A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations: implications for peptide-based analgesics

Abstract

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor.

TrkA Co-Receptors: The Janus Face of TrkA?

“…Nevertheless, to avoid these side effects, anti-NGF inhibitors with different binding modes than those of tanezumab have been developed, such as peptide A2 or BVNP-0197 (peptide that binds loop II/IV of NGF) [ 16 , 17 ].…”

N-Acetylcysteine Antagonizes NGF Activation of TrkA through Disulfide Bridge Interaction, an Effect Which May Contribute to Its Analgesic Activity

“…The action of NGF is complex and may impact acutely both in sensitizing to pain, through actions involving glutamate and Transient Receptor Potential Vanilloid 1 (TRPV1) receptors [ 7 ], and with more indirect immune-mediated mechanisms involving the NGF-induced production of prostaglandin D2 (PGD2) in joint mast cells [ 8 ], both of them increasing the risk of developing chronic pain. Accordingly, several anti-NGF strategies have been developed among which are the use of monoclonal antibodies against NGF and of small antagonist molecules at the TrkA receptor [ 9 , 10 , 11 , 12 ]. In the latter case, the search for peptides capable of blocking the interaction of NGF with TrkA is of particular interest as a possible antinociceptive strategy [ 12 ].…”

“…Accordingly, several anti-NGF strategies have been developed among which are the use of monoclonal antibodies against NGF and of small antagonist molecules at the TrkA receptor [ 9 , 10 , 11 , 12 ]. In the latter case, the search for peptides capable of blocking the interaction of NGF with TrkA is of particular interest as a possible antinociceptive strategy [ 12 ]. It should be noted that the cysteine residue at the C-terminus of such peptides is required to inhibit the NGF/TrkA interaction, despite not being involved in NGF binding, and that peptide dimerization enhances the inhibitory effect [ 12 ].…”

“…In the latter case, the search for peptides capable of blocking the interaction of NGF with TrkA is of particular interest as a possible antinociceptive strategy [ 12 ]. It should be noted that the cysteine residue at the C-terminus of such peptides is required to inhibit the NGF/TrkA interaction, despite not being involved in NGF binding, and that peptide dimerization enhances the inhibitory effect [ 12 ]. Interestingly enough, in previous research on novel peptide compounds with TrkA agonist/antagonist activities, Fantucci et al [ 13 ], searching for the smallest representative of the TrkA-NGF complex, found that the highest number of short-range interactions occur between NGF and TrkA, involving, respectively, the amino acid residues from 2 to 24 of NGF and the sequence 250–348 of TrkA, where the latter sequence contains a disulfide bond between cysteine 300 and cysteine 345.…”

Computational Insights into the Sequence-Activity Relationships of the NGF(1–14) Peptide by Molecular Dynamics Simulations

“…Moreover, NGF exerts a protective and healing effect on the corneal tissue that led to the approval of recombinant human NGF (Oxervate, Dompé Farmaceutici SpA, Milan, Italy) for the topical treatment of neurotrophic keratopathy, a rare corneal disorder induced by trigeminal nerve impairments which causes epithelial damage and corneal ulcerations [ 6 ]. In addition, the activation of NGF-TrkA pathway is also implicated in the transmission of pain signals [ 7 ] as well as in tumorigenesis and metastasis formation in different types of cancer [ 8 ].…”