Targeting the Nerve Growth Factor (NGF) Pathway in Drug Discovery

Perspective pubs.acs.org/jmc

Targeting the Nerve Growth Factor (NGF) Pathway in Drug Discovery. Potential Applications to New Therapies for Chronic Pain Bryan H. Norman*,† and Jeff S. McDermott‡ †

Discovery Chemistry Research and Technologies and ‡Neurophysiology, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Lilly Corporate Center, Indiana 46285, United States ABSTRACT: The neurotrophin nerve growth factor (NGF) has been implicated as a key mediator of chronic pain. NGF binds the tropomysin receptor kinase A (TrkA) and p75, resulting in the activation of downstream signaling pathways that have been linked to pro-nociception. While anti-NGF antibodies have demonstrated analgesia both preclinically and in patients, the mechanism of action of these agents remains unclear. We describe ligands targeting NGF, its receptors, and downstream/related targets. This Perspective highlights large and small molecule approaches to targeting the NGF−TrkA pathway both extra- and intracellularly. In addition, we present a strategic framework for future drug discovery efforts in this pathway beyond the targeting of NGF or its receptors. While existing tools have greatly informed NGF-mediated signaling, ongoing and future pathway research may help focus new drug discovery efforts on key novel targets and mechanisms. This may result in highly differentiated therapeutics with greater efficacy and/or improved safety profiles.

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INTRODUCTION Pain is a subjective term that is used to describe sensations associated with a multitude of conditions, ranging from minor injuries to serious pathological disorders.1 Acute pain, resulting from some specific event that causes tissue damage (such as surgery or injury),2 is generally mitigated when the condition has resolved and/or the tissue has healed. Chronic pain, on the other hand, may persist long after the resolution of injury or may be associated with an ongoing chronic condition such as osteoarthritis (OA) or cancer.3,4 Additionally, chronic pain may or may not be associated with ongoing inflammation, one of the body’s innate responses to tissue injury, which can exacerbate the painful response.5 While pain is an important human response that provides avoidance signals for our protection, the management of uncontrollable chronic pain remains a significant unmet medical need.6 In recent years, efforts to identify novel analgesic agents has been disappointing and drug discovery researchers have been confounded by the discovery of preclinical analgesic agents that did not transfer sufficient pain efficacy to humans.7,8 Thus, some of the most prescribed analgesics today represent drug classes, such as opioids and NSAIDs, that have been known for many decades. Unfortunately, these agents frequently either do not provide sufficient pain relief, have unacceptable side effects, or both.1,9 New, transformative analgesic therapies are greatly needed to combat many chronic pain conditions for which no meaningful therapy currently exists. Preclinical and clinical research over the past 20−25 years has demonstrated a role for the neurotrophin nerve growth factor (NGF) in the transmission of both acute and chronic pain signals.10,11 Because of recently reported clinical efficacy of NGF neutralizing antibodies,12 the pain © XXXX American Chemical Society

research community is hopeful that modulation of the NGF signaling pathway may provide significant analgesia for multiple painful conditions. In this Perspective, we will provide an overview of NGF target validation in pain and try to connect the many NGF signaling pathways to pain mechanisms. We will also review the current methods that have been used to interrogate the NGF pathway in drug discovery and how these approaches may result in new analgesic agents with a superior efficacy and safety profile relative to the current standards of care.

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BACKGROUND NGF was discovered by Levi-Montalcini and Cohen during their collaboration at Washington University in the late 1950s. Their work grew from earlier collaborations between LeviMontalcini and Hamburger, a colleague and mentor who remains an underappreciated contributor to the NGF discovery story.13 Levi-Montalcini and Cohen’s original trilogy of manuscripts, published in 1960,14−16 described the discovery and characterization of NGF. This work was recognized in 1986 when they were awarded the Nobel Prize in Medicine. In these seminal publications, they revealed a protein derived from the mouse salivary gland that was responsible for the development of a subset of neural crest cells, mainly primary afferent and sympathetic neuronal populations. As part of their experimental strategy, they employed the use of polyclonal antibodies to NGF and showed that sequestering NGF had its most dramatic Received: July 1, 2016

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DOI: 10.1021/acs.jmedchem.6b00964 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Neurotrophin dimers NGF, BDNF, NT-4, and NT-3 bind to their cognate receptors TrkA, TrkB, and TrkC, as well as to the common p75 receptor. Binding to the extracellular receptors triggers downstream intracellular signaling pathways.

terminal21 in addition to its role as an active coreceptor with p75, where it is thought to play a key role in proNGF/p75/ sortilin signaling.22 NGF is synthesized as a prepro-form, which is cleaved to the 25 kDa pro-form upon release from the endoplasmic reticulum.23 Upon dimerization, this 50 kDa pro-form dimer of NGF (proNGF) can be cleaved within the cell to release the 26 kDa mature NGF dimer, which shows picomolar binding affinity to TrkA receptors (see Figure 2). Unprocessed proNGF may also be secreted.24 Cells that respond to NGF include primary afferent neurons, keratinocytes, pancreatic β-islet cells, osteal cells, vascular epithelial cells, and immune cells (mast cells, eosinophils, monocytes) among others.25−27

effects on neuronal survival during prenatal development. Interestingly, they also noted morphological changes in sympathetic ganglia neurons with NGF sequestration in adult rodents. This latter finding is noteworthy in that it may have foretold the sympathetic neuronal changes observed in the preclinical toxicology studies with the contemporary anti-NGF antibodies still in late stage development for chronic pain conditions. (vide infra). NGF is the founding member of a group of neurotrophins that has classically included brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin-4 (NT-4). NT-4 is also sometimes referred to as NT-5, and there has been an NT-6 described in teleost fish.17 Glial cell line-derived neurotrophic factor (GDNF), as well as several other trophic factors such as ciliary neurotrophic factor (CNTF), are not grouped classically with the neurotrophins but belong to a wider group of neurotrophic factors. As shown in Figure 1, neurotrophins bind, as dimers, to several related receptors: a unique high affinity tropomysin receptor kinase (Trk) as well as to a common 75 kDa low affinity neurotrophin receptor known as p75. Trks are receptor tyrosine kinases (RTKs), a class of proteins that have extracellular, intracellular, and membrane spanning domains. Upon binding its extracellular ligand, RTKs dimerize, autophosphorylate on various tyrosine residues, and begin a cascade of downstream intracellular signaling events that are responsible for a multitude of physiological effects.18 In the case of NGF, its unique binding partner is the 140 kDa TrkA receptor (encoded by the NTRK1 gene). BDNF and NT4 bind preferentially to TrkB (NTRK2), and NT-3 is the high affinity ligand for TrkC (NTRK3) but has shown some affinity for TrkA and TrkB. All four neurotrophins bind with similar affinity to p75 (NGFR), a member of the tumor necrosis factor (TNF) receptor family of receptors.19 It should be pointed out that this two receptor view of each neurotrophin, as depicted in Figure 1, is likely overly simplistic and not reflective of the more complex underlying biology. The p75 protein has been shown to act as a coreceptor to the various Trks,20 affecting binding affinities. Likewise, the sortilin protein has been implicated in both the anterograde trafficking of the NGFbound TrkA signaling endosome to the primary afferent nerve

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NGF PATHWAY VALIDATION Validation for Pain. For 30 years after the reported discovery of NGF in 1960, academic work in the field continued to focus almost exclusively on its role in neuronal development. However, two unrelated events occurred in the early 1990s that led to a key breakthrough in pain research. The first of these was the initiation of clinical trials using recombinant human NGF to treat painful diabetic neuropathies.28 In the course of these early clinical studies, it was noted that there were consistent reports of injection site reactions and hyperalgesia lasting weeks or even months. Around this time, the seminal work of Mendell and co-workers was just beginning. Mendell and his colleagues, Lewin and Ritter, were in the process of doing developmental studies on the effects of NGF on adult rat nociceptors when they noticed that some rodents seemed to be experiencing profound hyperalgesia. While behavioral end points were not the original goal of these studies, the group was nevertheless the first to report a detailed description of the hyperalgesia induced by NGF in rodents in 1993.29 Combined, these reports led to a rapid expansion of academic and industrial work on the relationship between NGF and pain. Some key events in the latter part of the decade were the failure of the phase 3 trial of recombinant human NGF for neuropathy, the genetic linkage of mutations in both NGFB and NTRK1 to several congenital insensitivity to pain with anhidrosis (CIPA) populations,30 as B



