In animal models of acute and chronic neurodegeneration, a TrkB agonist affords long-lasting neuroprotection by causing sustained TrkB activation. The use of structural end points could have prognostic value to evaluate neuroprotection. This work contributes to the understanding of neurotrophic mechanisms underlying RGC death in glaucoma and optic nerve axotomy.

This study was initiated to find small molecule ligands that would induce a functional response when docked with neurotrophin Trk receptors. "Minimalist" mimics of β-turns were designed for this purpose. These mimics are: (i) rigid, yet easily folded into turn-like conformations, and (ii) readily accessible from amino acids bearing most of the natural side chains. Gram quantities of sixteen of these turn mimics were prepared, then assembled into 152 fluorescein-labeled bivalent peptidomimetics via a solution-phase combinatorial method. Fluorescence-based screening of these molecules using cells transfected with the Trk receptors identified 10 potential ligands of TrkC, the receptor for neurotrophin-3 (NT-3). Analogs of these bivalent peptidomimetics with biotin replacing the fluorescein label were then prepared and tested to confirm that binding was not due to the fluorescein. Several assays were conducted to find the mode of action of these biotinylated compounds. Thus, direct binding, survival and neuritogenic, and biochemical signal transduction assays showed 8 of the original 10 hits were agonistic ligands binding to the ectodomain of TrkC. Remarkably, some peptidomimetics afford discrete signals leading to either cell survival or neuritogenic differentiation. The significance of this work is three fold. First, we succeeded in finding small, selective, proteolytically stable ligands for the TrkC receptor; there are very few of these in the literature. Second, we show that it is possible to activate distinct and biased signaling pathways with ligands binding at the ectodomain of wild type receptors. Third, the discovery that some peptidomimetics initiate different modes of cell signaling increases their potential as pharmacological probes and therapeutic leads.The neurotrophins are dimeric protein growth factors that help regulate the peripheral and central nervous systems and other tissues that express the Trk and p75 neurotrophin receptors.(1,2) Trk receptors are relatively selective for, and bind with high affinity (∼K d 10 -11 M) to the neurotrophin. Nerve growth factor (NGF) docks with TrkA, Brain Derived Neurotrophic Factor (BDNF) interacts with TrkB(3), and Neurotrophin-3 (NT-3) interacts preferentially with TrkC but can also bind TrkA and TrkB ( Figure 1a). Ligand binding induces phosphorylation (pTyr) of Trk receptors and associated signaling partners and activation of the neurotrophic biological signals: cell growth, cell survival under stress (trophic activity), and/or cellular differentiation (neuritogenic or neurogenic activity).(4-6) * Correspondence regarding design and synthesis of the peptidomimetics to burgess@mail.tamu.edu, and on the biology and pharmacology to uri.saragovi@mcgill.ca. There is also a second receptor for the neurotrophins, p75, a member of the tumor necrosis factor (TNF) receptor superfamily. All the neurotrophins also bind the p75 receptor, but with lesser affinities (10 -9 to 10 -11 M) than for the Trk receptors. (7-9) The p75 receptor can transduce signals which ...

The Combi-Targeting concept postulates that a molecule termed combi-molecule (C-molecule) with binary epidermal growth factor receptor (EGFR) targeting/DNA-damaging properties and with the ability to be hydrolyzed to another EGFR inhibitor should induce sustained antiproliferative activity in cells overexpressing EGFR. Because we postulate that the EGFR affinity of the C-molecule and that of its hydrolytic metabolites are critical parameters for sustained potency against EGFR-overexpressing cells, we synthesized BJ2000 (IC 50 ϭ 0.1 M, competitive binding at ATP site), a novel C-molecule that can decompose into a 6-amino-4-anilinoquinazoline FD105 (IC 50 ϭ 0.2 M). Studies using the EGFR-overexpressing A431 cells revealed that BJ2000 could damage DNA and block epidermal growth factor-stimulated EGFR autophosphorylation by a partially irreversible mechanism. Blockade of EGFR autophosphorylation subsequently induced inhibition of mitogen-activated protein kinase activation and c-fos gene expression. Enzyme-linked immunosorbent assay and growth factormediated stimulation of proliferation assays in the EGFRexpressing NIH3T3HER14 demonstrated the preferential EGFR-targeting properties of BJ2000, and more importantly suggest that blockade of EGFR phosphorylation by this drug translate into significant growth inhibitory effects. These properties culminated into irreversible antiproliferative effects as confirmed by a sulforhodamine B assay. Five days after a 2-h treatment, BJ2000 retained significant antiproliferative effect in A431 cells, whereas its reversible metabolite FD105 almost completely lost its activity. This result in toto lend support to the Combi-Targeting concept according to which a molecular conjugate kept small enough to interact with EGFR and designed to degrade into another inhibitor of the same target plus a DNAdamaging species may induce sustained growth inhibitory effect in EGFR-overexpressing cells.

Piperidine-functionalized, 1,4-disubstituted-1,2,3-triazoles of generic structure 1 were conceived as "minimalist" mimics of peptidic beta-turn structures. Key features of these molecules include (i) the possibility of incorporating amino acid side chains corresponding to many of the protein amino acids; (ii) a close correspondence of separations of these side chains to i + 1 to i + 2 residues in turns; (iii) facile adjustment of the side-chain vectors on docking while only influencing two critical degrees of freedom; and (iv) some electrostatic polarity. Fifteen monomers of this type were made via copper-mediated cycloaddition reactions. Solution-phase methodologies were devised to assemble these monomers into bivalent compounds in high purity states (typically >85%) so that they could be used in first-pass biological assays without further purification. The skeleton for forming these bivalent compounds is triazine-based. There is a third site which allowed for introduction of a fluorescent label (library of compounds 2) or an alkyne-functionalized triethylene glycol chain (library of compounds 3) included to promote water-solubility and to allow incorporation of probes via copper-mediated cycloaddition reactions. In the event, two 135-membered libraries were prepared, one consisting of compounds 2 and the other of 3. No protecting groups or coupling agents were required; these attributes of the method were important to allow most of the products to be obtained in over 85% purities. The fluorescein-tagged library of compounds 2 was screened in a fluorescence-activated cell sorting (FACS) assay using cells transfected to overexpress one of the following neurotrophin receptors: TrkA, TrkC, and p75. Preliminary findings indicate four compounds 2gm, 2gn, 2gi, and 2gj bound the TrkA receptor selectively; all of these contain a threonine-lysine turn mimic. Thus, a pharmacological probe for the TrkA receptor has been developed.