High-Density Lipoproteins at the Interface between the NLRP3 Inflammasome and Myocardial Infarction.

Despite significant therapeutic advancements, morbidity and mortality following myocardial infarction (MI) remain unacceptably high. This clinical challenge is primarily attributed to two significant factors: delayed reperfusion and the myocardial injury resulting from coronary reperfusion. Following reperfusion, there is a rapid intracellular pH shift, disruption of ionic balance, heightened oxidative stress, increased activity of proteolytic enzymes, initiation of inflammatory responses, and activation of several cell death pathways, encompassing apoptosis, necroptosis, and pyroptosis. The inflammatory cell death or pyroptosis encompasses the activation of the intracellular multiprotein complex known as the NLRP3 inflammasome. High-density lipoproteins (HDL) are endogenous particles whose components can either promote or mitigate the activation of the NLRP3 inflammasome. In this comprehensive review, we explore the role of inflammasome activation in the context of MI and provide a detailed analysis of how HDL can modulate this process.

Drug discovery: In silico dry data can bypass biological wet data?

The recent and extraordinary increase in computer power, along with the availability of efficient algorithms based on artificial intelligence, has prompted a large number of inexperienced scientists to challenge the complex and yet competitive world of drug discovery, by pretending to identify new hits through the sole use of computer aided drug design (CADD). Does the golden era of dry data run the risk of overshadowing the importance of wet data and, in doing so, forget that in silico and biological data need each other in successful preclinical drug discovery programmes?

Molecular Determinants of EphA2 and EphB2 Antagonism Enable the Design of Ligands with Improved Selectivity.

With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.

A Pharmacological Investigation of Eph-Ephrin Antagonism in Prostate Cancer: UniPR1331 Efficacy Evidence.

The Eph kinases are the largest receptor tyrosine kinases (RTKs) family in humans. PC3 human prostate adenocarcinoma cells are a well-established model for studying Eph-ephrin pharmacology as they naturally express a high level of EphA2, a promising target for new cancer therapies. A pharmacological approach with agonists did not show significant efficacy on tumor growth in prostate orthotopic murine models, but reduced distal metastasis formation. In order to improve the comprehension of the pharmacological targeting of Eph receptors in prostate cancer, in the present work, we investigated the efficacy of Eph antagonism both in vitro and in vivo, using UniPR1331, a small orally bioavailable Eph-ephrin interaction inhibitor. UniPR1331 was able to inhibit PC3 cells' growth in vitro in a dose-dependent manner, affecting the cell cycle and inducing apoptosis. Moreover, UniPR1331 promoted the PC3 epithelial phenotype, downregulating epithelial mesenchymal transition (EMT) markers. As a consequence, UniPR1331 reduced in vitro PC3 migration, invasion, and vasculomimicry capabilities. The antitumor activity of UniPR1331 was confirmed in vivo when administered alone or in combination with cytotoxic drugs in PC3-xenograft mice. Our results demonstrated that Eph antagonism is a promising strategy for inhibiting prostate cancer growth, especially in combination with cytotoxic drugs.

Pharmacological characterization of second generation FXR agonists as effective EphA2 antagonists: A successful application of target hopping approach.

It is well demonstrated the key role of Eph-ephrin system, specifically of EphA2 receptor, in supporting tumor growth, invasion, metastasis and neovascularization. We previously identified FXR agonists as eligible antagonists of Eph-ephrin system. Herein we characterize new commercially available FXR (Farnesoid X Receptor) agonists as potential Eph ligands including Cilofexor, Nidufexor, Tropifexor, Turofexorate isopropyl and Vonafexor. Our exploration based on molecular modelling investigations and binding assays shows that Cilofexor binds specifically and reversibly to EphA2 receptor with a Ki value in the low micromolar range. Furthermore, Cilofexor interferes with the phosphorylation of EphA2 and the cell retraction and rounding in PC3 prostate cancer cells, both events depending on EphA2 activation. In conclusion, we can confirm that target hopping can be a successful approach to discover new moiety of protein-protein inhibitors.

A photosensitizing fusion protein with targeting capabilities.

The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed.

Protein-Protein Interaction Inhibitors Targeting the Eph-Ephrin System with a Focus on Amino Acid Conjugates of Bile Acids.

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings.

Cholenic acid derivative UniPR1331 impairs tumor angiogenesis via blockade of VEGF/VEGFR2 in addition to Eph/ephrin.

Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3ß-hydroxy-Δ5-cholenic acid derivative, which is already known to inhibit Eph-ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ5-cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds.

UniPR1331: Small Eph/Ephrin Antagonist Beneficial in Intestinal Inflammation by Interfering with Type-B Signaling.

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn's disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses.

Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Metabolic Soft Spot and Pharmacokinetics: Functionalization of C-3 Position of an Eph-Ephrin Antagonist Featuring a Bile Acid Core as an Effective Strategy to Obtain Oral Bioavailability in Mice.

UniPR129, an L-ß-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph-ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2-M7, mono-hydroxylated metabolites, M8-M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph-ephrin antagonists.