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Figure 2. NGF−TrkA canonical pathway, showing potential mechanisms for pharmacological intervention. Dimers of proNGF are converted to mature NGF dimers, which can initiate receptor dimerization and bind to TrkA and p75. In neuronal cells, NGF−TrkA binding induces the phosphorylation of multiple tyrosines and triggers downstream signaling through the PI3K, Ras, and PLCγ pathways. This leads to neurite outgrowth and other pharmacodymanic effects. Intervention of NGF−TrkA mediated signaling can occur via several mechanisms. Mechanism A: Sequestration of the NGF dimer. Mechanism B: Antagonism of the TrkA receptor. Mechanism C: Antagonism of the p75 receptor. Mechanism D: Inhibition of the intracellular TrkA kinase.

well as two key preclinical pharmacology studies revealing that in vivo sequestration of NGF was efficacious in animal models of inflammatory pain. The first of these two preclinical studies utilized a sheep anti-NGF polyclonal antibody, while the second employed a soluble TrkA receptor. Both successfully sequestered NGF in vivo and demonstrated analgesia. This evidence led to the development of a mouse anti-NGF antibody, muMab911 by Genentech, which was later humanized by Rinat to become the human anti-NGF antibody RN624.10 Rinat was the first to initiate testing of the NGF sequestration hypothesis in a clinical pain population. The early clinical work by Rinat was followed by several companies entering clinical studies with additional anti-NGF antibodies. A string of positive phase 2 data in multiple pain indications was encouraging. Unfortunately, reports of drug-related joint necrosis began to emerge in 2010, leading to a class hold by the FDA on further clinical testing, except in terminal cancer studies. After much adjudication, the vast majority of these cases were more accurately diagnosed as rapidly progressing osteoarthritis (RPOA). With the adoption of a risk mitigation strategy, which included dosing restrictions and a recommendation against concomitant NSAID use in osteoarthritis patients, the FDA lifted this clinical hold in 2012.31 However, a short time thereafter, a second clinical hold was put in place in late 2012 based on preclinical toxicology findings of sympathetic neuronal changes. Extensive additional preclinical investigation into these sympathetic effects demonstrated no sympathetic neuronal loss, only small changes in neuronal size,

which was reversible upon cessation of treatment. The clinical hold related to these findings was lifted by the FDA in the spring of 2015.32 Phase 3 trials for anti-NGF antibodies are ongoing in OA, chronic low back pain, and cancer pain. Researchers and clinicians have become increasingly optimistic that anti-NGF therapies can become novel and highly efficacious treatments for chronic pain. What remains less clear is the precise mechanism or mechanisms underlying the efficacy of these agents. Answering these questions will require additional detailed pathway analysis and likely the availability of specific new pharmacological tools. Validation for Other Diseases. While the focus of this perspective is NGF’s role in chronic pain, other areas of ongoing therapeutic interest include oncology, neurodegeneration, and allergic airway disease. In oncology, an in-frame deletion in the NTRK1 gene and a splice variant in the TrkA receptor have been linked to a form of acute myeloid leukemia and neuroblastoma, respectively.33,34 However, most of the recent interest in TrkA from the oncology field lies in numerous reports of Trk fusion proteins as oncogenes. These Trk fusions often lack an extracellular neurotrophin binding domain, and the resulting constitutively active isoforms have led to small molecule approaches targeting the TrkA kinase domain.35 Numerous efforts in the Trk kinase inhibitor space are ongoing for oncology, and many of these inhibitors would likely have analgesic potential as well. These efforts will be covered in depth in a following section. C



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suffer from weak potency and/or pharmacokinetic deficits that would be expected of small therapeutic peptides.43 There have been several reports of functional antibodies to the p75 receptor (mechanism C, Figure 2). The first was a rabbit polyclonal antibody from the Reichardt Lab.44−46 Additionally, Rogers and co-workers developed several mouse monoclonal antibodies to p75,47 although none of these ligands has been characterized extensively in the literature. Khodorova and co-workers have described evidence that some NGFmediated mechanisms of peripheral neuronal sensitization are likely driven through the p75 receptor.46 Thus, a well characterized p75 in vivo tool would certainly be of interest to the field. A more recent and active clinical program has been disclosed around an anti-TrkA antibody (mechanism B, Figure 2) that is currently being developed by Glenmark and reported to be completing phase 1 testing. This was derived from an earlier rodent antibody, MNAC13, described by Lay-Line Genomics.48,49 This antibody may provide the first clinical reports of the effects of TrkA antagonism and could reveal a potentially different safety and efficacy profile compared to the more clinically advanced anti-NGF antibodies.

A second area of interest for NGF lies in neurodegeneration. Studies in the late 1980s and early 1990s showed that icv NGF injections improved basal forebrain cholinergic neuron survival in aged rodents as well as improved performance in several models of cognition.36,37 However, attempts at NGF therapy to date have been hampered by delivery and/or pharmacokinetic concerns. In addition, in small human pilot studies where NGF was injected icv, severe spinal pain in the lower back resulted, halting the studies.38 In the case of neurodegeneration, both TrkA agonists and antagonists of proNGF/p75 apoptotic signaling may be of interest and are under current investigation.19 However, not unlike the earlier NGF efforts, there will likely need to be a strategy in place to avoid the hyperalgesic effects of TrkA agonism. These areas of investigation are not within scope of this perspective. Finally, NGF and its receptors have also been implicated in airway inflammatory disease. Many of the pathways involved in antigen-triggered allergic asthma involve the neurotrophins and particularly NGF. An excellent review of this biology may be found elsewhere.39

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NGF PATHWAY MODULATION APPROACHES In a phenotypic sense, anti-NGF antibodies provide strong validation for the disruption of NGF signaling leading to analgesic effects both preclinically and in patients.40 However, clarity around TrkA vs p75 signaling pathways and their relative contributions to these effects is still evolving.19,40,41 There are data that would support the importance of each and these pathways, and interpretations are further complicated by potential cross-talk between the TrkA and p75 pathways. Figure 2 shows the various potential mechanisms whereby disruption of NGF-mediated signaling could be achieved pharmacologically. NGF neutralizing antibodies and NGF binding small molecules would function extracellularly and likely disrupt both TrkA and p75 mediated signaling (mechanism A). Receptor antibodies and small molecule antagonists (mechanisms B and C) would target the extracellular domain of the receptors and disrupt NGF binding to TrkA and p75, respectively. This approach could potentially provide specificity for each target receptor. Finally, TrkA kinase inhibitors (mechanism D) would target the intracellular kinase domain. Both large and small molecule tools have provided opportunities to probe these mechanisms, and thus both will be reviewed.

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SMALL MOLECULES Historically, most approaches to the discovery of small molecule NGF-targeted analgesics have focused on the inhibition of signaling through the TrkA pathway. Figure 2 shows a simplified version of the NGF−TrkA canonical signaling pathway. Briefly, upon binding of the NGF dimer to the TrkA receptor tyrosine kinase dimer, three tyrosines in the TrkA activation domain (Y670, Y674, and Y675) undergo autophosphorylation. This enables the autophosphorylation of additional tyrosines, specifically Y490 and Y785, which trigger activation of the PI3K, Ras, and phospholipase C-γ-1 (PLC-γ1) pathways.50,51 Activation of these pathways in vivo has been linked to multiple pharmacodynamic end points, including pain.52 In vitro, a commonly measured end point for the activation of these pathways in neuronal cells is neurite outgrowth.51 Another likely relevant downstream effect of NGF−TrkA mediated signaling (not shown in Figure 2) is the rapid increase in membrane expression and activation of the transient receptor potential vanilloid I (TRPV1) channel, an important nociceptive mediator. It was shown that TrkAmediated PI3K pathway activation is responsible for TRPV1 phosphorylation, resulting in membrane localization and activation of the TRPV1 channel.53 This may explain the acute thermal nociceptive effects of NGF. Small molecule interrogation of the NGF−TrkA pathway has focused on two primary approaches, each with different challenges. Extracellular antagonism of the NGF−TrkA binding event (mechanisms A or B in Figure 2) is one approach that may modulate TrkA-mediated downstream signaling and was reviewed by Longo and Massa in 2013.19 One potential benefit to this strategy may be greater likelihood of TrkA specificity. Since TrkB and TrkC bind different neurotrophins (BDNF, NT-3, NT-4), receptor homology is not as great in the extracellular region.54 Thus, the medicinal chemist has a greater likelihood of finding a unique binding motif on the neurotrophin extracellular binding domain of TrkA vs the TrkB and TrkC receptors. However, disruption of this sort of protein− protein interaction has historically proven difficult.55 Thus, it is unsurprising that to date, only a few compounds with this mechanism of action have been identified, all with unremark-

