Blockade of Spinal EphA4 Reduces Chronic Inflammatory Pain in Mice.

Background: Erythropoietin-producing hepatocellular (Ephs) receptor and their ligands, ephrins, orchestrate the induction of cell proliferation and migration, axonal guidance, synaptic genesis and synaptic plasticity in the central nervous system. Previous studies demonstrated that EphBs/ephrinBs participate in the pathophysiology of neuropathic pain, inflammatory pain and bone cancer pain, but the role of EphA4 in the regulation of pain in the spinal cord is unknown. Therefore, we explored the role of EphA4 receptor in regulating chronic inflammatory pain.Methods: We established a mouse model of chronic inflammatory pain through plantar injection of complete freund's adjuvant (CFA) and assessed EphA4 expression in spinal cord by western blotting. EphA4 receptor was blocked by intrathecal injection of EphA4-Fc, an EphA4 antagonist, and pain behaviors were measured by assessing thermal hyperalgesia and mechanical allodynia. Finally, immunohistochemistry was performed to analyze the changes in the expression of Fos protein in spinal cord after blocking EphA4 receptor.Results: Plantar injection of CFA produced persistent thermal hyperalgesia and mechanical allodynia, which was accompanied by significant increases in spinal EphA4 and Fos expression. Blocking spinal EphA4 receptor suppressed CFA-induced pain behaviors and reduced the expression of Fos protein in spinal cord.Conclusions: Our study demonstrated that EphA4 receptor is involved in the generation and maintenance of CFA-induced chronic inflammatory pain and that blocking the spinal EphA4 receptor could relieve persistent pain behaviors in mice.

Environmental enrichment during the chronic phase after experimental stroke promotes functional recovery without synergistic effects of EphA4 targeted therapy.

Worldwide, stroke is the main cause of long-term adult disability. After the initial insult, most patients undergo a subacute period with intense plasticity and rapid functional improvements. This period is followed by a chronic phase where recovery reaches a plateau that is only partially modifiable by rehabilitation. After experimental stroke, various subacute rehabilitation paradigms improve recovery. However, in order to reach the best possible outcome, a combination of plasticity-promoting strategies and rehabilitation might be necessary. EphA4 is a negative axonal guidance regulator during development. After experimental stroke, reduced EphA4 levels improve functional outcome with similar beneficial effects upon the inhibition of EphA4 downstream targets. In this study, we assessed the effectiveness of a basic enriched environment in the chronic phase after photothrombotic stroke in mice as well as the therapeutic potential of EphA4 targeted therapy followed by rehabilitation. Our findings show that environmental enrichment in the chronic phase improves functional outcome up to 2 months post-stroke. Although EphA4 levels increase after experimental stroke, subacute EphA4 inhibition followed by environmental enrichment does not further increase recovery. In conclusion, we show that environmental enrichment during the chronic phase of stroke improves functional outcome in mice with no synergistic effects of the used EphA4 targeted therapy.

Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs.

The receptor tyrosine kinase, erythropoietin-producing hepatocellular A4 (EphA4), was recently identified as a molecular target for Alzheimer's disease (AD). We found that blockade of the interaction of the receptor and its ligands, ephrins, alleviates the disease phenotype in an AD transgenic mouse model, suggesting that targeting EphA4 is a potential approach for developing AD interventions. In this study, we identified five FDA-approved drugs-ergoloid, cyproheptadine, nilotinib, abiraterone, and retapamulin-as potential inhibitors of EphA4 by using an integrated approach combining virtual screening with biochemical and cellular assays. We initially screened a database of FDA-approved drugs using molecular docking against the ligand-binding domain of EphA4. Then, we selected 22 candidate drugs and examined their inhibitory activity towards EphA4. Among them, five drugs inhibited EphA4 clustering induced by ephrin-A in cultured primary neurons. Specifically, nilotinib, a kinase inhibitor, inhibited the binding of EphA4 and ephrin-A at micromolar scale in a dosage-dependent manner. Furthermore, nilotinib inhibited the activation of EphA4 and EphA4-dependent growth cone collapse in cultured hippocampal neurons, demonstrating that the drug exhibits EphA4 inhibitory activity in cellular context. As demonstrated in our combined computational and experimental approaches, repurposing of FDA-approved drugs to inhibit EphA4 may provide an alternative fast-track approach for identifying and developing new treatments for AD.