Excipient-free pulmonary insulin dry powder: Pharmacokinetic and pharmacodynamics profiles in rats.

A novel pure insulin spray-dried powder for DPI product (Ins_SD) was studied with respect to physico-chemical stability, in vitro respirability, bioavailability, activity and tolerability. Ins_SD powder exhibited a very high in vitro respirability, independently of the DPI product preparation (manual or semi-automatic). Physico-chemical characteristics of Ins_SD powder remained within the pharmacopoeia limits during 6 months of storage at room temperature. PK/PD profiles were measured in rats that received the pulmonary powders by intratracheal insufflation and compared with Afrezza inhalation insulin. Due to the low drug powder mass to deliver, both insulin powders were diluted with mannitol. Insulin from Ins_SD was promptly absorbed (tmax 15 min and Cmaxx4.9 ±â€¯1.5 mU/ml). Afrezza had a slower absorption (tmax 30 min and Cmax of 1.8 ±â€¯0.37 mU/ml). After glucose injection, Ins_SD determined a rapid reduction of glucose level, similar to Afrezza. As reference, insulin subcutaneous injection showed a long-lasting hypoglycemic effect due to the slow absorption that prolonged insulin plasma level. In summary, Ins_SD product is suitable for post-prandial glucose control, providing a convenient and compliant product, in particular in the event of using a disposable device. Albeit the product has to be stored in fridge, its stability at room temperature allows the diabetic individual to carry the daily dose in normal conditions.

Evaluation of the Anti-Tumor Activity of Small Molecules Targeting Eph/Ephrins in APC min/J Mice.

The Eph receptors are the largest receptors tyrosine kinases (RTKs) family in humans and together with ephrin ligands constitute a complex cellular communication system often dysregulated in many tumors. The role of the Eph-ephrin system in colorectal cancer (CRC) has been investigated and different expression of Eph receptors have been associated with tumor development and progression. In light of this evidence, we investigated if a pharmacological approach aimed at inhibiting Eph/ephrin interaction through small molecules could prevent tumor growth in APC min/J mice. The 8-week treatment with the Eph-ephrin antagonist UniPR129 significantly reduced the number of adenomas in the ileum and decreased the diameter of adenomas in the same region. Overall our data suggested as UniPR129 could be able to slow down the tumor development in APC min/J mice. These results further confirm literature data about Eph kinases as a new valuable target in the intestinal cancer and for the first time showed the feasibility of the Eph-ephrin inhibition as a useful pharmacological approach against the intestinal tumorigenesis. In conclusion this work paves the way for further studies with Eph-ephrin inhibitors in order to confirm the Eph antagonism as innovative pharmacological approach with preventive benefit in the intestinal tumor development.

Ephrin or not? Six tough questions on Eph targeting.

Introduction: The Eph-ephrin is a cell-cell communication system generating a forward signal in cell expressing Eph receptors and a reverse signal in ephrin-ligand expressing cells. While clearly involved in the insurgence and progression of cancer, the understanding of the molecular mechanisms regulated by this system needs development; this is a hurdle to the development of therapeutic strategies that can target the Eph receptors and/or their ephrin ligands.Areas covered: We have taken the opportunity to share some key questions on the most effective strategies to target the Eph-ephrin system. This article is based on our experience of the field and therefore is a Perspective and not comprehensive examination of the literature.Expert opinion: Targeting of the Eph-ephrin system has emerged as a potentially valuable approach for cancer therapy. Pharmacological tools have been reported in the last 15 years and these include forward signaling blockers such as kinases inhibitors and antagonists of forward and reverse signaling. Also, biologics including antibodies and recombinant proteins have been developed and some have reached early clinical stages. Data deem the Eph-ephrin system as a signaling axis that is an elusive target. A better understanding of the basic pharmacology behind the activity of available agents and a comprehensive knowledge of the ephrin biology are necessary. We are looking forward to knowing the opinion of the readers.

Optimization of EphA2 antagonists based on a lithocholic acid core led to the identification of UniPR505, a new 3α-carbamoyloxy derivative with antiangiogenetic properties.

The EphA2 receptor has been validated in animal models as new target for treating tumors depending on angiogenesis and vasculogenic mimicry. In the present work, we extended our current knowledge on structure-activity relationship (SAR) data of two related classes of antagonists of the EphA2 receptor, namely 5ß-cholan-24-oic acids and 5ß-cholan-24-oyl l-ß-homotryptophan conjugates, with the aim to develop new antiangiogenic compounds able to efficiently prevent the formation of blood vessels. As a result of our exploration, we identified UniPR505, N-[3α-(Ethylcarbamoyl)oxy-5ß-cholan-24-oyl]-l-ß-homo-tryptophan (compound 14), as a submicromolar antagonist of the EphA2 receptor capable to block EphA2 phosphorylation and to inhibit neovascularization in a chorioallantoic membrane (CAM) assay.

Drug-gut microbiota metabolic interactions: the case of UniPR1331, selective antagonist of the Eph-ephrin system, in mice.