LARGE MOLECULES

While the history of the anti-NGF antibodies was covered in the previous section, there have also been additional ligands developed within the large molecule/peptide space. These have included TrkA receptor truncation proteins, such as the second immunoglobulin (Ig2) domain for TrkA, which is known as TrkAIg2 or TrkA domain 5 (TrkAd5).42 This protein is believed to bind to and sequester NGF in a mechanism not unlike the anti-NGF antibodies (however with potentially differing effects on proNGF sequestration, mechanism A, Figure 2). While the TrkAd5 construct demonstrated preclinical analgesic activity,42 human clinical testing has not been reported, likely due to less than optimal pharmacokinetic properties. Many other peptide-based approaches have been described that either block or inhibit NGF/TrkA interactions, NGF/p75 interactions, or both. However, none of these efforts have moved beyond the tool generation stage and typically D



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able potency.19 Alternatively, inhibition of the TrkA kinase activity (mechanism D in Figure 2), an intracellular event, should also result in the modulation of downstream NGF− TrkA mediated signaling. This approach has been more successful in finding drug-like small molecules, many with good selectivity across the kinome. 56,57 However, the identification of TrkA kinase inhibitors with isoform selectivity vs TrkB and TrkC has proven much more difficult. It has been postulated that improved subtype specificity may improve the safety profile of Trk inhibitors. Both extracellular antagonism and intracellular kinase inhibition approaches are described in greater detail below. Extracellular Antagonism of NGF−TrkA Binding. As was previously described, the binding of NGF to the extracellular binding domain of TrkA results in a cascade of signaling events that are believed to be responsible for (at least) some of the pro-nociceptive properties of NGF. Thus, antagonism of NGF−TrkA binding is expected to reduce TrkA activation and subsequent downstream signaling. Similar to the large molecule approach, disruption of this protein− protein interaction with a small molecule could be accomplished via two specific mechanisms of action. First, the small molecule antagonist could bind directly to NGF (similar to NGF sequestering antibodies) in a fashion that renders the NGF protein dimer incapable of making a productive contact with the TrkA receptor. It should be pointed out that, similar to NGF antibodies, this approach would have the potential to disrupt NGF−p75 interactions as well. Alternatively, a small molecule antagonist could bind to the TrkA receptor (similar to TrkA receptor antibodies), thus preventing NGF from appropriately accessing the receptor. This approach would be more likely to leave NGF-mediated p75 signaling intact. Both types of modulators have been described. It turns out that most reported functional antagonists have been shown to function via the former mechanism. In contrast, compounds reported to bind the TrkA receptor have generally been NGF mimetics (agonists), producing NGF-like effects. It may be possible to optimize a TrkA binding agonist into an antagonist, especially if there is structural information to inform the binding site and binding mode on TrkA relative to NGF itself. For the purpose of this Perspective, we will describe a few of the better characterized compounds in each class. NGF Binders. Several NGF binders have been described and are shown in Figure 3. In PC12 cells, ALE-0540 (1, Figure 3) inhibited both 125I-NGF binding to TrkA (IC50 = 5.88 μM) and NGF-induced TrkA autophosphorylation (EC50 = 22 μM).58 Furthermore, in DRG neurons, 1 dose dependently inhibited NGF-mediated neurite outgrowth. The authors commented that preliminary mechanistic results using NGFaffinity chromatography suggested that 1 did not bind NGF directly and likely bound to receptors TrkA and p75. However, in later reports, it was determined that 1 did, indeed, bind directly to NGF (vide infra). In vivo, 1 blocked mechanical allodynia in the L5/L6 nerve ligation model (rats) after ip dosing (ED50 = 38 mg/kg). When dosed it, 1 blocked mechanical allodynia in the same model and also blocked tactile allodynia produced by thermal sensitization in the rat hindpaw (ED50 = 34.6 and 50 μg, respectively). While these data are consistent with functional antagonism of the NGF−TrkA interaction, one cannot rule out the potential contributions due to p75 modulation. Functional studies to specifically assess p75mediated events were not reported in this manuscript. Later in this Perspective, we will discuss the role of NGF−p75-mediated

effects on intracellular ceramide−sphingolipid signaling that has been associated with hypersensitivity.

Figure 3. Chemical structures of NGF binders.

In addition to 1, PD90780 (2, Figure 3)59 and Ro 08-2750 (3, Figure 3)60 have been shown to bind NGF and prevent NGF association with both TrkA and p75. These studies also demonstrated functional antagonism of downstream TrkAmediated signaling events, such as TrkA autophosphorylation and neurite outgrowth, as well as p75-mediated events, such as apoptosis, when tested in the p75-positive/TrkA-negative cell line SK-N-MC. Another small molecule, PQC 083 (4, Figure 3),61 as well as several related analogues and compound 1−3, were shown to disrupt cell differentiation, another downstream NGF-mediated signaling event62 in PC12 cells (4: IC50 = 7 μM).61 Through the use of 3D docking analysis, the authors hypothesized that all four antagonists bound similarly to NGF (in the loop I/IV cleft). Recently, surface plasmon resonance (SPR) spectroscopy was used to assess all four compounds’ ability to inhibit NGF binding to immobilized TrkA and p75 receptors.63 Under the conditions, the binding affinities of NGF−TrkA and NGF−p75 were determined to be 13 and 15 nM, respectively. Table 1 summarizes the ability of each compound to inhibit 10 nM NGF binding to both receptors. It should be pointed out that the studies using 4 were confounded due to nonspecific binding of that molecule to NGF and both receptors. This property rendered 4 inappropriate for use in SPR assays. Of the other three compounds, it is interesting to note that none achieved maximum inhibition of NGF−p75 binding and all showed more potent inhibition of NGF−TrkA association vs NGF− p75. Furthermore, it was shown that 1, 2, and 3 did not show any direct binding to TrkA nor p75. Table 1. SPR Derived IC50s for Small Molecule Inhibitors of NGF Binding to p75 and TrkAa IC50 (max inhib) 1 2 3 4

P75 (μM)

TrkA (μM)

>300 (6.2%) 110 (65%) 244 (37%) n.d. (70%)

149 (44%) 47 (100%) 33 (100%) nd (nd)

a

Table 1 used with permission from Sheffield, K. S.; Kennedy, A. E.; Scott, J. A.; Ross, G. M. Characterizing nerve growth factor-p75(NTR) interactions and small molecule inhibition using surface plasmon resonance spectroscopy. Anal. Biochem. 2016, 493, 21−26, Elsevier.63

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TrkA Binders/NGF Mimetics. Several compounds have been reported to bind directly to TrkA.19 Interestingly, none of these agents have demonstrated convincing antagonist function and are thus better referred to as NGF mimetics. However, none can truly recapitulate NGF-like agonist function. This perspective will highlight the data for the most characterized TrkA binder, antidepressant amitriptyline (5, Figure 4).64

More recently, Shoemark and co-workers have used nuclear magnetic resonance (NMR) to inform a solution structure of a 13 C, 15N-labeled TrkAIg2 analogue, known as TrkAIg2-NMR.69 This construct demonstrated wild-type TrkAIg2 activity such as NGF binding and inhibition of NGF-induced neurite outgrowth. 1H−15N heteronuclear single quantum correlation (HSQC) spectra showed significant chemical shift perturbations (CSPs) or line broadening in the presence of NGF, with most of the spectral changes occurring in the region that binds the N-terminal helix of NGF. This is consistent with the 2.2 Å X-ray crystal structure of NGF bound to TrkAIg2.66 Similar 1 H−15N HSQC studies using amitriptyline and TrkAIg2-NMR revealed over 20 CSPs, 13 of which were common with the NGF structure and map to the binding site of the N-terminal helix of NGF. This is consistent with 5 and NGF having overlapping binding sites on TrkAIg2. Using a docking program, the group hypothesized a putative 5−TrkAIg2 binding pose. They confirmed 5−TrkAIg2 binding (IC50 = 60 μM) in a competition binding assay using 125I-NGF in HEK cells. Interestingly, they also showed that 5 inhibited NGFinduced phospho-MAPK formation in HEK293 cells (IC50 = 86 μM), in contrast to the previously reported NGF agonist-like function in different cells/tissues such as PC12 and DRGs. Further exploration will be required to better understand the function of 5 in tissues most relevant to pain states. TrkA Kinase Inhibitors. Because of the NGF−TrkA target validation story in the early 2000s, many research groups have targeted TrkA with small molecule kinase inhibitors. As one might expect, finding potent inhibitors of the TrkA kinase has not proven as difficult as finding selective inhibitors. Two excellent reviews on TrkA inhibitors have been published. The first covered the work from publications and patent applications from 2002 to 200856 and the second from 2009 to 2013.57 Because these are exhaustive reviews with associated references, we would refer the readers to these papers for details. As previously mentioned, there has been much interest in the development of TrkA kinase inhibitors for oncology indications and clinical progress in oncology is probably more advanced, compared to pain indications. The majority of these inhibitors are multitargeted kinase inhibitors, although a few have been reported as pan-Trk inhibitors with good selectivity vs the rest of the kinome. While oncologic indications are not within the scope of this perspective, we will provide a brief update on clinical oncolytic agents targeting TrkA in a later section. Here, we will provide an overview of the evolution of TrkA inhibitors through the years from a more historical and strategic viewpoint and highlight the more recent identification of novel inhibitors with specific emphasis on applications to pain research. In pharma, initial work to discover novel TrkA inhibitors was probably opportunistic, likely emerging from kinase inhibitor libraries from previous kinase target efforts. Most of these compounds likely bound in the conserved ATP binding site and engaged the hinge residues in a manner that allowed the activation loop residues Asp-Phe-Gly (DFG) to be in a DFG-in conformation, which is similar to the unliganded (apo) conformation. These are known as type I inhibitors. Some, however, bound to the ATP site but also extended into a hydrophobic pocket that is accessible when the DFG residues fold out (DFG-out conformation). These are known as type II inhibitors. Several papers have described the nature of type I, type II, type III (ATP site/nonhinge binders), and type IV (allosteric) kinase inhibitors, and the reader is encouraged to

