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.

Probing the effects of MR120 in preclinical chronic colitis: A first-in-class anti-IBD agent targeting the CCL20/CCR6 axis.

Concerning the growing interest in the role played by the CCL20/CCR6 axis in IBD pathogenesis and in the search for novel anti-IBD small molecules, we have recently discovered the first small-molecule (MR120) endowed with protective action against TNBS-induced colitis and zymosan-induced peritonitis. This protective action occurs through interference with the CCL20/CCR6 signaling. The aim of the present work is to expand the preclinical investigation of MR120, evaluating its beneficial anti-inflammatory effect on a model of chronic colitis obtained by cyclically exposing C57BL/6 mice to 3% DSS. Subcutaneous administration of MR120 at 1 mg/kg, the same dose effective against acute inflammation, helped attenuate several systemic and local inflammatory responses induced by DSS. Besides significantly improving murine health conditions, MR120 counteracted mucosal macroscopic injury, the increase of colonic edema and neutrophils oxidative activity, and mitigated spleen enlargement, while not significantly lowering intestinal IL-6 concentration. Overall, repeated daily treatment with MR120 for approximately 30 days was well tolerated and showed moderate protection in a relevant model of chronic colitis, in line with the beneficial effect previously observed in acute models of intestinal inflammation. Although more potent analogues of MR120 will be needed to more fully evaluate their clinical translatability, the present work provides a valuable example of in vivo efficacy of CCL20/CCR6 modulators in a chronic model of IBD.

Recombinant Alpha-1 Antitrypsin as Dry Powder for Pulmonary Administration: A Formulative Proof of Concept.

Alpha-1 antitrypsin (AAT) deficiency is a genetic disorder associated with pulmonary emphysema and bronchiectasis. Its management currently consists of weekly infusions of plasma-purified human AAT, which poses several issues regarding plasma supplies, possible pathogen transmission, purification costs, and parenteral administration. Here, we investigated an alternative administration strategy for augmentation therapy by combining recombinant expression of AAT in bacteria and the production of a respirable powder by spray drying. The same formulation approach was then applied to plasma-derived AAT for comparison. Purified, active, and endotoxin-free recombinant AAT was produced at high yields and formulated using L-leucine and mannitol as excipients after identifying compromise conditions for protein activity and good aerodynamic performances. An oxygen-free atmosphere, both during formulation and powder storage, slowed down methionine-specific oxidation and AAT inactivation. This work is the first peer-reviewed report of AAT formulated as a dry powder, which could represent an alternative to current treatments.

Discovery of small-molecules targeting the CCL20/CCR6 axis as first-in-class inhibitors for inflammatory bowel diseases.

The CCL20/CCR6 axis is implicated in the migration of CCR6+ immune cells towards CCL20, its sole ligand, whose expression is increased during inflammatory processes and is known to play a pivotal role in triggering different autoimmune-mediated inflammatory diseases. Herein, we report a drug discovery effort focused on the development of a new pharmacological approach for the treatment of inflammatory bowel diseases (IBDs) based on small-molecule CCR6 antagonists. The most promising compound 1b was identified by combining in silico studies, sustainable chemistry and in vitro functional/targeted assays, and its efficacy was finally validated in a classic murine model of colitis (TNBS-induced) and in a model of peritonitis (zymosan-induced). These data provide the proof of principle that a pharmacological modulation of the CCL20/CCR6 axis may indeed represent the first step for the development of an orally bioavailable drug candidate for the treatment of IBD and, potentially, other diseases regulated by the CCL20/CCR6 axis.

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.

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.

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.

Increased Mucosal Thrombin is Associated with Crohn's Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation.

BACKGROUND AND AIMS: Thrombin levels in the colon of Crohn's disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn's disease. METHODS: Thrombin activity was studied in tissues from Crohn's disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid. RESULTS: Active forms of thrombin were increased in Crohn's disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models. CONCLUSIONS: Our data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Impact of S1P Mimetics on Mesenteric Ischemia/Reperfusion Injury.

Mesenteric ischemia/reperfusion (I/R), following the transient deprivation of blood flow to the gut, triggers an acute flogistic process involving the disruption of endothelial and epithelial barriers integrity, the activation of immune cells, and the abundant release of inflammatory mediators. Among them, the lipid mediator sphingosine-1-phosphate (S1P) is involved in maintaining epithelial and endothelial barrier integrity and in governing the migration of immune cells through the interaction with S1P1-5 receptors. Therefore, the present work aims to investigate the involvement of S1P signaling in intestinal I/R-induced injury by studying the effects of FTY720, the non-selective S1P1,3-5 agonist, and comparing them with the responses to ozanimod, selective S1P1,5 agonist, in a murine model of gut I/R. Intestinal edema, gut and lung neutrophil infiltration, and oxidative stress were evaluated through biochemical and morphological assays. The collected results highlight the protective action of FTY720 against the inflammatory cascade elicited by mesenteric I/R injury, mainly through the control of vascular barrier integrity. While these beneficial effects were mimicked by ozanimod and can be therefore attributed largely to the effects exerted by FTY720 on S1P1, the recruitment of myeloid cells to the injured areas, limited by FTY720 but not by ozanimod, rather suggests the involvement of other receptor subtypes.

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.

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIß and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.

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.