Functional mimetics of neurotrophins and their ... - Semantic Scholar

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Biochemical Society Transactions (2006) Volume 34, part 4

Functional mimetics of neurotrophins and their receptors J. Peleshok*† and H.U. Saragovi*†‡1 *Lady Davis Institute-Jewish General Hospital, 3755 Cote St. Catherine, F-223, Montreal, QC, Canada H3T 1E2, †Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, QC, Canada H3G 1Y6, and ‡Oncology and the Cancer Center, McGill University, 3655 Drummond Street, Montreal, QC, Canada H3G 1Y6

Abstract Neurotrophins regulate cell survival, death, differentiation and growth. Neurotrophins and their receptors have been validated for pathologies including neurodegenerative disorders of the central nervous system and the peripheral nervous system, certain types of cancers, asthma, inflammation and others. Development of neurotrophin-based therapeutics is important due to the limitations of using whole neurotrophins as pharmacological agents. The use of mimicry has proven to be an alternative. Mimetics can be developed through a number of different approaches. To develop receptor-binding agents, we have used anti-receptor antibody mimicry and neurotrophin mimicry. To develop ligand-binding agents, we have used antiligand antibody mimicry and receptor mimicry. High-throughput screening can be incorporated to complement any of these approaches. The end result is small molecule peptidomimetics with properties favourable over proteins. The present review will offer a general overview of these strategies with a few proven examples from our laboratory.

Neurotrophins and their receptors Neurotrophins have been well characterized in terms of their ability to positively and negatively regulate cell survival, growth, selection and differentiation [1]. They are a family of approx. 25 kDa polypeptides which function as homodimers [2,3]. This family consists of NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3) and NT-4. The neurotrophins mediate their effects through the Trk (tropomyosin receptor kinase) family of receptors and the p75 receptor, which is a member of the tumour necrosis factor receptor family. The neurotrophins bind Trk receptors with 10−11 M affinities (NGF–TrkA, BDNF–TrkB and NT-3–TrkC) and high selectivity, with the exception of NT-3, which binds TrkA and TrkB with lower affinity. Mature neurotrophins bind p75 with relatively lower affinity (∼10−9 M). Trk receptor activation is generally associated with the prosurvival effects of neurotrophins, whereas activation of the p75 receptor can elicit distinct and often opposite biological functions such as pro-survival or pro-apoptosis depending on the cell type and receptor density [4,5].

Neurotrophin receptors are validated targets for therapeutics Neurotrophin receptors have been validated as a pharmacological target for pathological states. In a simplistic way, it Key words: antibody, mimicry, nerve growth factor (NGF), neurotrophin, tropomyosin receptor kinase (Trk). Abbreviations used: BDNF, brain-derived neurotrophic factor; CDR, complementaritydetermining region; DRG, dorsal root ganglion; HTS, high-throughput screening; mAb, monoclonal antibody; NGF, nerve growth factor; NT-3, neurotrophin-3; RGC, retinal ganglion cell; SAR, structure–activity relationship; Trk, tropomyosin receptor kinase. 1 To whom correspondence should be addressed (email [email protected]).

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may be stated that hypoactivity or low receptor expression can lead to neurodegeneration, whereas hyperactivity or high receptor overexpression can lead to transformation. In the central nervous system, the disorders most studied are Alzheimer’s disease and related diseases involving memory and cognition. For example, TrkA expression correlates inversely with neurodegeneration, cognitive impairment and progression towards full Alzheimer’s dementia [6,7]. Similar results have been obtained for age-associated cognitive decline. Moreover, NGF acting as an agonist of TrkA can reverse or delay cognitive decline in experimental models, although it has several drawbacks that make it an inadequate pharmacological agent (see below). In the peripheral nervous system, neuropathic pain and chronic pain including ‘phantom limb’ syndromes are characterized by inappropriate neuronal growth or sprouting involving Trk signalling. Efficacy in animal models of neuropathic pain has been demonstrated with agonists such as NGF, and with antagonists such as anti-NGF antibodies or TrkA receptor bodies for NGF sequestration in chronic and inflammatory pain. However, to date, human clinical trials have not been successful. Also several types of cancer, particularly those of neuroectoderm origin, overexpress Trk receptors. In some cancers (e.g. neuroblastoma), neurotrophin receptor expression correlates with good prognosis; in others (e.g. prostate, gliomas, melanomas and medulloblastomas), neurotrophin receptor expression correlates with bad prognosis. More recently, the roles of neurotrophin mechanisms in glaucoma have also been explored. Glaucoma is a ‘neurodegenerative’ disease characterized by loss of vision concomitant with high intraocular pressure. Progressive death of retinal neurons [RGCs (retinal ganglion cells)] leads to

