Designing Nerve Growth Factor Analogues to Suppress Pain Signal Transduction Mediated by the p75NTR-NGF-TrkA Complex: A Structural and Biophysical Perspective

Introduction

Chronic pain imposes a substantial burden on global healthcare systems, affecting millions of individuals and severely compromising their quality of life [1–4]. Unraveling the intricate mechanisms underlying pain signal transduction is essential for developing targeted and effective therapeutic interventions [5–7]. The p75 neurotrophin receptor (p75NTR)-nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex has emerged as a critical nexus in the modulation of nociceptive signaling, presenting a promising target for innovative analgesic strategies [8–11].

Historically, nerve growth factor (NGF) was first discovered approximately 60 years ago by Rita Levi-Montalcini as a protein that induces the growth of nerves [12–15]. It is now known that NGF is also associated with Alzheimer’s disease and intractable pain, and hence, it, along with its high-affinity receptor, tropomyosin receptor kinase (Trk) A, is considered to be 1 of the new targets for therapies being developed to treat these diseases [16–22]. Anti-NGF antibody and TrkA inhibitors are known drugs that suppress NGF/TrkA signalling, and many drugs of these classes have been developed thus far. Interestingly, local anaesthetics also possess TrkA inhibitory effects. This manuscript describes the development of an analgesic that suppresses NGF/TrkA signalling, which is anticipated to be one of the new methods to treat intractable pain [23–28].

While the implication of NGF and its receptors, TrkA and p75NTR (referred to as the NGF system) in CNS autoimmune neuroinflammation is not fully elucidated, the necessity of designing nerve growth factor (NGF) analogues to suppress pain signal transduction mediated by the p75NTR-NGF-TrkA complex arises from the intricate and multifaceted nature of the neurotrophic signalling pathways involved in CNS autoimmune neuroinflammation [29–34]. Since native NGF plays a pivotal role in both promoting neuronal survival and contributing to pain sensitization through its interactions with the p75NTR and TrkA receptors, the delicate balance between these contrasting effects underscores the need for targeted interventions that selectively modulate the signalling cascade to alleviate pain without compromising essential neurotrophic functions [35–38]. Therefore, in this article, designing NGF analogues provides a strategic approach to fine-tune the molecular interactions within the p75NTR-NGF-TrkA complex, aiming to attenuate the nociceptive signals while preserving the beneficial aspects of NGF-mediated neurotrophic support [39–46].

Materials and Methods

According to a structure search of the Protein Data Bank (PDB) [47], as of March 28, 2024, there are a total of sixteen experimentally determined NGF-related structures deposited into the PDB, as listed in Table 1.

Table 1: Experimentally Determined NGF-Related Structures (released newest from oldest) in the Protein Data Bank (PDB [47]) as of March 28, 2024, QUERY code: Additional Structure Keywords HAS EXACT PHRASE "NERVE, GROWTH FACTOR".

Among the sixteen, PDB ID 2IFG [48] is the only experimental structure of the extracellular segment of human TRKA in complex with nerve growth factor, as shown by Figure 1. Therefore, in this article, PDB ID 2IFG is chosen here as a structural template for the design of NGF analogues to suppress pain signal transduction mediated by the p75NTR-NGF-TrkA complex [48].

Figure 1: A Overview of the Experimental Structure of the Extracellular Segment of Human TRKA in Complex with Nerve Growth Factor (PDB ID 2IFG [48]). This Figure is Prepared by PyMol [49].

Specifically, of the structure of the extracellular segment of hu- man TRKA in complex with nerve growth factor with PDB ID 2IFG, a set of relevant amino acid sequences are listed in italics in fasta format as below [48],