Antisense-mediated reduction of EphA4 in the adult CNS does not improve the function of mice with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal adult onset motor neuron disease characterized by progressive denervation and subsequent motor impairment. EphA4, a negative regulator of axonal growth, was recently identified as a genetic modifier in fish and rodent models of ALS. To evaluate the therapeutic potential of EphA4 for ALS, we examined the effect of CNS-directed EphA4 reduction in preclinical mouse models of ALS, and assessed if the levels of EPHA4 mRNA in blood correlate with disease onset and progression in human ALS patients. We developed antisense oligonucleotides (ASOs) to specifically reduce the expression of EphA4 in the central nervous system (CNS) of adult mice. Intracerebroventricular administration of an Epha4-ASO in wild-type mice inhibited Epha4 mRNA and protein in the brain and spinal cord, and promoted re-innervation and functional recovery after sciatic nerve crush. In contrast, lowering of EphA4 in the CNS of two mouse models of ALS (SOD1G93A and PFN1G118V) did not improve their motor function or survival. Furthermore, the level of EPHA4 mRNA in human blood correlated weakly with age of disease onset, and it was not a significant predictor of disease progression as measured by ALS Functional Rating Scores (ALSFRS). Our data demonstrates that lowering EphA4 in the adult CNS may not be a stand-alone viable strategy for treating ALS.

Exosomal MicroRNA-10a Is Associated with Liver Regeneration in Rats through Downregulation of EphA4.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play vital roles in liver regeneration. Previous studies mainly focused on the functions of intracellular miRNAs, while the functions of circulating exosomal miRNAs in liver regeneration remain largely unknown. The aim of this study was to identify the key exosomal miRNA that played vital roles in liver regeneration. METHODS: The Sprague-Dawley male rats were assigned to 70% partially hepatectomized group (n = 6) and sham surgery group (n = 6). The peripheral blood of both groups was collected 24 h after surgery. The exosomal miRNAs were extracted, and microarray was used to find out the key miRNA implicated in liver regeneration. Adenovirus was used to overexpress the key miRNA in rats, and proliferating cell nuclear antigen (PCNA) staining was applied to study the effect of key miRNA overexpression on liver regeneration. Western blotting was used to validate the predicted target of the key miRNA. RESULTS: Exosomal miR-10a was upregulated more than nine times in hepatectomized rats. The level of miR-10a was increased in the early phase of liver regeneration, reached the top at 72 h postsurgery, and decreased to perioperative level 168 h after surgery. Moreover, enforced expression of miR-10a by adenovirus facilitated the process of liver regeneration as evidenced by immunohistochemical staining of PCNA. Erythropoietin-producing hepatocellular receptor A4 (EphA4) has been predicted to be a target of miR-10a. The protein level of EphA4 was decreased in the early phase of liver regeneration, reached the bottom at 72 h postsurgery, and rose to perioperative level 168 h after surgery, which was negatively correlated with miR-10a, confirming that EphA4 served as a downstream target of miR-10a. Moreover, inhibition of EphA4 by rhynchophylline could promote the proliferation of hepatocytes by regulating the cell cycle. CONCLUSION: Exosomal miR-10a might accelerate liver regeneration through downregulation of EphA4.

EPHA4-FC TREATMENT REDUCES ISCHEMIA/REPERFUSION-INDUCED INTESTINAL INJURY BY INHIBITING VASCULAR PERMEABILITY.