Figure 4. Chemical structures of potential TrkA binders.

Jang and co-workers showed that 5 could bind to the TrkA and TrkB extracellular domains (ECDs) with Kis of 3 and 14 μM, respectively.64 Similar to NGF, 5 triggered dimerization and activation of downstream signaling such as TrkA autophosphorylation and MAP kinase and PI3K/AKT pathway activation in primary hippocampal and cortical cultures that express TrkA. Other related tricyclic antidepressants, such as imipramine (6, Figure 4),64 did not show these activities. Similar to NGF, amitriptyline induced neurite outgrowth in PC12 cells and this effect was blocked by inhibitors of TrkA kinase, PI3K and MEK1. In vivo, 5 (15 mg/kg, ip) induced tyrosine phosphorylation of TrkA and TrkB in mouse brain. All of these activities are consistent with neurotrophin mimetic behavior. However, in addition to binding TrkB with similar affinity as TrkA, 5 demonstrated some non-NGF-like activities both in vitro and in vivo. Specifically, 5 induced TrkA phosphorylation of Tyr751 but not Tyr490 in hippocampal neurons. Additionally, after 5 days of dosing, 5 promoted TrkATrkB heterodimerization in mouse brain. This effect was also observed in vitro in HEK293 cells that had been cotransfected with TrkA and TrkB.64 Clearly, 5 represents an interesting small molecule in that it binds to TrkA. It may represent a good starting point for the optimization of more potent analogues with either NGF−TrkA agonist or antagonist function. Of course, for analgesic activity, TrkA antagonism would be desired. Thus, optimizing this molecule into a selective TrkA antagonist would be significantly assisted by structural information describing its binding mode to both TrkA and TrkB. Jang and co-workers began to map possible binding sites for 5 on TrkA. They showed that deletion of the immoglobulin domains Ig1, Ig2, and Ig1+Ig2 in TrkA had no effect on the binding of 5, while removal of the entire extracellular domain significantly diminished binding. TrkA truncation studies showed that the first leucine-rich motif (LRM1) was required for binding 5. This is a bit surprising due to the evidence that suggests that the TrkAIg2 domain, also known as TrkA domain 5 (TrkAd5), represents the NGF binding domain of the receptor. This evidence includes mutagenesis/domain deletion65 and structural information such as protein−protein X-ray crystal structures of the NGF dimer associated with two molecules of TrkAIg2.66,67 Furthermore, as already described, TrkAIg2 functions as a soluble NGF receptor with picomolar affinity. It was shown to sequester NGF and was analgesic in multiple preclinical pain models.42,68 F



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review those manuscripts for a more thorough explanation of kinase enzyme inhibition modes.70,71 Most of the early TrkA inhibitors inhibited multiple additional off-target kinases. Furthermore, it was realized that it may be unlikely to see high TrkA selectivity vs TrkB and TrkC with these types of inhibitors due to the very high sequence homology in the kinase domains. Using an analysis previously described by Cherry and Williams72 for ATP binding site kinase inhibitors, there are 49 residues in each Trk kinase domain that could potentially interact with an inhibitor binding in either a type I or type II fashion. Of those, all but two residues are common among the three isoforms and those two differences are quite conservative. Specifically, two arginines in TrkA (R593/R599) are replaced by lysines (K638/K644 and K622/K630) in TrkB and TrkC, respectively.73 In the absence of ligand−protein Xray crystal structures in these early years, some groups built homology models to help understand and visualize inhibitor binding modes. Nonetheless, the compounds reported during the first decade of TrkA inhibitor research remained largely pan-Trk inhibitors with significant off-target kinase activity. Many of these compounds were reported to have efficacy in preclinical models for pain and oncology end points, albeit with less than desirable safety profiles. Starting in 2012, several groups reported ligand−protein Xray crystal structures of all three Trk isoforms.73−77 This advance allowed medicinal chemists to use structure-based drug design to rapidly identify compounds with improved potency and selectivity vs non-Trk kinases. Initially, these methods did not seem to provide a route to TrkA selectivity vs TrkB and TrkC. Figure 5 shows an overlay of several ligand−TrkA X-ray crystal structures from the Protein Data Bank (PDB). All of these structures engage the hinge and are either type I or type II inhibitors. It can be observed that the two unique TrkA residue side chains, R593 and R599, do not seem to make significant productive interactions with any of these ligands and generally project their side chain atoms outside of the kinase binding pocket. Thus, it is apparent why all of the early reports described only pan-Trk inhibitors. Nonetheless, with improved potency and selectivity vs non-Trk kinases, several groups were able to optimize properties and pharmacokinetic parameters and demonstrate efficacy in multiple rodent pain models. Below, we will highlight several specific demonstrations of this approach. A few years prior to 2012, AstraZeneca reported on the optimization of a screen hit to give AZ-23 (7, Figure 6),78 a potent (15:1 peripheral to CNS exposed), ataxia was observed at higher doses that resulted in brain target coverage. Thus, another medicinal chemistry goal would be to design compounds with even less CNS exposure.

Figure 9. Structure of 17.

The Array group has recently reported on the design of TrkA inhibitors with cellular potency less than 1 nM and >1000-fold selectivity vs TrkB and TrkC. They have shown that selective TrkA inhibitors have similar efficacy to pan-Trk inhibitors in multiple models of both acute and chronic pain, including the rat CFA model, at similar doses and exposures. A selective TrkA inhibitor did not induce hyperphagia in rats, which supports the hypothesis that this adverse effect was BDNF/

Figure 10. Array TrkA inhibitors. I



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and other patent applications describe related compounds that are less selective or nonselective.91−98 Four of these structures are highlighted in Figure 10. All four compounds were reported to have IC50s < 100 nM in a TrkA kinase binding assay and were tested in a panel of 230 off target kinases at 10 μM. It was reported that 18 and 19 showed no inhibition of any off target kinases,89,90 including TrkB, while examples 20 and 21 were similarly selective vs non-Trk kinases but showed near-maximal inhibition of TrkB at 10 uM.91,93 TrkC data was not reported. It is particularly interesting to note the subtle differences in structures between some of the TrkA selective and nonselective (vs TrkB) inhibitors. Array recently announced a collaboration with the Asahi Kasei Pharma Corporation to co-develop their selective TrkA inhibitors for pain and inflammation.99 Very recently, the Merck group reported on the discovery of selective TrkA inhibitors, unrelated to their previous pan-Trk inhibitors.76 The most characterized molecule, compound 22, is shown in Figure 11.100 The group described TrkA in vitro potency (cellular) of 6 nM, with 230- and 190-fold selectivity vs TrkB and TrkC, respectively. Similar to the Pfizer approach, the Merck group increased Pgp substrate potential to reduce central exposure and assessed peripheral to central target engagement by measuring phospho-TrkA levels in skin and brain. Compound 22 demonstrated a 6.8-fold preference for skin vs brain target engagement.