Neurotrophins – Mechanisms in Disease and Therapy

irreversible blindness. Because RGCs express TrkA, agonists are expected to afford neuroprotection.

Neurotrophin proteins as therapeutics Whole neurotrophins have been shown to be inappropriate pharmacological agents. The likely reasons for failure include (i) pleiotropic effects as a consequence of whole receptor activation; (ii) undesired interactions with both receptors (i.e. NGF can cause activation of TrkA and/or p75), resulting in multiple signals and complex biological outcomes; (iii) unintended activation of cells expressing one or the other receptor; (iv) short half-lives; and (v) unsuccessful tissue targeting [8]. Thus, while the receptors are validated targets, the pharmacological agents used to date can be improved. Problems associated with using the neurotrophins as therapeutics can be averted by developing highly selective receptor ligands (e.g. either binding Trk or p75). Ligands can be developed with chemical and pharmacological properties such as proteolytic stability, small size, ability to penetrate tissue barriers etc. and the ability to agonize or antagonize the receptors. One approach that meets these criteria has been to develop biologically active neurotrophin receptor peptidomimetic ligands. The pharmacological advantages of mimetics over protein therapeutics are well documented, and include lack of immunogenicity, low molecular mass, low manufacturing costs and good pharmacokinetic profiles [9]. Mimetics are small molecules, generally rationally designed, that either resemble (or mimic) a domain of a protein of interest or target and bind to a domain of a protein of interest. If the domain is directly involved in protein function, then the mimetic might be functional. Thus, to develop functionally active mimics, it is necessary to first identify the region of the protein that has the associated desired function.

Mimicry Our general criteria for defining a ‘mimetic’ include structural, binding and functional mimicry. In the first criterion alone, a structural mimetic would be a compound that mimics structural characteristics such as conformation, charges and steric aspects of a protein. These mimetics may be developed from structural analysis of the polypeptide ligand using X-ray crystallography or, if the crystal structure is unavailable, through development of mAbs (monoclonal antibodies) directed against a region of the receptor. Mimics of key structural components of the three-dimensional structure, or the CDR (complementaritydetermining region) of the anti-receptor antibody that is made up of a series of β-turns, may afford a mimic of the structural characteristics of the protein ligand. There are several examples of mAbs being structural mimics of a ligand. For example, the generation of engineered mAbs in which a domain of a ligand is engineered into the mAb itself, or the generation of anti-idiotypic antibodies. In the second criterion alone, the mimetic binds to the natural protein target. These compounds share binding characteristics with a ligand, endogenous or otherwise, but may

not share any structural features with this ligand. This may be the case for small molecules resulting from HTS (high-throughput screening) using an assay based on binding competition (i.e. receptor–ligand inhibition). This compound may show extremely high affinities towards the protein yet share none of the structural characteristics of an endogenous ligand or anti-receptor antibody, nor the ability to elicit function after binding. In the third criterion alone, mimetics may act as agonists or antagonists. Functional mimicry may be mimetics that afford a defined biological effect or measurable biological function similar to that afforded by the polypeptide that they mimic. These compounds need not even bind to the same receptor as the ligand they mimic. For example, many ‘insulinomimetics’ do not bind to the insulin receptor but affect some signal pathway linked to the insulin receptor, or they can induce the release of insulin. In addition, they need not be structural mimics of the ligand. For these reasons, we aim to develop mimetics that meet all three criteria: structural, binding and functional.