The inflammatory response is characterized by increased endothelial permeability, which permits the passage of fluid and inflammatory cells into interstitial spaces. The Eph/ephrin receptor ligand system plays a role in inflammation through a signaling cascade, which modifies Rho-GTPase activity. We hypothesized that blocking Eph/ephrin signaling using an EphA4-Fc would result in decreased inflammation and tissue injury in a model of ischemia/reperfusion (I/R) injury. Mice undergoing intestinal I/R pretreated with the EphA4-Fc had significantly reduced intestinal injury compared to mice injected with the control Fc. This reduction in I/R injury was accompanied by significantly reduced neutrophil infiltration, but did not affect intestinal inflammatory cytokine generation. Using microdialysis, we identified that intestinal I/R induced a marked increase in systemic vascular leakage, which was completely abrogated in EphA4-Fc-treated mice. Finally, we confirmed the direct role of Eph/ephrin signaling in endothelial leakage by demonstrating that EphA4-Fc inhibited tumor necrosis factor-α-induced vascular permeability in human umbilical vein endothelial cells. This study identifies that Eph/ephrin interaction induces proinflammatory signaling in vivo by inducing vascular leak and neutrophil infiltration, which results in tissue injury in intestinal I/R. Therefore, therapeutic targeting of Eph/ephrin interaction using inhibitors, such as EphA4-Fc, may be a novel method to prevent tissue injury in acute inflammation by influencing endothelial integrity and by controlling vascular leak.

Inhibition of Eph receptor A4 by 2,5-dimethylpyrrolyl benzoic acid suppresses human pancreatic cancer growing orthotopically in nude mice.

Ephrin receptor A4 (EphA4) is overexpressed in human pancreatic adenocarcinoma (PDAC) and activate cell growth. Recent studies have identified small molecules that block EphA4. In this study, we investigated the correlation between EphA4 expression and the prognosis of patients with PDAC. We also examined the cytostatic efficacy of 2,5-dimethylpyrrolyl benzoic acid (compound 1), a small molecule that blocks EphA4, in PDAC cells. Overall survival of patients with EphA4 positivity was significantly shorter than that of patients with EphA4 negativity (P = 0.029). In addition, multivariate analysis revealed that EphA4 expression was an independent prognostic factor in PDAC patients (P = 0.039). Compound 1 showed a cytostatic efficacy in PDAC cells expressing EphA4 in vitro and in vivo. Our study indicated that compound 1 suppressed both EphA4 and Akt phosphorylations, and induced apoptosis in PDAC cells expressing EphA4. In conclusion, compound 1 has a high potential as a therapeutic agent for patients with PDAC.

Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells.

Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.

High-Throughput Screening by Nuclear Magnetic Resonance (HTS by NMR) for the Identification of PPIs Antagonists.

In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.

Dynamic principle for designing antagonistic/agonistic molecules for EphA4 receptor, the only known ALS modifier.

Additional to involvement in diverse physiological and pathological processes such as axon regeneration, synaptic plasticity, and cancers, EphA4 receptor has been recently identified as the only amyotrophic lateral sclerosis (ALS) modifier. Previously, we found that two small molecules bind the same EphA4 channel at almost equivalent affinities but mysteriously trigger opposite signaling outputs: one activated but another inhibited. Here, we determined the solution structure of the 181-residue EphA4 LBD, which represents the first for 16 Eph receptors. Further NMR dynamic studies deciphered that the agonistic and antagonistic effects of two small molecules are dynamically driven, which are achieved by oppositely modulating EphA4 dynamics. Consequently, in design of drugs to target EphA4, the dynamic requirement also needs to be satisfied in addition to the classic criteria. For example, to increase the survival of ALS patients by inhibiting EphA4, the drugs must enhance, or at least not suppress, the EphA4 dynamics.

Are we using the right pharmacological tools to target EphA4?

The EphA4 receptor has been proposed to be a key actor in neurodegenerative diseases. In the last years, several research groups focused their efforts on the discovery of small molecules capable of blocking EphA4 activity by binding its extracellular domain. However, none of the compounds so far identified possess adequate chemical and/or pharmacological profiles to assess the "druggability" of EphA4 in animal models. New efforts are required to deliver a new generation of suitable pharmacological tools.

Development and structural analysis of a nanomolar cyclic peptide antagonist for the EphA4 receptor.

