Structural,biological, and pharmacological strategies for the inhibition of nerve growth factor

Nerve growth factor (NGF) is critical for the development and maintenance of sympathetic and sensory neurons in the developing nervous system, including nociceptors. In the adult nervous system, NGF is known to produce significant pain signals by binding to the TrkA and p75NTR receptors. Several pathological pain disorders are associated with nerve growth factor dysregulation, including neuropathic pain, osteoarthritic pain, and hyperalgesia. Currently, clinical management of these pathologies has relied on the use of opioid and non-steroidal anti-inflammatory drugs (NSAID). However, several chronic pain conditions demonstrate insensitivity to NSAID treatment or the development of detrimental opioid-related side effects, including addiction. As NGF plays an important role in pain generation; antibodies, small molecules and peptides have been designed to antagonize NGF. In this review, we discuss the structural biology of NGF ligand/receptor interaction, and we review current biological and pharmacological strategies to modulate NGF-related pathologies.     

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza- adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)- dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF- dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.     

Chick embryo nerve growth factor : Fractionation and biological activity

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.     

Wheat germ agglutinin blocks the biological effects of nerve growth factor

The binding of nerve growth factor (NGF) to specific cell surface receptors initiates a variety of effects that lead to the morphological and biochemical differentiation of clonal pheochromocytoma, PC12, cells. The lectin wheat germ agglutinin (WGA) alters the characteristics of NGF-receptor interaction. We have found that treatment of PC12 cells with WGA dramatically and reversibly inhibits the ability of NGF to elicit three distinct biological effects characteristic of NGF action. Two of these events, the rapid ruffling of cell-surface membranes and the stimulation of the phosphorylation of a 250-kD cytoskeletal protein in situ, occur rapidly and are an immediate consequence of receptor occupancy. Both of these effects are blocked by pretreatment of the cells with WGA. WGA was also found to inhibit the NGF-stimulated regeneration of neurites that occurs over 1- 2 d. Both the WGA inhibition of neurite outgrowth and the phosphorylation of the 250-kD cytoskeletal protein were reversed upon addition of the specific sugar N-acetylglucosamine. These data demonstrate that the WGA-induced changes in the NGF-receptor interaction reflect important alterations in the ability of the receptor to transmit biological signals, resulting in the abrogation of the biological effects of NGF on these cells.     

Multiple biological activities for two peptides derived from the nerve growth factor precursor

ProNGF can be cleaved proteolytically at dibasic residues and liberates two other peptides beside NGF, LIP1 a 29 amino acid (aa) peptide and LIP2 a 38 aa peptide. These peptides were found present in the rat intestine and shown to induce rapid phosphorylation of the Trk receptor in cell lines. The present study describes several novel biological properties for these peptides. They exert an anti-proliferative effect on the mitogenic activity of estrogen and IGF in MCF-7 cells. They protect against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. They bind to murine microglial cells and induce phosphorylation of Akt. These results suggest a role for LIP1 and LIP2 in cell survival.     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate

Conjugation of poly(ethylene glycol) derivatives with therapeutic proteins is a promising approach for enhancing protein stability and, therefore, effectiveness. An N-hydroxysuccinimidyl ester of fluorescein-PEG 2000 was used for chemical modification of mouse nerve growth factor (mNGF), a dimeric protein with therapeutic potential for Alzheimer's disease. The mNGF-PEG2000-fluorescein conjugate was characterized by RP-HPLC, spectrofluorometry, and SDS-PAGE and was biologically active, as determined by two independent NGF-specific assays (enhancement of ChAT activity in fetal neurons and neurite outgrowth in PC12 cells). The conjugate was not detectable by a standard NGF ELISA, suggesting a fortuitous reduction in protein recognition by antibodies.     

Capsaicin-induced inhibition of axoplasmic transport is prevented by nerve growth factor

Summary Capsaicin injected into the scrotal skin of rats was observed to induce a decrease in the amount of horseradish peroxidase (HRP) transported in the pudendal nerve to the sixth lumbar dorsal root ganglion on the pretreated side. This was seen as a decrease in the number of HRP-labelled neurones compared to the control side. A morphometric study confirmed that the effect of capsaicin was exerted predominantly on the small neurones. Injection of nerve growth factor (NGF) into the pudendal nerve prevented the deleterious effects of capsaicin, thereby suggesting a possible site of action and mechanism for the effect of capsaicin on peripheral nerves.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

蛇毒神经生长因子生物活性研究

从蛇毒中分离的神经生长因子（ＮＧＦ）,经用鸡胚背根神经节做生物活性测定,发现能促进神经节树突最大生长的最小ＮＧＦ浓度为２．５ｎｇ／ｍｌ。半成品的毫克蛋白（ｍｇＰ）比活性在１．５４×１０５～５．３５×１０５Ｕ／ｍｇＰ之间。经用ＨＰＬＣ和ＳＤＳ－ＰＡＧＥ电泳做纯度分析,主峰面积在９５％以上。并从四种ＮＧＦ成品赋形剂中选出了理想的配方     

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.     

Processing of the native nerve growth factor precursor to form biologically active nerve growth factor

In the mouse submaxillary gland beta nerve growth factor (beta-NGF) forms a complex with two members of the kallikrein family of serine proteases, termed the alpha- and gamma-subunits of NGF. We demonstrate that the beta-NGF precursor produced in mammalian cells via a recombinant vaccinia virus can be cleaved by stoichiometric quantities of the gamma-subunit to produce beta-NGF. Trypsin in catalytic quantities also produces native beta-NGF. Proper cleavage depends critically on the conformation of the precursor. beta-NGF has at least 10-fold more biological activity than its precursor.     

Human nerve growth factor: Lack of immunocrossreactivity with mouse nerve growth factor

Human β-nerve growth factor (hNGF) was purified from term human placenta. The biological potency of hNGF in the chick dorsal root ganglion assay did not differ significantly from that of mouse NGF (mNGF). Molecular weight determinations of mNGF and hMGF were also similar. No immunological crossreactivity was noted between hNGF, at a concentration of 100 μg/ml, and mNGF in a radioimmunoassay for mNGF using 6 different antisera to mNGF. hNGF shares several properties with mNGF but is immunological distinct. The results of studies in man using antisera to mNGF should be interpreted with caution.     

Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones

Local protein synthesis in nerve growth cones has been suggested, but how it is controlled remains largely unknown. We found eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, in growth cones by immunocytochemistry. While phosphorylated eEF2 was weakly distributed in advancing growth cones, eEF2 phosphorylation was increased by high potassium-evoked calcium influx. In the growth cone, calcium elevation increased eEF2 kinase (EF2K), a calcim-calmodulin-dependent enzyme. Calcium also decreased the level of phosphorylated p70-S6 kinase (S6K), a kinase known to inhibit EF2K. Moreover, calcium elevation decreased total eEF2 in growth cones. Since phosphorylated eEF2 inhibits mRNA translation, calcium elevation appears to inhibit mRNA translation in growth cones by a synergistic mechanism involving regulation of EF2K, S6K, and eEF2 itself. Time-lapse imaging showed that calcium elevation induced growth arrest of neurites. The inhibitory effect on mRNA translation may thus be involved in the regulation of neurite outgrowth.     

Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors

Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.