A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain.

The Eph receptor tyrosine kinases and their ephrin ligands regulate many physiological and pathological processes. EphA4 plays important roles in nervous system development and adult homeostasis, while aberrant EphA4 signaling has been implicated in neurodegeneration. EphA4 may also affect cancer malignancy, but the regulation and effects of EphA4 signaling in cancer are poorly understood. A correlation between decreased patient survival and high EphA4 mRNA expression in melanoma tumors that also highly express ephrinA ligands suggests that enhanced EphA4 signaling may contribute to melanoma progression. A search for EphA4 gain-of-function mutations in melanoma uncovered a mutation of the highly conserved leucine 920 in the EphA4 sterile alpha motif (SAM) domain. We found that mutation of L920 to phenylalanine (L920F) potentiates EphA4 autophosphorylation and signaling, making it the first documented EphA4 cancer mutation that increases kinase activity. Quantitative Föster resonance energy transfer and fluorescence intensity fluctuation (FIF) analyses revealed that the L920F mutation induces a switch in EphA4 oligomer size, from a dimer to a trimer. We propose this switch in oligomer size as a novel mechanism underlying EphA4-linked tumorigenesis. Molecular dynamics simulations suggest that the L920F mutation alters EphA4 SAM domain conformation, leading to the formation of EphA4 trimers that assemble through two aberrant SAM domain interfaces. Accordingly, EphA4 wild-type and the L920F mutant are affected differently by the SAM domain and are differentially regulated by ephrin ligand stimulation. The increased EphA4 activation induced by the L920F mutation, through the novel mechanism we uncovered, supports a functional role for EphA4 in promoting pathogenesis.

Glycoengineering of EphA4 Fc leads to a unique, long-acting and broad spectrum, Eph receptor therapeutic antagonist.

Eph receptors have emerged as targets for therapy in both neoplastic and non-neoplastic disease, however, particularly in non-neoplastic diseases, redundancy of function limits the effectiveness of targeting individual Eph proteins. We have shown previously that a soluble fusion protein, where the EphA4 ectodomain was fused to IgG Fc (EphA4 Fc), was an effective therapy in acute injuries and demonstrated that EphA4 Fc was a broad spectrum Eph/ephrin antagonist. However, a very short in vivo half-life effectively limited its therapeutic development. We report a unique glycoengineering approach to enhance the half-life of EphA4 Fc. Progressive deletion of three demonstrated N-linked sites in EphA4 progressively increased in vivo half-life such that the triple mutant protein showed dramatically improved pharmacokinetic characteristics. Importantly, protein stability, affinity for ephrin ligands and antagonism of cell expressed EphA4 was fully preserved, enabling it to be developed as a broad spectrum Eph/ephrin antagonist for use in both acute and chronic diseases.

Identification and characterization of Nanobodies targeting the EphA4 receptor.

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role.

Potent and Selective EphA4 Agonists for the Treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity. The molecules exhibit excellent selectivity and display efficacy in a SOD1 mutant mouse model of ALS. Interestingly, the molecules appear to act as agonists for the receptor in certain surrogate cellular assays. While the exact mechanisms responsible for the therapeutic effect of the new agonists remain to be elucidated, we believe that the described agent represents both an invaluable pharmacological tool to further decipher the role of the EphA4 in ALS and potentially other human diseases, and a significant stepping stone for the development of novel treatments.

Antagonistic molecular interactions of photosynthetic pigments with molecular disease targets: a new approach to treat AD and ALS.

Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are progressive neurodegenerative diseases that affect the neurons in the brain and the spinal cord. Neuroinflamation and apoptosis are key players in the progressive damage of the neurons in AD and ALS. Currently, there is no drug to offer complete cure for both these diseases. Riluzole is the only available drug that can prolong the life time of the ALS patients for nearly 3 months. Molecules that offer good HIT to the molecular targets of ALS will help to treat AD and ALS patients. P53 kinase receptor (4AT3), EphA4 (3CKH) and histone deacetylase (3SFF) are the promising disease targets of AD and ALS. This paper discusses on a new approach to combat neurodegenerative diseases using photosynthetic pigments. The docking studies were performed with the Autodock Vina algorithm to predict the binding of the natural pigments such as ß carotene, chlorophyll a, chlorophyll b, phycoerythrin and phycocyanin on these targets. The ß carotene, phycoerythrin and phycocyanin had higher binding energies indicating the antagonistic activity to the disease targets. These pigments serve as a potential therapeutic molecule to treat neuroinflammation and apoptosis in the AD and ALS patients.

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.

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.

Sizes of interface residues account for cross-class binding affinity patterns in Eph receptor-ephrin families.

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell-cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin-As (Bowden et al., Structure 2009;17:1386-1397); and EphB2, which can bind ephrin-A5 in addition to the ephrin-Bs (Himanen et al., Nat Neurosci 2004;7:501-509). A crystal structure is available of the interacting domains of the EphA4-ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386-1397). In this complex, the ligand-binding domain of EphA4 adopts an EphB-like conformation. To understand why other cross-class EphA receptor-ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4-ephrinB1, (2) EphA4-ephrinB3, and (3) EphA2-ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity.

Discovery and characterization of a novel cyclic peptide that effectively inhibits ephrin binding to the EphA4 receptor and displays anti-angiogenesis activity.

The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.

Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5.

Eph receptor tyrosine kinases and their ephrin ligands mediate cell signaling during normal and oncogenic development. Eph signaling is initiated in a multistep process leading to the assembly of higher-order Eph/ephrin clusters that set off bidirectional signaling in interacting cells. Eph and ephrins are divided in two subclasses based on their abilities to bind and activate each other and on sequence conservation. EphA4 is an exception to the general rule because it can be activated by both A- and B-class ephrin ligands. Here we present high-resolution structures of the complete EphA4 ectodomain and its complexes with ephrin-A5. The structures reveal how ligand binding promotes conformational changes in the EphA4 ligand-binding domain allowing the formation of signaling clusters at the sites of cell-cell contact. In addition, the structural data, combined with structure-based mutagenesis, reveal a previously undescribed receptor-receptor interaction between the EphA4 ligand-binding and membrane-proximal fibronectin domains, which is functionally important for efficient receptor activation.