The EphA4 receptor is highly expressed in the nervous system, and recent findings suggest that its signaling activity hinders neural repair and exacerbates certain neurodegenerative processes. EphA4 has also been implicated in cancer progression. Thus, EphA4 inhibitors represent potential therapeutic leads and useful research tools to elucidate the role of EphA4 in physiology and disease. Here, we report the structure of a cyclic peptide antagonist, APY, in complex with the EphA4 ligand-binding domain (LBD), which represents the first structure of a cyclic peptide bound to a receptor tyrosine kinase. The structure shows that the dodecameric APY efficiently occupies the ephrin ligand-binding pocket of EphA4 and promotes a "closed" conformation of the surrounding loops. Structure-guided relaxation of the strained APY ß-turn and amidation of the C terminus to allow an additional intrapeptide hydrogen bond yielded APY-ßAla8.am, an improved APY derivative that binds to EphA4 with nanomolar affinity. APY-ßAla8.am potently inhibits ephrin-induced EphA4 activation in cells and EphA4-dependent neuronal growth cone collapse, while retaining high selectivity for EphA4. The two crystal structures of APY and APY-ßAla8.am bound to EphA4, in conjunction with secondary phage display screens, highlighted peptide residues that are essential for EphA4 binding as well as residues that can be modified. Thus, the APY scaffold represents an exciting prototype, particularly since cyclic peptides have potentially favorable metabolic stability and are emerging as an important class of molecules for disruption of protein-protein interactions.

Acute delivery of EphA4-Fc improves functional recovery after contusive spinal cord injury in rats.

Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury. Using a ledged tapered balance beam and open-field testing, we observed significant improvements in recovery of locomotor function after EphA4-Fc treatment. Consistent with functional improvement, using high-resolution ex vivo magnetic resonance imaging at 16.4T, we found that rats treated with EphA4-Fc had a significantly increased cross-sectional area of the dorsal funiculus caudal to the injury epicenter compared with controls. Our findings indicate that EphA4-Fc promotes functional recovery following contusive spinal cord injury and provides further support for the therapeutic benefit of treatment with the competitive antagonist in acute cases of spinal cord injury.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Inhibition of Eph receptor-ephrin ligand interaction by tea polyphenols.

Tea contains a variety of bioactive chemicals, such as catechins and other polyphenols. These compounds are thought to be responsible for the health benefits of tea consumption by affecting the function of many cellular targets, not all of which have been identified. In a high-throughput screen for small molecule antagonists of the EphA4 receptor tyrosine kinase, we identified five tea polyphenols that substantially inhibit EphA4 binding to a synthetic peptide ligand. Further characterization of theaflavin monogallates from black tea and epigallocatechin-3,5-digallate from green tea revealed that these compounds at low micromolar concentrations also inhibit binding of the natural ephrin ligands to EphA4 and several other Eph receptors in in vitro assays. The compounds behave as competitive EphA4 antagonists, and their inhibitory activity is affected by amino acid mutations within the ephrin binding pocket of EphA4. In contrast, the major green tea catechin, epigallocatechin-3-gallate (EGCG), does not appear to be an effective Eph receptor antagonist. In cell culture assays, theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced tyrosine phosphorylation (activation) of Eph receptors and endothelial capillary-like tube formation. However, the wider spectrum of Eph receptors affected by the tea derivatives in cells suggests additional mechanisms of inhibition besides interfering with ephrin binding. These results show that tea polyphenols derived from both black and green tea can suppress the biological activities of Eph receptors. Thus, the Eph receptor tyrosine kinase family represents an important class of targets for tea-derived phytochemicals.

High-content analysis of proapoptotic EphA4 dependence receptor functions using small-molecule libraries.

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.

Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.

The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. In the present study we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrated that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical-shift changes observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4, binding. The peptides exhibit a long half-life in cell culture medium which, with their substantial binding affinity and selectivity for EphA4, makes them excellent research tools to modulate EphA4 function.

Fragment based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase.

The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in silico screening procedure which was focused on finding novel, EPHA4 hinge binding fragments. This resulted in the identification of 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine derivatives as EPHA4 inhibitors. Hit exploration yielded a compound with 2 µM (IC(50)) affinity for the EPHA4 receptor tyrosine kinase domain. Soaking experiments into a crystal of the EPHA4 kinase domain gave a 2.11Å X-ray structure of the EPHA4 - inhibitor complex, which confirmed the binding mode of the scaffold as proposed by the initial in silico work. The results underscore the strength of fragment based in silico screening as a tool for the discovery of novel lead compounds as small molecule kinase inhibitors.

Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state.

The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors.

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice.

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.