Mimetics binding at hot spots The pharmacophore of the mimetic analogues can be studied to make rational improvements to structure, binding and function. A pharmacophore is the compiled steric and electronic features (e.g. hydrogen-bonding and electrostatic interactions) of the mimetic that is necessary to ensure optimal interactions with a specific biological target and to either agonize or antagonize the biological response [10]. Upon identification of a pharmacophore, chemical libraries making analogues of the pharmacophore can yield interesting compounds, and aid in the refinement of SARs (structure– activity relationships) [11]. SAR studies show what structural characteristics of a compound are responsible for the ensuing activity at the receptor. Generally, these focused libraries yield a high percentage of ‘hits’ and can afford second generation compounds for further optimization towards the construction of lead agents. As stated above, it is necessary to identify important regions of a protein to mimic so that the resulting agents are more likely to possess biological function. These target regions of proteins are termed ‘hot spots’ [9]. Hot spots are regions of a protein that when ‘tickled’ by a ligand will cause an identifiable biological signal. Note that endogenous ligands bind at receptor hot spots, but not all the binding sites of endogenous ligands are hot spots. Moreover, receptor domains not bound by endogenous ligands may also be hot spots acting through conformational or allosteric mechanisms [9].

p75 regulation of TrkA activity As an example of targeting receptor hot spots, we will discuss the development of selective peptidomimetic ligands of neurotrophin receptors that would target the extracellular domain of TrkA over p75 [4,5,12–14]. These agents would elicit biological signalling exclusively through the TrkA receptor as opposed to p75/TrkA or p75 alone. C 2006

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Neurotrophin binding of p75 can elicit distinct and often opposite biological functions depending on the cell type and receptor density [4,5]. Co-expression of p75 and Trk has been shown to influence the binding affinity of neurotrophins, but more importantly p75 also regulates functional responses of Trks to endogenous neurotrophin ligands and even to artificial ligands [4,15,16]. To study receptor hot spots, receptor chimaeras were made by swapping each of the five domains of the extracellular region of TrkA (leucine-rich motif D2, two cysteine-rich clusters D1 and D3 and two immunoglobulinlike domains D4 and D5) with those from TrkB. Because TrkB does not respond to NGF, we studied gain of function to NGF agonism in the TrkB chimaeras, and the influence of p75 in the utilization of domains as hot spots [15]. The results illustrate a model of p75–NGF–TrkA interaction in which NGF binds TrkA at the D5 domain and induces a conformational change at the D4 domain. Expression of the p75 receptor allows for the high-affinity binding of TrkA to NGF by presentation of an otherwise cryptic second binding site on the TrkA receptor at the D1 domain. Thus, in this example, three potential hot spots can be targeted for ligand mimicry: the natural ligand hot spot (D5), the conformational regulatory hot spot (D4) or the cryptic hot spot regulated by p75 (D1). It is expected that selective agonists that bind to only one hot spot of TrkA may be able to elicit distinct signals as opposed to NGF. Indeed, many of the NGF and NT-3 mimics we made can elicit neuritogenesis (neuritic extension) without trophic effects (survival under stress), or trophic effects without neuritogenesis, and some can elicit both activities associated with full NGF signalling [11,14].

Figure 1 Comparison of minimized average structure calculated for each family with the confirmation of the C–D loop in NGF available from crystallographic data Upper panel: structures in family 1 adopt β-turn type I conformation and correlate with the conformation of the C–D loop from McDonald et al. [2]. Reprinted by permission from Macmillan Publishers Ltd: Nature, McDonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A. and Blundell, T.L. (1991) New protein fold revealed by a 2.3-Å resolution crystal structure of nerve growth factor, vol. 354, pp. 411–414, copyright 1991. Hydrogen bond distances between Asp4 (carbonyl or side-chain carboxyl) are 2.7 Å in NAc-C-(92–96) and 3.45 and 2.75 Å in the NGF structure. Lower panel: structures in family 2 adopt a type γ L-αR β-turn and are compared with the structure of subunit A reported by Holland et al. [3]. Reprinted from Journal of Molecular Biology, 239, Holland, D.R., Cousens, L.S., Meng, W. and Matthews, B.W., ‘Nerve growth factor in different crystal forms displays structural flexibility and reveals zinc binding sites’, pp. 385–400, Copyright (1994), with permission from Elsevier. No hydrogen-bonding partner of Gln7 could be identified in the second family of NAc-C-(92–96) structures.

