Small-Molecule Ligands that Bind the RET Receptor Activate Neuroprotective Signals Independent of but Modulated by Coreceptor GFRα1.

Glial cell line-derived neurotrophic factor (GDNF) binds the GFRα1 receptor, and the GDNF-GFRα1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFRα1-RET signaling complex, agents that bind and activate RET directly and independently of GFRα1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFRα1 coexpression. However, RET activation by these ligands is constrained by GFRα1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.

Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors.

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy.

Many neurodegenerative retinal diseases are treated with monoclonal antibodies (mAb) delivered by invasive intravitreal injection (IVT). In Diabetic Retinopathy there is a scarcity of effective agents that can be delivered using non-invasive methods, and there are significant challenges in the validation of novel therapeutic targets. ProNGF represents a potential novel target, and IVT administration of a function-blocking anti-proNGF mAb is therapeutic in a mouse model of DR. We therefore compared invasive IVT to less invasive systemic intravenous (IV) and local subconjunctival (SCJ) administration, for therapy of Diabetic Retinopathy. The IV and SCJ routes are safe, afford sustained pharmacokinetics and tissue penetration of anti-proNGF mAb, and result in long-term therapeutic efficacy that blocks retinal inflammation, edema, and neuronal death. SCJ may be a more convenient and less-invasive approach for ophthalmic use and may enable reduced frequency of intervention for the treatment of retinal pathologies.

Subconjunctival Delivery of p75NTR Antagonists Reduces the Inflammatory, Vascular, and Neurodegenerative Pathologies of Diabetic Retinopathy.

Purpose: The p75NTR is a novel therapeutic target validated in a streptozotocin mouse model of diabetic retinopathy. Intravitreal (IVT) injection of small molecule p75NTR antagonist THX-B was therapeutic and resolved the inflammatory, vascular, and neurodegenerative phases of the retinal pathology. To simplify clinical translation, we sought a superior drug delivery method that circumvents risks associated with IVT injections. Methods: We compared the pharmacokinetics of a single 40 µg subconjunctival (SCJ) depot to the reported effective 5 µg IVT injections of THX-B. We quantified therapeutic efficacy, with endpoints of inflammation, edema, and neuronal death. Results: The subconjunctival depot affords retinal exposure equal to IVT injection, without resulting in detectable drug in circulation. At week 2 of diabetic retinopathy, the SCJ depot provided therapeutic efficacy similar to IVT injections, with reduced inflammation, reduced edema, reduced neuronal death, and a long-lasting protection of the retinal structure. Conclusions: Subconjunctival injections are a safe and effective route for retinal delivery of p75NTR antagonists. The subconjunctival route offers an advantageous, less-invasive, more compliant, and nonsystemic method to deliver p75NTR antagonists for the treatment of retinal diseases.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9ß1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9ß1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and ßNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9ß1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of ßNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and ßNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9ß1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9ß1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

Block-copolymer micelles as carriers of cell signaling modulators for the inhibition of JNK in human islets of Langerhans.

Here we investigate the potential of PCL-b-PEO micelles in preventing the cell death of isolated human islets of Langerhans. PCL-b-PEO micelles were loaded with c-Jun NH2-terminal kinases inhibitor SP600125 to rescue the isolated islets. Mechanistic studies of the uptake were conducted in PC12 cells. Incorporation of SP600125 afforded 8.2 fold greater solubility of SP600125 in micelle suspension. To investigate the effectiveness of micelle-incorporated SP600125 in preventing the islet cell death, we challenged the islets with TNF-alpha, IL-1, and IFN gamma. Micelle-incorporated SP600125 did not lose its inhibitory activity during incorporation into micelles, and it protected the islets against cytokine-induced loss of viability to the same extent as control SP600125. Moreover, the concentration of micelle-incorporated SP600125 used was 13-fold lower, demonstrating the greater efficacy of micelle delivered SP600125. Micelles maintained their cytoplasmic distribution without detectable nuclear localization in islets. The inhibition of JNK was confirmed by western blots. This study suggests that micelle-based intracellular delivery of potent, poorly water soluble, cell-death-pathway inhibitors may represent a valuable addition to established delivery of cytocidal block-copolymer micelle-incorporated bioactives.

Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase: a potential drug delivery route for tumor cells.

BACKGROUND: Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway. METHODOLOGY/PRINCIPAL FINDINGS: Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was negligible both in vitro and following intravenous injection in vivo where AMF/PGI-FITC was selectively internalized by subcutaneous B16-F1 tumor cells. CONCLUSIONS/SIGNIFICANCE: The raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells.

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors.

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.

HER2-mediated internalization of a targeted prodrug cytotoxic conjugate is dependent on the valency of the targeting ligand.

HER2 is a validated therapeutic target for cancer. There are no natural ligands, but monoclonal antibodies and peptides that bind HER2 act as artificial ligands, selectively affecting HER2-overexpressing tumors. One reported mechanism for this effect is receptor downregulation, but the expected correlation of ligand-dependent HER2 internalization and tumor inhibition remain poorly characterized. Moreover, HER2 ligands have limited therapeutic efficacy and often they require adjuvant treatment with the chemotherapeutic Taxol. Here, we generated a series of HER2 ligands (Anti-HER2/neu peptide ligands, AHNPmonovalent and AHNPbivalent) with different valency and correlated their internalization-promoting ability to biological potency. Since AHNPbivalent (but not AHNPmonovalent) induces rapid receptor internalization, we exploited this feature to deliver cytotoxic conjugates coupling AHNPbivalent and Taxol (Taxol . AHNPbivalent). The prodrug conjugate releases Taxol after receptor-mediated internalization, and cytotoxicity can be used as a marker of internalization. Taxol . AHNPbivalent is significantly more cytotoxic than free Taxol + free AHNPbivalent. Hence, the Taxol x AHNP(bivalent) prodrug binds to HER2, induces receptor internalization and downregulation, and the subsequent release of free Taxol inside the targeted cell results in synergistic toxicity, The effect is selective towards HER2- expressing cells. This work links HER2 receptor internalization and growth arrest, and the chemical conjugation strategy may yield improved and HER2 selective therapeutics.