Sonia Covaceuszach

The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.

Nerve growth factor (NGF) is involved in pain transduction mechanisms and plays a key role in many persistent pain states, notably those associated with inflammation. On this basis, both the NGF ligand and its receptor TrkA (tyrosine kinase A) represent an eligible target for pain therapy. Although the direct involvement of NGF in pain modulation is well established, the effect of a direct functional block of the TrkA receptor is still unknown. In this study, we have demonstrated that MNAC13, the only anti-TrkA monoclonal antibody for which function neutralizing properties have been clearly shown both in vitro and in vivo, induces analgesia in both inflammatory and neuropathic pain models, with a surprisingly long-lasting effect in the latter. The formalin-evoked pain licking responses are significantly reduced by the MNAC13 antibody in CD1 mice. Remarkably, treatment with the anti-TrkA antibody also produces a significant antiallodynic effect on neuropathic pain: repeated i.p. injections of MNAC13 induce significant functional recovery in mice subjected to sciatic nerve ligation, with effects persisting after administration. Furthermore, a clear synergistic effect is observed when MNAC13 is administered in combination with opioids, at doses that are not efficacious per se. This study represents a direct demonstration that neutralizing antibodies directed against the TrkA receptor may display potent analgesic effects in inflammatory and chronic pain.

Development of a Non Invasive NGF-Based Therapy for Alzheimer's Disease

Nerve growth factor (NGF) deficits are linked to Alzheimers Disease (AD), due to the role of NGF on basal forebrain cholinergic neurons (BFCN). We have further established that a disequilibrium in NGF signaling and/or processing from its precursor proNGF is also directly and causally related to the aberrant activation of an amyloidogenic route to neurodegeneration. The therapeutic potential of using human NGF to provide a long-lasting cholinergic trophic support, thereby preventing or slowing cognitive decline in AD patients, has therefore a strong rationale. However, a simple and practical means of delivering NGF to the brain in a safe and long-term manner, limiting the undesired adverse effects of NGF in activating nociceptive responses, has represented a significant challenge. For this reason, pilot clinical studies have been performed so far with invasive approaches requiring neurosurgery. We obtained a proof of principle, in neurodegeneration animal models, of an alternative, non-invasive delivery of NGF through an intranasal route, which facilitates access of NGF to the central nervous system (CNS), while minimizing the biodistribution of NGF to compartments where it activates undesired effects, such as pain. The ideal NGF product for a non invasive NGF-based therapy would be a recombinant NGF that, while exhibiting an identical biological activity to that of human NGF, can be traced, against the endogenous NGF, in order to optimize the therapeutical dose range and meet the required therapeutic window. We describe an engineered mutein of hNGF, hNGF-61, that is selectively recognized, against endogenous NGF, by a specific antibody. hNGF-61 mutein has an identical potency and bioactivity profile as hNGF, in vitro and in vivo. Moreover, hNGF-61 and hNGF are equally effective in rescuing the behavioral and neurodegenerative phenotype in adult and aged AD11 anti-NGF mice. Finally, we demonstrated that intranasally delivered hNGF-61 is significantly more effective than ocularly applied hNGF-61, to determine phenotypic rescue in AD11 mice. The development of hNGF-61 towards clinical applications in AD patients is under way.

Taking Pain Out of NGF: A “Painless” NGF Mutant, Linked to Hereditary Sensory Autonomic Neuropathy Type V, with Full Neurotrophic Activity

During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8–10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Dissecting NGF Interactions with TrkA and p75 Receptors by Structural and Functional Studies of an Anti-NGF Neutralizing Antibody

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

Delivery of NGF to the brain: intranasal versus ocular administration in anti-NGF transgenic mice.

Nerve growth factor (NGF) has a great potential for the treatment of Alzheimers disease. However, the therapeutic administration of NGF represents a significant challenge, due to the difficulty to deliver relevant doses to the brain, in a safe and non-invasive way. We previously demonstrated the efficacy of a non-invasive delivery of NGF to the brain in animal models, by an intranasal route. Recently, topical eye application of NGF was proposed, as an option for the delivery of NGF to the brain. Here, we compare the efficacy of the two delivery routes of hNGF-61, a recombinant traceable form of human NGF, in the mouse neurodegeneration model AD11. The intranasal administration appeared to be significantly more effective than the ocular one, in rescuing the neurodegenerative phenotypic hallmarks in AD11 mice. The ocular administration of hNGF-61 showed a more limited efficacy, even at higher doses. Thus, NGF nasal drops represent a viable and effective option to successfully deliver therapeutic NGF to the brain in a non-invasive manner.

Intrinsic structural disorder of mouse proNGF

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm‐proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X‐ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti‐NGF antibodies and the soluble forms of TrkA and p75NTR receptors. SAXS measurements revealed the rm‐proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular “crab‐like” and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the “crab‐like” to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75NTR receptors and a panel of neutralizing anti‐NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners. Proteins 2009.

Structural and functional properties of mouse proNGF

The unprocessed pro-form of the NGF (nerve growth factor), proNGF (NGF precursor, without signal peptide), has been suggested to have additional functions distinct from its role as a promoter of protein folding, i.e. apoptosis and/or neurotrophic activity. Aiming to gain insights into the specific molecular interactions that mediate proNGF biological activity and into the structural determinants stabilizing its pro-region, rm-proNGF (recombinant mouse proNGF) was expressed in Escherichia coli, refolded in vitro and characterized by physicochemical methods. X-ray solution scattering measurements (small angle X-ray scattering) revealed that rm-proNGF is dimeric in solution and appears to be anisometric when compared with the compact structure of the NGF dimer. Two structural models, a globular crab-like shape and an elongated rod-like shape, equally fit to the experimental results, pointing to an intrinsically structural disordered pro-region of NGF. The models obtained allowed the interpretation of TrkA (tropomyosin receptor kinase A) binding and activation assays in cell cultures, shedding new light on the key role of proNGF in neuronal survival and neurodegeneration.

Neutralization of NGF‐TrkA receptor interaction by the novel antagonistic anti‐TrkA monoclonal antibody MNAC13: A structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA‐mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy‐terminal region of the first immunoglobulin‐like domain (d4) of TrkA. The X‐ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 Å resolution. The antigen‐binding site is characterized by a crevice, surrounded by hydrophilic‐charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure‐based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states. Proteins 2005.

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

Purification, crystallization and preliminary X-ray analysis of the Fab fragment from MNAC13, a novel antagonistic anti-tyrosine kinase A receptor monoclonal antibody

The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of nerve-growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems. Structural studies of the FabMNAC13 fragment were performed to gain insights into the mechanism of action of this potentially therapeutic monoclonal antibody. The optimal conditions for crystallization of FabMNAC13 were determined. Crystals appeared as prismatic bundles, displayed P2(1)2(1)2(1) space-group symmetry and diffracted to a resolution of 1.8 A. The unit-cell parameters were determined to be a = 52.73, b = 67.55, c = 111.43 A. The data set was 99.5% complete. Molecular replacement was performed, resulting in a correlation coefficient of 0.55 and an R value of 0.40. The structure refinement is now in progress.