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TRKA KINASE INHIBITORS IN THE CLINIC

While the preclinical evaluation of multiple TrkA kinase inhibitors for pain conditions have been described, there have been very few reports on the clinical experiences with TrkA inhibitors for pain-related conditions and no published clinical POCs. While the intent of this perspective is to focus on analgesia, we felt that it would be helpful to include some of the TrkA inhibitors currently being studied in the clinic for other indications such as oncology and psoriasis. It should be pointed out that some of the agents described in the previous sections have demonstrated preclinical antitumor efficacy and may be on a path toward clinical investigations in oncology. Conversely, some of the drug candidates being pursued for oncological indications may prove beneficial for analgesia. For pain and psoriasis, some investigators have explored alternative routes of administration of TrkA inhibitors in an effort to maximize local exposures and minimize systemic exposures of the agents. Creabilis has demonstrated that CT327 (structure not disclosed), a pegylated analogue of K252a (23, Figure 12) and potent pan-Trk inhibitor,101 was effective in reducing the pruritis associated with psoriasis after topical administration in patients.102 Creabilis has also communicated that they are investigating an additional topical TrkA inhibitor, CT340 (structure not disclosed), for neuropathic pain and inflammation.103 In another example of local delivery investigations, SanofiGenzyme is studying the intra-articular delivery of the TrkA inhibitor GZ389988 (structure not disclosed). Intra-articular injection of this agent in two preclinical rat models of joint pain, the streptococcal peptidoglycan polysaccharide (PGPS) and monoiodoacetate (MIA) models, demonstrated gait improvement and joint analgesia, respectively.104 In 2015, clinical studies began with intra-articular GZ389988 in patients with painful knee OA.105 While not specifically disclosed, the structure of GZ389988 may be related to compound 24 (Figure 12), which was described in a recent patent application.106 This compound was reported as a potent TrkA and TrkB inhibitor, with selectivity over TrkC, in a kinase assay. In 2015, Purdue Pharma acquired the selective TrkA inhibitor VM-902A (structure not disclosed) from VM Pharma.107 VM-902A was described as an orally bioavailable, allosteric inhibitor with peripheral only distribution. VM-902A may be related to the compounds described in patent applications by VM Pharma, such as compound 25 (Figure 12).108,109 Compound 25 was reported to inhibit TrkA, but not

Figure 11. Merck selective TrkA inhibitor.

Figure 12. Clinical TrkA inhibitors for pain and psoriasis. J



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Figure 13. Clinical TrkA inhibitors for oncological indications.

inhibitors. It should be pointed out that 17 is the only drug candidate in this group that has been reported to be selective for the Trks vs a large collection of other kinases.83 Drug candidates MGCD-516 (26),112b RXDX-101 (27),115,117 DCC2701 (28),112c TSR-011 (29),112a,113 and XL-184 (30)119 are all reported to inhibit a range of other kinase targets such as cMet, ALK, Tie2, ROS1, VEGFR2, and others. Thus, any reported clinical outcomes will not provide definitive validation for the NGF−TrkA pathway in oncology. Nonetheless, we will review a few valuable and interesting learnings from these studies that may help inform the role of TrkA inhibition in cancer and analgesia. It has recently been reported that some NTRK chromosomal rearrangements result in oncogenic fusion proteins.113 These Trk-overexpressing fusions are constitutively active, with increased canonical Trk pathway signaling, and are responsive to Trk kinase inhibitors both in vitro and in vivo. In fact, because these fusion proteins lack the Trk extracellular binding domains, small molecule kinase inhibitors would clearly be the most appropriate intervention approach. Several clinical studies have reported robust efficacy with the Trk kinase inhibitors 17 and 27 in patients with oncogenic TrkA fusion proteins. These reports have described responses in patients with multiple tumor types containing Trk fusions such as lung,114 colorectal,115 and soft tissue sarcoma.116 In the case of 27, which also targets the anaplastic lymphoma kinase (ALK) and the protooncogene tyrosine−protein kinase ROS1, it was noted that after a remarkable initial response, a patient experienced drug resistance to 27 and disease progression. It was determined that this patient’s resistance was the result of two TrkA kinase domain mutations. Specifically, G595 was mutated to arginine (G595R) and G667 was mutated to cysteine (G667C). This exact resistance mechanism could be recapitulated in mice by

TrkB nor TrkC, in a cell-based functional assay. Additionally, it demonstrated efficacy in multiple rat pain models, including the CFA-induced inflammatory pain model and the chronic constriction injury (CCI) model after either sc or po dosing.108 Purdue Pharma has reported that this compound has completed phase I studies and phase II studies will commence in 2016 for the potential treatment of chronic pain.107 Finally, Ono Pharmaceuticals has announced the development of Ono-4474 (structure not disclosed) for pain indications. Ono-4474 was described as a selective pan-Trk inhibitor with low CNS exposure. Preclinical efficacy in the rat monoiodoacetate (MIA) model after oral administration was recently described. Similar to the hyperphagic effects described by some Array pan-Trk inhibitors, Ono also described adverse effects associated with increased food consumption, albeit at exposures 36-fold higher than the efficacy exposures.110 Ono has announced the start of phase I clinical trials to assess the safety, tolerability, and pharmacokinetic properties of Ono4474 in humans. Additionally, they intend to assess effects on NGF-induced hyperalgesia in healthy volunteers.111 Several Trk kinase inhibitors have been explored clinically for cancer indications and there have been several recent publications on this topic,35,112a,113 highlighting some of the recent clinical outcomes. One important difference between oncological and pain indications for TrkA inhibitors is safety assessment. Because phase I trials in oncology utilize cancer patients, there exists no information on adverse effects of these agents in healthy volunteers. Furthermore, the tolerance for drug related adverse effects for nononcological indications would be significantly less than for oncology indications. Figure 13 shows the structural diversity identified in Trk kinase inhibitors in current clinical development in oncology. All of these drug candidates are described as potent pan-Trk K



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Figure 14. X-ray crystal structures of type I and type II inhibitors in the TrkA active site. These structures demonstrate the relative proximity of mutant TrkA residues to the ligands. (A) X-ray crystal structure of 31 bound to TrkA (PDB ID code 4YPS), with highlighted mutations. (B) X-ray crystal structure of 32 bound to TrkA (PDB ID code 4PMM), with highlighted mutations. (C) Chemical structures of 31 and 32.

implanting tumor cells from the patient (pretreatment) and dosing the animal with 27 until resistance developed. While there exist no published X-ray crystal structures of the 27− TrkA complex, there was a recent disclosure of 27 bound to ALK in the conserved hinge region of the ATP binding site.117 Thus, it seems likely that it would bind similarly to the hinge in TrkA. In fact, Doebele and co-workers modeled 27 in the TrkA kinase binding site, showing the potential impact of each mutation on ligand binding. This model predicted that the G595R mutation would confer greater resistance than the G667C mutation based on the proximity of the mutation relative to the inhibitor in the putative modeled binding site. It should be pointed out that kinase inhibitor-induced acquired resistance was also observed in related tyrosine kinase targets (ALK, ROS1, EGFR, and KIT), with mutations occurring at the same conserved kinase domain residues.116 Experimentally, using engineered Ba/F3 TPM3-TrkA fusion protein containing cells, it was indeed shown that wild-type (WT), G595R, and G667C mutants differentiated in vitro with respect to sensitivity toward this inhibitor. Compound 27 reduced cell viability in WT cells (IC50 = 0.006 μM) but required about 10× higher concentrations to achieve similar effects in the G667C mutants (IC50 = 0.061 μM). In the G595R mutants, 27 was inactive (IC50 > 1.0 μM). Similar results were described for both 17 and 29, both of which likely bind similarly in the ATP pocket. To help with the visualization of these mutants in the TrkA kinase binding pocket, we have included two structures from the Protein Data Bank. Figure 14A shows the structure of a typical type I inhibitor (31, 4YPS)77 bound to TrkA while Figure 14B represents a typical type II inhibitor (32, 4PMM).76 The chemical structures of the ligands are shown in Figure 14C. As can be seen in Figure 14A,B, the residue at position 595 sits almost directly below the hinge. Thus, one would expect that the mutation of glycine to arginine would almost certainly disrupt hinge binding inhibitors. It is this mutation that confers the greatest resistance to the inhibitors that were tested, which were all hinge binders. Alternatively, residue 667 is located just

before the DFG motif and thus might be less likely to display broad resistance among some type I and II inhibitors. Type III inhibitors, which bind in the hydrophobic pocket in a DFG-out conformation, but not in the hinge, may not be impacted by the G595R mutation and may be more likely to be resistant to the G667C mutation. Finally, allosteric (type IV) inhibitors may not be impacted by either mutation. Of course, nonkinase domain mutations could always occur and confer resistance to allosteric inhibitors as well. In addition to responses of patients harboring oncogenic NTRK1−3 fusions, there was another interesting report of clinical efficacy with cabozantinib (30, Figure 13) in castrationresistant prostate cancer patients with bone metastases.118,119 Bone pain is a debilitating condition associated with bone metastases, and most patients who have bone metastases require narcotics for pain control. Compound 30 is a potent pan-Trk inhibitor with multikinase activity, so the specific role of TrkA in the response cannot be determined. However, in addition to significant positive responses on bone scan and survival end points, there was a clinically meaningful improvement in pain relief. It is impossible to know the source of this analgesic response, and it is quite possible that it is solely attributable to the patients’ improved condition as represented by the bone scan data. Nonetheless, the analgesia is encouraging and may suggest a role for TrkA in cancer pain.