>TRKAchainA

CPDACCPHGSSGLRCTRDGALDSLHHLPGAENLTELYIEN-

QQHLQHLELRDLRGLGELRNLTIV KSGLRFVAPDAFHFT-

PRLSRLNLSFNALESLSWKTVQGLSLQELVLSGNPLHCS-

CALRWLQRWEEEGL GGVPEQKLQCHGQGPLAHMPNASCGVPTLKVQVPNAS-

VDVGDDVLLRCQVEGRGLEQAGWILTE

LEQSATVMKSGGLPSLGLTLANVTSDLNRKNVTCWAEND-

VGRAEVSVQVNVSFPASVQLHTAVEM

HHWCIP FSVDGQ PA P SLR WLF NGSV LNETS -

FIFTEFLEPAANETVRHGCLRLNQPTHVNNGNYTLLA

ANPFGQASASIMAAFMDNP

>TRKAchainB

CPDACCPHGSSGLRCTRDGALDSLHHLPGAENLTELYIEN-

QQHLQHLELRDLRGLGELRNLTIV KSGLRFVAPDAFHFT-

PRLSRLNLSFNALESLSWKTVQGLSLQELVLSGNPLHCS-

CALRWLQRWEEEGL

GGVPEQKLQCHGQGPLAHMPNASCGVPTLKVQVPNAS-

VDVGDDVLLRCQVEGRGLEQAGWILTE

LEQSATVMKSGGLPSLGLTLANVTSDLNRKNVTCWAEND-

VGRAEVSVQVNVSFPASVQLHTAVEM

HHWCIP FSVDGQ PA P SLR WLF NGSV LNETS - FIFTEFLEPAANETVRHGCLRLNQPTHVNNGNYTLLA ANPF- GQASASIMAAFMDNP

>NGFchainE

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVL-

GEVNINNSVFKQYFFETKCRDPNPV DSGCRGIDSKHWN-

SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR-

RA >NGFchainF

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVL-

GEVNINNSVFKQYFFETKCRDPNPV DSGCRGIDSKHWN-

SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR-

RA

To further elucidate the mechanism of the design of NGF an- alogues to suppress pain signal transduction mediated by the p75NTR-NGF-TrkA complex here, Figure 2 shows a closer look of the homodimer of two native NGF molecules, as experimen- tally determined by X-ray diffraction in 2007 (PDB ID 2IFG), as the core of the idea is that NGF analogues are to be still able to bind two neurotrophin receptors: p75 neurotrophin receptor (p75NTR) and tyrosine kinase receptor A (TrkA), yet, their abil- ity to form a homodimer (Figure 2) is inhibited via disruptions of a set of key inter-residue interactions at the binding interface of native NGF homodimers (Figure 1) [48,50].

Figure 2: A Closer Look of the Homodimer of Two Native NGF Molecules as Determined by X-ray Diffraction (PDB ID 2IFG [48]). This Figure Is Prepared by PyMol [49].

A Comprehensive Structural and Biophysical Analysis of the p75NTR-NGF-TrkA Complex

As described previously in, a comprehensive structural and bio- physical analysis was conducted for the structure of the extra- cellular segment of human TRKA in complex with nerve growth factor with PDB ID 2IFG [48,50–52]. Specifically, the salt bridge analysis was conducted with an in-house python script only for titrateable residues (Asp, Glu, Lys, Arg and His), 4.0 Å was used as the cutoff distance for the two oppositely charged groups [50]. The hydrogen bond analysis was also conducted for only side chain nuclei with an in-house python script, and employed two geometric criteria: (a) a cutoff value of the angle formed by acceptor (A), donor (D) and hydrogen (H) (∠ADH) of 30°; (b) a cutoff value of donor-acceptor distance at 3.0 Å. That is, a hydrogen bond is only considered to be formed if ∠ADH is no larger than 30° and the donor-acceptor distance is not larger than 3.0 Å [50].

The Design of NGF Analogues to Suppress Pain Signal Transduction Mediated by the p75NTR-NGF-TrkA Com- plex

In combination with the comprehensive structural and biophys- ical analysis as described above, the key amino acid residues at the p75NTR-NGF-TrkA complex binding interface (PDB ID: 2IFG) were examined carefully [? ] in PyMol, and the inter-res- idue distances were calculated by PyMol to identify potential neighbouring residue pair(s) to modulate the structural stability of the p75NTR-NGF-TrkA complex structure, leading to the de- sign of a set of NGF analogues with reduced affinity such that ability of NGF monomers to form a homodimer (Figure 2) is inhibited via disruptions of a set of key inter-residue interactions at the binding interface of native NGF homodimers (Figure 1). Specifically, after homology structural modeling with Modeller, the binding affinity between rt-PA analogue and PAI-1 was cal- culated using Prodigy [49,50,53-55] .

Results

As described above, the core of the idea is that NGF analogues are to be still able to bind two neurotrophin receptors: p75 neu- rotrophin receptor (p75NTR) and tyrosine kinase receptor A (TrkA), yet, their ability to form a homodimer (Figure 2) is in- hibited via disruptions of a set of key inter-residue interactions at the binding interface of native NGF homodimers (Figure 1) [50]. Essentially, the design of NGF analogues here is the con- struction of an n-dimensional NGF dimer-based mini GIBAC [52] towards the suppression of chronic NGF-related pain sig- naling, where n represents the number of site-specific mutations introduced to the amino acid sequence of NGF, as listed below:

>NGFchainE

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVL-

GEVNINNSVFKQYFFETKCRDPNPV DSGCRGIDSKHWN-

SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR-

RA >NGFchainF

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVL-

GEVNINNSVFKQYFFETKCRDPNPV DSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWR-

FIRIDTACVCVLSRKAVRRA

Table 2: Single Site-Specific Mutation-Based Analysis of the Binding Affinities Between Two Nerve Growth Factor Monomers. Prodigy is Used to Calculate The Binding Energy and Kd Between Two Nerve Growth Factor Monomers. In this table, the row for Nddm (yellow background) Represents the Binding Affinity Between Two Native Nerve Growth Factor Monomers [55].

Given that the length of monomeric NGF is 121, n is not to be beyond six to ensure that the NGF analogues is more than 95% homologous to its native counterpart, and the accuracy of the homology structural modeling using the experimental structure of the extracellular segment of human TRKA in complex with nerve growth factor (PDB ID 2IFG [48]).

Here, in this article, a total of three occasions were reported, i.e., n = 3, specifically,

1. n = 1, i.e., a one-dimensional NGF dimer-based mini GIBAC [51,52] for the design of NGF analogues towards the suppression of NGF-related chronic pain signalling, as partly listed in Table 2.