Ligands of receptor hot spots Peptidomimetic ligands can be obtained through a number of different methods such as rational design, HTS, mimicry of endogenous ligands and mimicry of anti-receptor mAbs [8,9,11].

Endogenous ligand mimicry NGF has been crystallized by two independent groups, Holland [3] and McDonald [2]. The C–D β-loop region of the NGF protomer, which has been hypothesized to confer the functionality of NGF on to the TrkA receptor, was different in each study. The loop configuration proposed by McDonald [2] is as a hairpin loop which adopts a type I β-turn conformation and is stabilized through a hydrogen bond between the Gln96 and Asp93 . The model proposed by Holland is a type γ L-αR β-turn [2,3]. C-(92–96) is a bioactive cyclic peptide mimic with the sequence N-Ac-Y-C-T-D-E-K-Q-C-Y in which the T-D-EK-Q sequence comprises amino acids 92–96 of the C–D loop of NGF and the two cysteine residues are disulphide bonded to create a β-loop [12,13]. The solution structure of C-(92– 96) was determined by NMR and shows that this mimic can adopt two preferred conformations that are consistent with both different conformations proposed in the crystallography work [12,13]. We propose that the C–D loop of the NGF C 2006

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molecule can exist in a form that spends approximately equal amount of time in each of the two proposed configurations. Whether one of them is bioactive or whether the molecule binds TrkA in an induced-fit fashion is not known. Figure 1 shows the solution structure of both C-(92– 96) peptidomimetic and the comparable C–D β-turn region of the NGF protein. The C-(92–96) peptidomimetic is biologically active solely as a cyclic monomer, as the linear form has no biological activity [12,13]. The lack of function attributed to linearized C-(92–96) peptide confirms the


relevance of the secondary structure of the peptide. C-(92–96) functions as a univalent and monomeric ligand to TrkA [5]. C-(92–96) is able to induce cellular differentiation and afford trophic effects to embryonic DRG (dorsal root ganglion) cells in culture [5]. These effects were synergized by the addition of MC192 (a functional anti-p75 mAb). C-(92–96) is defined as a competitive antagonist of NGF to TrkA and an inverse antagonist of TrkA in its ability to stabilize TrkA in a conformation that is favourable to subsequent TrkA–TrkA interactions [5]. These results suggest that the rational design of peptidomimetics of neurotrophins can possess the key requirements of a mimetic, namely structural homology, selective binding and desired function. The second proposed method of generating mimetics is discussed below, namely antibody mimicry.

Mimicry based on anti-receptor antibody The region of the antibody conferring specific binding recognition is within β-loops found within the antibody CDR [9]. In our laboratory, we have been successful in the development of a series of anti-receptor mAbs targeting receptor hot spots. One of them, termed 5C3, is a full agonist of TrkA by binding the D5 domain of TrkA [16]. D5 was previously shown to be responsible for ligand-specific binding and selective induction of receptor activation. As also shown for NGF, the function of mAb 5C3 is influenced by co-expression of the p75 receptor; but the binding of mAb 5C3 is not influenced by p75 [17]. The pharmacophore of 5C3 was identified and a mimetic of 5C3, termed D3, was developed [18]. In contrast with the C-(92–96) cyclic peptide, the D3 compound is a macrocyclic ring with amino acid side chains corresponding to the βturn of the 5C3 CDR incorporated into the cyclic backbone. D3 is a 540 Da, proteolytically stable molecule. The D3 molecule is univalent and monomeric, yet it can activate TrkA. Biotinylated-D3 was used for binding studies and selectivity to TrkA was determined. Exclusive activity of D3 at TrkA is desired due to the complex and sometimes contradictory signalling afforded by p75–TrkA interactions. D3 is characterized as a partial agonist to TrkA in its ability to confer cell survival and induce cellular differentiation in DRG neurons. Given the D3 pharmacophore, a focused library was developed to optimize these characteristics. The same backbone was used to develop a library of potential NT-3 mimetics as well.