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EMERGING POTENTIAL DRUG TARGETS WITHIN THE NGF PATHWAY As was demonstrated in Figure 2, much is known about the canonical signaling pathways downstream of the NGF-induced TrkA activation event. Many of these downstream effectors are well characterized historic drug targets for oncology and a host of additional indications. These include kinase targets such as Ras, Raf, MEK, MAPK, PI3K, AKT, and others.70 On the basis of past clinical investigations, it seems unlikely that inhibitors of such ubiquitous signaling mechanisms would have the necessary safety profile required for analgesic therapies. L



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However, inhibitor tools of these targets provide extraordinarily valuable preclinical information regarding NGF pathway mechanisms in pain states. NGF-mediated changes in various ion channel targets have been described.120 One well characterized downstream drug target of TrkA is the TRPV1 channel.121 Agonists and antagonists of this target have been studied for pain, both preclinically and clinically.122 In fact, TRPV1 is considered a highly validated target for several pain indications. Unfortunately, adverse effects related to thermoregulation have precluded the successful registration of TRPV1 modulators.122 Because of the existing extensive literature treatment committed to both the canonical kinase pathways and TRPV1, we have not included them in this Perspective and the reader is encouraged to refer to the references cited herein. In recent years, several additional targets associated with preclinical and clinical pain end points have been linked to the NGF pathway, albeit with some incomplete understanding. Thus, we felt it was appropriate to include a few of these in this Perspective. Angiotensin II Type 2 Receptor Modulators. Over the past several decades, significant understanding of the renin− angiotensin system (RAS) has emerged with most of the attention being toward the role of the small peptide angiotensin II (Ang II) in cardiovascular disease.123 The RAS pathway is shown in Figure 15. Several targets along this pathway, specifically renin inhibitors, angiotensin converting enzyme (ACE) inhibitors, and Ang II receptor antagonists have demonstrated preclinical and clinical cardiovascular benefit.124 Drugs that modulate this pathway are among the most prescribed agents in the world.125 While the pharmacological effects associated with cardiovascular end points, such as hypertension, are known to be mediated through the Ang II type 1 receptor (AT1R),126 the role of the Ang II type 2 receptor (AT 2 R) in human disease has been studied significantly less. AT1R and AT2R are class A G-protein coupled receptors (GPCRs), which share only 32% sequence homology.127 The role of the RAS pathway in multiple pain conditions has been studied both preclinically and clinically, and two excellent reviews have been recently published.128,129 However, understanding has been confounded by multiple paradoxical results. For example, modulators have been reported to have both analgesic and hyperalgesic effects in preclinical animals and in humans, depending upon the pain condition and specific target in the pathway.129 Specifically, ACE inhibitors can show hyperalgesic effects in humans.130 On the basis of preclinical studies in rodents, it has been hypothesized that this is due to the role of ACE in degradation of pain and inflammation mediators such as bradykinin.131 Research in recent years has demonstrated a potential role for Ang II, via AT2R, in neuronal hyperexcitability and neurite outgrowth.132 Ang II and AT2R are present in sensory neurons of rat and human DRG and nerve fibers in peripheral nerves, skin, bowel, and urinary bladder.133 In 2006, Plouffe and coworkers showed that Ang II induced TrkA receptor phosphorylation (Y490), MAPK phosphorylation, and neurite outgrowth in NG108-15 cells.134 These Ang II-mediated effects could be abrogated by pan-Trk inhibitor AG879 (33), consistent with modulation of the NGF−TrkA signaling pathway. Furthermore, MEK inhibitor PD98059 (34) similarly inhibited Ang II-induced MAPK phosphorylation and neurite outgrowth in NG108-15 cells, also consistent with cross-talk between AT2R and the NGF/TrkA pathway. Subsequently, it was shown that in DRGs, while Ang II induced neurite

Figure 15. Renin−angiotensin system (RAS) pathway and differentiation of some effects mediated by the AT1R and AT2R receptors.

outgrowth similar to NGF, AT2R antagonists were unable to modulate NGF-induced neurite outgrowth.135 Figure 16 highlights some of the effects that have been observed that are consistent with AT2R-mediated transactivation of the TrkA signaling pathway. More recently, Guimond and co-workers have shown that the subtype selective small molecule AT2R agonist C21/M024 (35, Figure 17)136 stimulated MAPK phosphorylation and neurite outgrowth, similar to Ang II, in NG108-15 cells.137 Interestingly, the isomeric C38/M132 (36, Figure 17),136 an AT2R antagonist, was shown to inhibit these downstream effects in a manner similar to another AT2R antagonist PD123319 (37, Figure 17).138 Collectively, these data seem consistent with pathway convergence at the TrkA phosphorylation event. Additional studies with these and other ligands should help clarify the connection between these two pathways. Several groups have described analgesic effects of AT2R antagonists in rodent pain models such as the chronic constriction injury (CCI) model of neuropathic pain,138,139 the prostate cancer induced bone pain model,140 and the CFA model for inflammatory pain141 in both rats and mice. It was shown that efficacy was abolished in AT2R knockout mice, consistent with the effect being AT2R mediated.139 In humans, it has been reported that the potent and selective AT2R antagonist EMA401 (38, (S)-enantiomer of PD126055, Figure 17) demonstrated clinical efficacy in patients with post herpetic neuralgia after 4 weeks of dosing (PO, 100 mg, BID). Compound 38 was superior to placebo and similar to active comparators, such as pregabalin (300 and 600 mg) and gabapentin (1200 mg), and showed no serious adverse effects.142 While this is an exciting proof of concept study, multiple gaps exist in our understanding of the RAS pathway as it relates to both the NGF pathway and analgesic effects. As this molecule progresses in the clinic, additional preclinical studies will likely prove valuable in further understanding of this mechanism of action. Sphingosine-1-phosphate (S1P) Receptor Modulators. The ceramide−sphingosine−sphingosine-1-phosphate (S1P) signaling pathway has been studied extensively in association with many biological processes and diseases.143 In addition to signaling activities of their own that initiate various inflammation pathways,144 ceramides are hydrolyzed by M



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ceramidases to produce sphingosine, which can be elaborated to S1P by two sphingosine kinases, SphK1 and SphK2.145 S1P can bind to and activate at least five G-protein coupled receptors S1PR1−5 (Figure 18).143 Over the past few decades, this pathway has been linked to multiple pathological conditions such as cancer, inflammation, and autoimmune disorders.146,147 Consequently, multiple approaches to modulation of the pathway have been explored, such as inhibition of sphingosine kinases,148 S1P neutralizing antibodies,149 and S1PR modulators.150 Perhaps the most clinically validated disease that is responsive to S1P pathway modulation is multiple sclerosis (MS), where numerous agents have demonstrated clinical improvement in MS patients.151 The S1PR modulator FTY720 (fingolimod, 39, Figure 19) is approved for the treatment of MS.152 In the case of many S1PR1 modulators, including 39, the agents are difficult to categorize as agonists or antagonists. Many of these pro-drugs are metabolized to a phosphorylated form by sphingosine kinases, rendering them S1P1 agonists. Subsequently, upon binding to the S1P1 receptor, they can cause receptor internalization, making them functional antagonists in some tissues.151 Because of the significant complexity and massive collection of literature dedicated to the study of the ceramide− S1P pathway in disease (see references cited above), we suggest that the readers consult many of the existing review articles on this pathway for nonpain indications. The role of the S1P pathway in peripheral pain circuits and, specifically, NGF-mediated end points, has been investigated through the years. As early as 1997, Edsall and co-workers

Figure 16. Ang II binds to the GPCR AT2R and triggers transactivation of the TrkA pathway, leading to end points such as neurite outgrowth. In a manner similar to NGF-stimulated downstream signaling, these effects can be inhibited by pan-Trk and MEK inhibitors.

