NMR-Guided Design of Potent and Selective EphA4 Agonistic Ligands.

In this paper, we applied an innovative nuclear magnetic resonance (NMR)-guided screening and ligand design approach, named focused high-throughput screening by NMR (fHTS by NMR), to derive potent, low-molecular-weight ligands capable of mimicking interactions elicited by ephrin ligands on the receptor tyrosine kinase EphA4. The agents bind with nanomolar affinity, trigger receptor activation in cellular assays with motor neurons, and provide remarkable motor neuron protection from amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Structural studies on the complex between EphA4 ligand-binding domain and a most active agent provide insights into the mechanism of the agents at a molecular level. Together with preliminary in vivo pharmacology studies, the data form a strong foundation for the translation of these agents for the treatment of ALS and potentially other human diseases.

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.

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.

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.

The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor.

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.

Eph receptor tyrosine kinases regulate astrocyte cytoskeletal rearrangement and focal adhesion formation.

EphA4 null mice have impaired astrocytic gliosis following spinal cord injury. This may be because of altered cytoskeletal regulation and is examined herein using cultured astrocytes from wildtype and EphA4 null mice. Under basal conditions EphA4 null astrocytes appeared relatively normal but following stimuli resulting in cytoskeletal rearrangement, EphA4 null cells responded more slowly. When F-actin stress fibers were collapsed using the Rho kinase inhibitor HA1077, fewer EphA4 null cells showed stress fiber collapse in response to HA1077 and recovered stress fibers more slowly following HA1077 removal. EphA4 null astrocytes were less adherent and had smaller focal adhesions, while activation of Eph receptors with ephrin-A5-Fc increased the numbers of focal adhesions in both wildtype and knockout astrocytes following serum starvation. Using scratch wound assays, EphA4 null astrocytes invading the scratch showed impaired glial fibrillary acidic protein expression, particularly in proliferative cells. Astrocytes did not express Ephexin, a major Eph-interacting Rho guanine exchange factor, but they expressed Vav proteins, with lower levels of phospho-Vav in EphA4 null compared to wildtype astrocytes. This may contribute to the slower cytoskeletal responses generally observed in the EphA4 null astrocytes. Eph receptor signaling therefore regulates astrocyte reactivity through modulation of cytoskeletal responses.