Focused small molecule libraries The approach taken for the rational development of C-(92– 96) and D3 prompted further investigation and development of a small molecule library to TrkA and TrkC [11]. The objective of the rational design of this compound library was to target the D5 domain of TrkC and TrkA. The developed compounds were designed to have improved functionality and binding while simultaneously decreasing size and peptidic nature. The small compound library (see Figure 2) was based on a cyclic backbone made up of a turn component and an organic fragment. The organic fragment

can be a cyclic amine, sulphide or ether with one of four amine substituents. The turn component can be decorated with dipeptide fragments to create focused combinatorial libraries towards the desired pharmacophore. Binding was assessed by FACS analysis (with the use of fluoresceinated analogues), and function was assessed by survival, differentiation and tyrosine phosphorylation assays. The results of the present study pointed to a multifaceted activity profile whereby TrkA/TrkC binding to known receptor hot spots may not necessarily confer function, but conformational changes of the receptor are necessary to induce functionality [11]. Interestingly, receptor tyrosine phosphorylation may occur, with either survival or differentiation being the resulting function. These results address the complex nature of receptor signalling, and also help develop the potential for selective, specific compounds for the further elucidation of the receptor signalling profile.

HTS The screening of a selective small molecule library consisting of rationally designed small molecules is applicable for a lowthroughput or medium-throughput screen focusing primarily on functionality of the compounds. However, the HTS for small molecules in large compound libraries from a range of backgrounds is relevant for the elucidation of functional compounds that may not have been conceived through rational design. The HTS is only as good as the assay used for the screen, and as such the prior identification of a hot spot to target is necessary. The premise of this is that a compound that binds to a previously identified hot spot of the receptor could possess function.

Preclinical efficacy of mimetics Age-associated cognitive impairment Treatment of cognitively impaired aged rats with D3 agonist of TrkA has been shown to act as a neuroprotective agent. Aged rats showed evidence of improved memory and improved cholinergic phenotype. The effect was long-lived, for 3 months after drug delivery. D3 performed equally or better than NGF. Even when NGF was used at optimal doses that yield efficacy with lowest toxicity, 25% of the aged rats suffered from weight loss and other severe side effects and results on these rats were discarded. Had these results been considered, D3 would have been significantly better than NGF as a whole [19]. Preliminary pharmacokinetic and toxicity profiles for D3 seem to be promising and are ongoing.

Glaucoma Treatment of glaucoma with the TrkA agonist D3 and related agonists have also yielded promising results. A rat model has been developed by episcleral cauterization in which the vessels that promote outflow of aqueous humour are cauterized, resulting in a build-up of fluid and subsequent increase in intraocular pressure [20]. Treatment of cauterized C 2006

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Figure 2 Focused small molecule compound library The library is derived from D3 and has three major components. The backbone structure can be based on either a cyclic amine, sulphide or ether. The turn component can be modified through the addition of dipeptide fragments at R1 and R2. The organic fragment can also be modified through the addition of an amine substitute at X. An example of one of these compounds is shown.

rats with D3 has shown a significant reduction of RGC death compared with untreated rats. The protective effect of TrkA agonism is additive to the effect of β-blockers which reduce humoral production and normalize intraocular pressure.

Cancer Given the direct correlation between TrkA mRNA and TrkA protein levels and favourable prognosis in neuroblastoma biopsies, non-invasive diagnostic detection of TrkA-expressing tumours was attempted. Radiolabelling the C-(92–96) mimetic with a technetium isotope [99m Tc (‘artificial’ short-half-life radioisotope of technetium)] was shown to specifically target TrkA-expressing tumours in nude mice injected with cells overexpressing the receptor. The mimetic was approx. 11 times better than an mAb at targeting and imaging solid tumours in vivo [21].

This work was funded by the CIHR (Canadian Institutes of Health Research). J.P. received a fellowship from the CIHR/MCETC (Montreal Centre for Experimental Therapeutics in Cancer)/FRSQ (Fonds de la Recherche en Sante´ der Quebec) ´ Strategic Training Program in Experimental Therapeutics.

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Received 13 April 2006

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