Figure 17. Selective AT2R modulators. N



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to the plasma membrane, where its colocalization with substrate sphingosine produced S1P.154 NGF-mediated SphK1 translocation to the plasma membrane was not observed in the TrkA negative mutant PC12 nnr5 cells, providing further evidence for the involvement of TrkA signaling in neuronal S1P synthesis.154 As previously described, NGF causes neurite outgrowth in PC12 cells in a TrkA-dependent fashion. Toman and coworkers further showed, via S1PR1 knock-down, that NGFinduced neurite outgrowth was S1PR1 dependent.154 A role for the NGF−TrkA−S1P pathway in chemotherapyinduced peripheral neuropathy (CIPN) has recently been described. Many patients on paclitaxel, which is indicated for the treatment of multiple types of cancer, experience painful CIPN, a significant dose-limiting adverse effect.155 The mechanism underlying this effect in humans is unknown. Preclinically, it has been shown that paclitaxel induces hypersensitivity and allodynia in rats and these effects correlate with increased expression of NGF, TrkA, and phospho-TrkA in DRGs. Additionally, pan-Trk inhibitor 23 partially reversed these effects in paclitaxel-treated rats.156 In a separate report, it was shown that, in rats, paclitaxel-induced neuropathic pain end points could be blocked by S1PR1 modulators 39, CYM5442 (40), ponesimod (41), and NIBR14 (42) in a dose-dependent fashion.157 These results are supportive of a role for the NGF− TrkA−S1P pathway modulators in paclitaxel-induced CIPN. Alternatively, there is an emerging story around the importance of NGF signaling, via the p75 receptor, to activate sphingomyelin signaling, leading to an increase in intracellular ceramides and sphingolipids and increased neuronal sensitization. Specifically, it was shown by Zhang and co-workers that NGF, via production of ceramide, was associated with an increase in action potential firing in isolated sensory neurons.158 This group also demonstrated that a p75 antibody could

Figure 18. Ceramide−S1P biosynthetic pathway. Ceramides are converted to S1P through the action of ceremidases and sphingosine kinases. S1P-mediated signaling occurs via five GPCRs S1PR1−5.

described NGF-induced increases in sphingosine kinase activity and S1P levels in PC12 cells.153 These end points could be inhibited by the pan-Trk inhibitor 23 (Figure 12), suggesting a Trk-mediated effect. Later, Toman and co-workers showed more specifically that in PC12 cells, NGF translocated SphK1

Figure 19. S1P1 modulators. O



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pathways.167 However, it has also been shown that proNGF can serve as a trophic factor in cellular contexts where TrkA is the dominant receptor vs p75. Similarly, NGF’s actions are more complex than simply TrkA receptor preference. NGF binding to TrkA may be influenced by p75 acting as a co-receptor. Furthermore, NGF appears to bind to p75 when sortilin is not present, activating a different kinase pathway than that modulated by proNGF/p75/sortilin activity.168 A good argument can be made for efficacy attributable to either NGF or proNGF sequestration, via either p75 or TrkA mediated signaling or a combination of all of the above. Clearly, selective ligands for each of these mechanisms will help answer some of these questions. Given the degree of activity currently directed toward the development of TrkA inhibitors and antagonists, this will likely be the first NGF receptor to achieve clinical proof of concept in a chronic pain population. In addition, there is certainly a reasonable hypothesis to develop p75 antagonists for pain, but these efforts have not yet been reported to be in development. Beyond modulating the more proximal receptors, there are a multitude of other points of intervention in the NGF pathway that may be relevant for analgesic drug discovery. The classical mechanisms of NGF-dependent hyperalgesia have typically included systemic (non-neuronal) pro-inflammatory effects of NGF, direct effects of NGF on primary afferent neuron sensitization, and central sensitization of the spinal cord dorsal horn circuits. Additionally, given the aforementioned effects of the anti-NGF antibodies on autonomic neuronal morphology, it is also worth considering this as an additional potential mode of action. Many chronic pain conditions have been shown to have a strong sympathetic component, often involving sympathetic postganglionic neuronal dendrites. These dendrites form basket-like structures around primary afferent DRG somas, directly sensitizing these neurons through increased adrenergic signaling.169 The anti-NGF therapies could be causing an atrophy of these sympathetic dendritic processes and reversing primary afferent sympathetic sensitization. Figure 20 shows four distinct areas through which NGF (and proNGF) may produce their pro-nociceptive effects. These include nonneuronal pro-inflammatory effects, central and peripheral neuronal sensitization, and sympathetic driven adrenergic effects. The pro-inflammatory effects of NGF are well described and have been reviewed in detail.170 NGF has the ability to trigger mast cell degranulation, is involved in some immune cell maturation, and is expressed on mononuclear cells. While there are certainly secondary effects of these pro-inflammatory mediators on neuronal sensitization, this mechanism of action and related drug targets have been reviewed in detail in many prior reports on general inflammation.171 For the purposes of this Perspective, we have chosen to focus on the direct neuronal actions of NGF and related targets. NGF-dependent primary afferent sensitization is typically discussed in terms of either thermal or mechanical hyperalgesia. However, in considering potential drug discovery opportunities, it may be more beneficial to consider acute/post-translational mechanisms versus transcriptionally dependent mechanisms of sensitization. These acute circuits have been described in great detail and usually involve experiments in isolated DRG neurons. NGF triggers multiple phosphorylation pathways resulting in up- or down-regulation of terminal proteins, usually ion channels or other cell surface receptors.172,173 There have been numerous reports of NGF acutely potentiating TRPV1

prevent NGF-induced neuronal excitability and sensitization, however, the effect was restored by the addition of exogenous ceramide.159 The group ultimately designed a collection of experiments to inform the mechanism of action for the enhancement of action potentials (APs) in sensory neurons. It was shown that both sphingosine and S1P, when intracellularly perfused, similarly enhanced APs and the effects of NGF, ceramide, and sphingosine could be inhibited by the SphK1/2 inhibitor dimethylsphingosine (DMS, 43).160 APs enhanced by the intracellular perfusion of S1P were unchanged by the addition of 43. These data are consistent with S1P, via one of the S1P receptors, playing a direct role in NGF−p75-induced neuronal sensitization. While the relative contributions of TrkA and p75 toward activating neuronal excitation pathways may be unclear, the evidence does seem to suggest a convergence toward S1P and the S1P receptors. Recently, multiple authors have published thorough reviews on the role of the ceramide−S1P pathway in pain.161−163 These reviews document many additional converging lines of evidence implicating this pathway in both peripheral and centrally mediated sensitization and highlight multiple studies, both in vitro and in vivo, that demonstrate how activation of the pathway can lead to neuron excitability and hyperalgesia in rodents. Correspondingly, many examples of pathway modulation have demonstrated the ability to block pain end points such as thermal and mechanical hyperalgesia. Perhaps one of the more convincing data sets describing the role of the S1P pathway in pain states was described by Doyle and coworkers.164 They showed that intraplantar injection of ceramide produced thermal hyperalgesia in rats, which could be attenuated by the SphK1/2 inhibitor SKI-I (44) in a dose response fashion. It was also shown that the anti-S1P antibody LT1002 had a similar effect, as did the S1PR1 antagonist W146 (45, Figure 19). Importantly, both the control antibody LT1017 and the inactive enantiomer of W146 (W140, 46) showed no effect on ceramide induced hyperalgesia, consistent with an S1PR mediated effect. Similarly, 39, a functional antagonist of S1PR1, showed dose-dependent antinociceptive activity in both the formalin and spared nerve ligation models.165 In spite of preclinical validation and pharmacological evidence for a role in multiple pain states, clinical studies with S1PR modulators for pain indications have not yet been reported. As previously discussed, several S1PR modulators are in development for MS and other autoimmune/immunemodulatory indications. Perhaps some of those agents will ultimately be investigated for uses in pain relief.