2. n = 2, i.e., a two-dimensional NGF dimer-based mini GIBAC [51,52] for the design of NGF analogues towards the suppression of NGF-related chronic pain signalling, as partly listed in Table 3.

3. n = 3, i.e., a three-dimensional NGF dimer-based mini GIBAC [51,52] (supplementary file trio.pdf) for the design of NGF analogues towards the suppression of NGF-related chronic pain signalling, as partly listed in Table 4.

Table 3: Double Site-Specific Mutation-Based Analysis of the Binding Affinities Between Two Nerve Growth Factor Monomers. Prodigy [55] is Used to Calculate the Binding Energy And Kd Between Two Nerve Growth Factor Monomers. In this table, the Row for Nddm (Yellow Background) Represents the Binding Affinity Between Two Native Nerve Growth Factor Monomers

As quantitatively described in Tables 2, 3 and 4, the binding affinity (Kd) between two native nerve growth factor monomers at 37 â?¦C is 1.2 × 10-9 M, while the binding affinity (K ) between two nerve growth factor analogue monomers at 37 â?¦C is reduced to as low as 5.8 × 10-6 M (i.e., the S4A_A98Q_V33A site-specific mutations for native NGF monomers) with a set of triple site- specific mutations introduced into the amino acid sequence of native NGF sequence, towards the disruption of the homodimer of NGF and the suppression of NGF-related chronic pain signalling mediated by the p75NTR-NGF-TrkA complex (PDB ID 2IFG [48]).

Table 4: Triple Site-Specific Mutation-Based Analysis of the Binding Affinities Between Two Nerve Growth Factor Monomers. Prodigy is Used to Calculate the Binding Energy and Kd Between Two Nerve Growth Factor Monomers. In this table, the Row for Nddm (Yellow Background) Represents the Binding Affinity Between Two Native Nerve Growth Factor Monomers [55]..

Conclusion and Discussion

Overall, this article for the first time puts forward a set of NGF analogues, including in particular NGF analogues with four site-specific mutations, for whose dimerization’s the Kd at 37 ºC were reduced by three orders of magnitude (from 10-9 M to 10-6 M) compared to the K at 37 ºC for the dimerization of native NGFs. As discussed above, a structural and biophysical perspective (Figure 3) is crucial in guiding the rational design of these analogues, ensuring a nuanced understanding of the intricate molecular mechanisms involved in pain transduction and enabling the development of therapeutics with enhanced specificity and efficacy [56–60]. Hence, the investigation here of the design of NGF analogues, targeted at suppressing pain signal transduction mediated by the p75 neurotrophin receptor (p75NTR)-NGF-tyrosine kinase receptor A (TrkA) complex, has yielded valuable insights into the potential for developing innovative analgesic interventions [61–65].

Of further pharmaceutical interest is the integration of experimental biophysical techniques complemented our computational predictions, offering a dynamic perspective on the interactions between the designed analogues and the complex [66–69]. In the realm of drug discovery and design, the quest for innovative therapeutic interventions to alleviate chronic pain has become a pivotal focus. Chronic pain, a pervasive and debilitating condition, poses a significant clinical challenge,necessitating the exploration of novel targets and strategies. Among the various molecular players implicated in pain signal transduction, the p75 neurotrophin receptor (p75NTR)-nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex has emerged as a promising nexus for intervention [70–74].

In short, the use of NGF analogues in drug discovery heralds a paradigm shift in the approach to pain modulation. By harnessing insights from structural and biophysical perspectives, researchers can tailor NGF analogues to disrupt specific interactions within the p75NTR-NGF-TrkA complex. This precision allows for the design of therapeutics with the potential to selectively dampen pain signalling pathways while minimizing off-target effects [75–82].

Figure 3: Flowchart of the Design of NGF Analogues to Disrupt the NGF Homodimer and Suppress Pain Signal Transduction Mediated by the p75NTR-NGF-TrkA Complex

In conclusion, the structural and biophysical perspective on designing NGF analogues to suppress pain signal transduction provides a robust foundation for the development of targeted analgesic therapies. By elucidating the structural biophysics underlying the p75NTR-NGF-TrkA complex, this article contributes valuable knowledge to the field, paving the way for the next generation of therapeutic strategies aimed at alleviating chronic pain [83–91].

Ethical Statement

No ethical approval is required.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the author used OpenAI’s ChatGPT in order to improve the readability of the manuscript, and to make it as concise and short as possible. After using this tool, the author reviewed and edited the content as needed and takes full responsibility for the content of the publication.

Author Contributions: Conceptualization, W.L.; methodology, W.L.; software, W.L.; validation, W.L.; formal analysis, W.L.; investigation, W.L.; resources, W.L.; data duration, W.L.; writing–original draft preparation, W.L.; writing–review and editing, W.L.; visualization, W.L.; supervision, W.L.; project administration, W.L.; funding acquisition, not applicable.

Funding: This research received no external funding.

Conflicts of Interest: The author declares no conflict of interest.

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