The interest on the role of gut microbiota in the biotransformation of drugs and xenobiotics has grown over the last decades and a deeper understanding of the mutual interactions is expected to help future improvements in the fields of drug development, toxicological risk assessment and precision medicine. In this paper, a microbiome drug metabolism case is presented, involving a lipophilic small molecule, N-(3ß-hydroxy-Δ5-cholen-24-oyl)-l-tryptophan, UniPR1331, active as antagonist of the Eph-ephrin system and effective in vivo in a murine orthotopic model of glioblastoma multiforme (GBM). Following the administration of a single 30 mg/kg dose (p.o.) to mice, maximal plasma levels were reached 30 min after dosing and rapidly declined thereafter. To explain the observed in vivo behaviour, in vitro phase I and II metabolism assays were conducted employing mouse and human liver subcellular fractions and profiling main metabolites by means of tandem (HPLC-ESI-MS/MS) and high resolution mass spectrometry (HPLC-ESI-HR-MS). In the presence of in vitro mouse liver fractions, UniPR1331 showed a low phase I metabolic clearance, despite the identification of a 3-oxo and several hydroxylated metabolites. Conversely, after oral administration of UniPR1331 to mice, a novel isobaric metabolite was detected that (i) was subjected, as parent UniPR1331, to enterohepatic circulation (ii) had not been previously identified in vitro in mouse liver microsomes and (iii) was not observed forming after intraperitoneal (i.p.) administration of UniPR1331. An in vitro faecal fermentation assay produced the same chemical entity supporting a major role of gut microbiota in the in vivo clearance of UniPR1331.

Enhanced photosensitizing properties of protein bound curcumin.

AIMS: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors. MATERIALS AND METHODS: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging. KEY FINDINGS: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case. SIGNIFICANCE: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors.

Targeting the Eph/Ephrin System as Anti-Inflammatory Strategy in IBD.

Besides their long-known critical role in embryonic growth and in cancer development and progression, erythropoietin-producing hepatocellular carcinoma type B (EphB) receptor tyrosine kinases and their ephrin-B ligands are involved in the modulation of immune responses and in remodeling and maintaining the integrity of the intestinal epithelial layer. These processes are critically involved in the pathogenesis of inflammatory-based disorders of the gut, like inflammatory bowel diseases (IBDs). Accordingly, our aim was to investigate the role of the EphB/ephrin-B system in intestinal inflammation by assessing the local and systemic effects produced by its pharmacological manipulation in 2,4,6-trinitrobenzenesulfonic acid (TNBS)- (Th1-dependent model) and dextran sulphate sodium (DSS)- (innate response model) induced colitis in mice. To this purpose, we administered chimeric Fc-conjugated proteins, allegedly able to uni-directionally activate either forward (ephrin-B1-Fc) or reverse (EphB1-Fc) signaling, and the soluble monomeric EphB4 extracellular domain protein, that, simultaneously interfering with both signaling pathways, acts as EphB/ephrin-B antagonist.The blockade of the EphB/ephrin-B forward signaling by EphB4 and EphB1-Fc was ineffective against DSS-induced colitis while it evoked remarkable beneficial effects against TNBS colitis: it counteracted all the evaluated inflammatory responses and the changes elicited on splenic T lymphocytes subpopulations, without preventing the appearance of a splice variant of ephrin-B2 gene elicited by the haptenating agent in the colon. Interestingly, EphB4, preferentially displacing EphB4/ephrin-B2 interaction over EphB1/ephrin-B1 binding, was able to promote Tumor Necrosis Factor alpha (TNFα) release by splenic mononuclear cells in vitro. On the whole, the collected results point to a potential role of the EphB/ephrin-B system as a pharmacological target in intestinal inflammatory disorders and suggest that the therapeutic efficacy of its blockade seemingly works through the modulation of immune responses, independent of the changes at the transcriptional and translational level of EphB4 and ephrin-B2 genes.

Hypericin-Apomyoglobin: An Enhanced Photosensitizer Complex for the Treatment of Tumor Cells.

Bioavailability of photosensitizers for cancer photodynamic therapy is often hampered by their low solubility in water. Here, we overcome this issue by using the water-soluble protein apomyoglobin (apoMb) as a carrier for the photosensitizer hypericin (Hyp). The Hyp-apoMb complex is quickly uptaken by HeLa and PC3 cells at submicromolar concentrations. Fluorescence emission of Hyp-apoMb is exploited to localize the cellular distribution of the photosensitizer. The plasma membrane is rapidly and efficiently loaded, and fluorescence is observed in the cytoplasm only at later times and to a lesser extent. Comparison with cells loaded with Hyp alone demonstrates that the uptake of the photosensitizer without the protein carrier is a slower, less efficient process, that involves the whole cell structure without preferential accumulation at the plasma membrane. Cell viability assays demonstrate that the Hyp-apoMb exhibits superior performance over Hyp. Similar results were obtained using tumor spheroids as three-dimensional cell culture models.