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CONCLUSIONS AND FUTURE APPROACHES FOR THERAPIES The unprecedented efficacy of the anti-NGF therapies currently in late stage clinical testing makes this pathway an intriguing opportunity for future drug development efforts. As is often the case when trying to deconvolute clinical efficacy, the precise mechanism of action of the anti-NGF therapies is not completely understood. While sequestration of NGF and the resulting dampening of NGF-dependent signaling is the presumed primary mechanism of action, even this could be called into question. There is good evidence that proNGF can play a prominent role in neuronal excitability.46,166 It is unclear if anti-NGF therapies may also sequester proNGF. ProNGF is the preferred ligand of p75, especially in the background of sortilin as a coreceptor and is often linked to pro-apoptotic P



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Figure 20. NGF and proNGF-mediated effects have been linked to inflammation, peripheral sensitization, central sensitization, and sympathetic neuron effects. All of these effects may play distinct roles in NGF-mediated hyperalgesia.

site, and small molecules directed at the internal kinase domain through both orthosteric and allosteric mechanisms. Targeting the external domain may prevent TrkA internalization and the formation of the signaling endosome. Alternatively, depending on the antibody, this approach may induce receptor internalization and lead to lysosomal degradation rather than formation of the signaling endosome. While a kinase directed mode of action may not prevent the formation of the endosome, it would be expected to modulate most of the transcriptional signaling, as the kinase domain would presumably remain occupied and inhibited throughout the retrograde transport process. In addition, it is not entirely clear if there may be other signaling mechanisms that are dependent on endosome formation but perhaps not dependent on the canonical kinase pathways currently targeted by the existing kinase inhibitors. As TrkA ligand drug discovery continues, some of these fundamental questions will likely be addressed. Central sensitization refers to the phenomena where nonpainful or lightly painful stimuli are transmitted to spinal cord circuits wherein a pathological amplification takes place, resulting in exaggerated perception.176 This occurs as a result of both synaptic changes at the first-order primary afferent synapse but also changes in the second-order neurons of the spinal cord dorsal horn. NGF has been shown to increase the transcription and transport of several key neurotransmitters to the proximal primary afferent synapses. These include CGRP, substance P, and glutamate, among others.172 In addition, it has been shown that chronic NGF activation of primary afferents can result in BDNF release at the spinal terminals.177 BDNF has multiple roles on the secondary spinal dorsal horn neuron, including potentiation of postsynaptic NMDA current178 as well as affecting the chloride reversal potential likely through action on the potassium−chloride transporter KCC2.179,180 This latter effect can lead to excitatory chloride conductances from GABA and/or glycine channels versus their typical inhibitory effects. Release of BDNF from primary afferent terminals is not the only source of BDNF within the spinal cord, as it has also been shown to be released from activated microglia.180 Nonetheless, the NGF/BDNF connections are an

currents, and while various mechanisms have been proposed for this effect, they all typically involve a variation of TrkA phosphorylation leading to activation of downstream kinases and the eventual up-regulation of TRPV1 function.53,174 Likewise, the acute effects of p75 activation have been studied in detail. An elegant set of experiments from Nicol and coworkers showed that NGF upregulates tetrodotoxin-resistant sodium current and decreases a delayed rectifier potassium current in isolated DRGs, both within a few minutes of NGF exposure.158 Many other similar experiments have implicated a plethora of other ion channels including KCNQ2/3, Nav1.7, calcium channels, etc.120 What remains unanswered is how relevant these more acute pathways may be to the observed clinical efficacy with the anti-NGF antibodies. The onset of observed efficacy would provide some insight into the mechanism, but, unfortunately, this is difficult to determine with antibodies due to their longer term exposure characteristics. If some of the TrkA targeted small molecule therapies demonstrate acute effects, it may implicate these proximal phosphorylation pathways as likely axes for efficacy. In some of the previously described preclinical pain assays, many TrkA ligands have shown efficacy within minutes to an hour, but it remains to be seen how this translates into the clinic. In the case of chronic peripheral sensitization, this is likely supported by new transcriptional activity dependent on the retrograde transport of the NGF bound TrkA signaling endosome.167 Many of the same ion channels mentioned for the acute circuits are also supported by increased transcriptional activity to maintain this sensitization over extended timeframes. However, while there is evidence of p75 having the ability to form signaling endosomes as well,175 this is less well described in the background of DRG signaling and certainly warrants further investigation. TrkA, or perhaps p75, receptor antagonism would be expected to inhibit these transcriptional events, but an open question is how different modes of attack may affect these transcriptional events. As mentioned previously, there are multiple TrkA receptor modulation approaches currently being assessed, including antibodies, peptides, small molecules targeting the external NGF binding Q



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intriguing and active area of research and future points of intervention may well emerge in this pathway. Key questions remain regarding the more relevant pathological ligand for chronic pain: NGF and/or proNGF. Likewise, full understanding of the importance of the TrkA and/or p75 receptors in the transmission of pain signals remains outstanding. Beyond these primary signaling questions, there also remain larger questions around the primary mechanism of analgesia for the current anti-NGF therapies. Reversal of inflammatory responses, dampening of peripheral and central sensitization, and sympathetic dendritic atrophy are all plausible candidates for the primary mechanism of action. As the anti-NGF antibodies have achieved thousands of patient exposures, it is clear these therapies are safe over months of exposure. With longer duration, late-stage trials currently ongoing, it always remains a possibility that a new safety signal may emerge. However, barring such an event, these therapies may prove to be excellent options for multiple chronic pain indications. From a drug discovery perspective, several key considerations remain. Any future therapy in this pathway that could be safely co-administered with NSAIDS would be desirable and may differentiate from the first-generation anti-NGF antibodies. Likewise, oral small molecules with both a more convenient route of administration and a shorter duration of action versus the current antibodies may be beneficial for certain indications and patient populations. One could imagine an acute acting therapy being useful for breakthrough pain. Another key question is whether the current antibodies have achieved maximum efficacy within this pathway. An argument could be made that sequestering NGF should inhibit nearly all NGF (or proNGF)-mediated signaling and that maximum efficacy has been achieved. However, there are many local autocrine-like circuits where NGF is released proximal to the active receptor(s). In these cases, receptor antagonism may be a preferred strategy for modulating these local circuits versus a ligand sequestration strategy. Likewise, if shutting down a particular component of the NGF pathway only results in a partial down- or up-regulation of a key signaling protein, then perhaps a more targeted therapy would be more efficacious. It is reasonable to expect that TrkA directed therapies will be the next to make it into clinical proof of concept trials. Beyond this, it will be essential to deconvolute the primary mechanism of action for the anti-NGF therapies. This will help focus future drug discovery efforts on a more narrow range of possible targets.

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Directors of the Medicinal and Bioorganic Chemistry Foundation. He has published over 40 papers and given many invited lectures at scientific conferences and universities. His current research interests are focused on the discovery of novel analgesic agents for use in chronic pain. Jeff S. McDermott is a Principal Research Scientist in Neuroscience Discovery at Eli Lilly and Company. He received his B.S. in Biology from the University of CaliforniaIrvine in 1994 and attended graduate and medical school at The Chicago Medical School, receiving an M.S. in Physiology in 1995. He joined Lilly in 2012 and currently leads multiple large and small molecule drug discovery efforts focused on chronic pain. He is a cellular electrophysiologist with over 25 years of research experience, including prior drug discovery positions with Abbott Laboratories and Amgen. He has presented numerous abstracts at national scientific conferences and coauthored 18 publications in the areas of ion channel physiology and pharmacology. His interests include chronic pain, ion channels, and mechanisms ion channel regulation.

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ACKNOWLEDGMENTS We thank Dr. David Evans, Senior Research Scientist at Eli Lilly and Company (Erl Wood, UK), for the generation of the protein−ligand structure images shown in Figures 5 and 14.

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ABBREVIATIONS USED Ang II, angiotensin II; AT1R, angiotensin receptor subtype 1; AT2R, angiotensin receptor subtype 2; BDNF, brain derived growth factor; CFA, complete freund’s adjuvant; CGRP, calcitonin gene related peptide; CIA, collagen-induced arthritis; CIPA, congenital insensitivity to pain with anhidrosis; CIPN, chemotherapy-induced neuropathic pain, CNTF, ciliary neurotrophic factor; CSP, chemical shift perturbations; DFG, AspPhe-Gly; DRG, dorsal root ganglion; ECD, extracellular domain; ER, efflux ratio; GDNF, glial cell line-derived neurotrophic factor; KCC2, potassium−chloride transporter; KID, kinase insert domain; LRM1, leucine rich motif; MIA, monoiodoacetate; MNX, meniscal transection; MS, multiple sclerosis; NGF, nerve growth factor; OA, osteoarthritis; PGPS, streptococcal peptidoglycan polysaccharide; RPOA, rapidly progressive osteoarthritis; RTK, receptor tyrosine kinase; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1-phosphate receptor subtype 1; SphK, sphingosine kinase; SPR, surface plasmon resonance; Trk, tropomysin receptor kinase; TRPV1, transient receptor potential vanilloid I

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AUTHOR INFORMATION

REFERENCES

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Corresponding Author

*Phone: 317-277-2235. Fax: 317-277-3652. E-mail: norman@ lilly.com. Notes

The authors declare no competing financial interest. Biographies Bryan H. Norman is a Senior Research Advisor in Discovery Chemistry at Eli Lilly and Company. He received his Ph.D. at Emory University and was an NIH Postdoctoral Fellow at Penn State University. After three years at Monstanto/Searle, Bryan joined Lilly in 1993, where he has led multiple drug discovery efforts through the years, many of which culminated in clinical candidates for endocrine and pain indications. Bryan has served on the Editorial Board of Burger’s Medicinal Chemistry and is currently on the Board of R



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