivalent inhibitors could impair migration and invasion through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and matrix metalloproteinases. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 was shown to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in the breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8.

Identification of histone deacetylase inhibitors with (arylidene)aminoxy scaffold active in uveal melanoma cell lines.

Uveal melanoma (UM) represents an aggressive type of cancer and currently, there is no effective treatment for this metastatic disease. In the last years, histone deacetylase inhibitors (HDACIs) have been studied as a possible therapeutic treatment for UM, alone or in association with other chemotherapeutic agents. Here we synthesised a series of new HDACIs based on the SAHA scaffold bearing an (arylidene)aminoxy moiety. Their HDAC inhibitory activity was evaluated on isolated human HDAC1, 3, 6, and 8 by fluorometric assay and their binding mode in the catalytic site of HDACs was studied by molecular docking. The most promising hit was the quinoline derivative VS13, a nanomolar inhibitor of HDAC6, which exhibited a good antiproliferative effect on UM cell lines at micromolar concentration and a capability to modify the mRNA levels of HDAC target genes similar to that of SAHA.

Activation of carbonic anhydrases from human brain by amino alcohol oxime ethers: towards human carbonic anhydrase VII selective activators.

The synthesis and carbonic anhydrase (CA; EC 4.2.1.1) activating effects of a series of oxime ether-based amino alcohols towards four human (h) CA isoforms expressed in human brain, hCA I, II, IV and VII, are described. Most investigated amino alcohol derivatives induced a consistent activation of the tested CAs, with KAs spanning from a low micromolar to a medium nanomolar range. Specifically, hCA II and VII, putative main CA targets when central nervous system (CNS) diseases are concerned, were most efficiently activated by these oxime ether derivatives. Furthermore, a multitude of selective hCA VII activators were identified. As hCA VII is one of the key isoforms involved in brain metabolism and other brain functions, the identified potent and selective hCA VII activators may be considered of interest for investigations of various therapeutic applications or as lead compounds in search of even more potent and selective CA activators.

The metalloproteinase ADAM10 requires its activity to sustain surface expression.

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.

Developments in Carbohydrate-Based Metzincin Inhibitors.

Matrix metalloproteinases (MMPs) and A disintegrin and Metalloproteinase (ADAMs) are zinc-dependent endopeptidases belonging to the metzincin superfamily. Upregulation of metzincin activity is a major feature in many serious pathologies such as cancer, inflammations, and infections. In the last decades, many classes of small molecules have been developed directed to inhibit these enzymes. The principal shortcomings that have hindered clinical development of metzincin inhibitors are low selectivity for the target enzyme, poor water solubility, and long-term toxicity. Over the last 15 years, a novel approach to improve solubility and bioavailability of metzincin inhibitors has been the synthesis of carbohydrate-based compounds. This strategy consists of linking a hydrophilic sugar moiety to an aromatic lipophilic scaffold. This review aims to describe the development of sugar-based and azasugar-based derivatives as metzincin inhibitors and their activity in several pathological models.

An overview of carbohydrate-based carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs) are metalloenzymes responsible for the reversible hydration of carbon dioxide to bicarbonate, a fundamental reaction involved in various physiological and pathological processes. In the last decades, CAs have been considered as important drug targets for different pathologies such as glaucoma, epilepsy and cancer. The design of potent and selective inhibitors has been an outstanding goal leading to the discovery of new drugs. Among the different strategies developed to date, the design of carbohydrate-based CA inhibitors (CAIs) has emerged as a versatile tool in order to selectively target CAs. The insertion of a glycosyl moiety as a hydrophilic tail in sulfonamide, sulfenamide, sulfamate or coumarin scaffolds allowed the discovery of many different series of sugar-based CAIs, with relevant inhibitory results. This review will focus on carbohydrate-based CAIs developed so far, classifying them in glycosidic and glycoconjugated inhibitors based on the conjugation chemistry adopted.