Design of Potent Peptide Mimetics of Brain-derived Neurotrophic Factor

Brain-derived neurotrophic factor (BDNF) has potential for the treatment of human neurodegenerative diseases. However, the general lack of success of neurotrophic factors in clinical trials has led to the suggestion that low molecular weight neurotrophic drugs may be better agents for therapeutic use. Here we describe small, dimeric peptides designed to mimic a pair of solvent-exposed loops important for the binding and activation of the BDNF receptor, trkB. The monomer components that make up the dimers were based on a monocyclic monomeric peptide mimic of a single loop of BDNF (loop 2) that we had previously shown to be an inhibitor of BDNF-mediated neuronal survival (O'Leary, P. D., and Hughes, R. A. (1998) J. Neurochem. 70, 1712–1721). Bicyclic dimeric peptides behaved as partial agonists with respect to BDNF, promoting the survival of embryonic chick sensory neurons in culture. We reasoned that the potency and/or efficacy of these compounds might be improved by reducing the conformational flexibility about their dimerizing linker. Thus, we designed a highly conformationally constrained tricyclic dimeric peptide and synthesized it using an efficient, quasi-one-pot approach. Although still a partial BDNF-like agonist, the tricyclic dimer was particularly potent in promoting neuronal survival in vitro (EC50 11 pm). The peptides described here, which are greatly reduced in size compared with the parent protein, could serve as useful lead compounds for the development of true neurotrophic drugs and indicate that the structure-based design approach could be used to obtain potent mimetics of other growth factors that dimerize their receptors. Brain-derived neurotrophic factor (BDNF) has potential for the treatment of human neurodegenerative diseases. However, the general lack of success of neurotrophic factors in clinical trials has led to the suggestion that low molecular weight neurotrophic drugs may be better agents for therapeutic use. Here we describe small, dimeric peptides designed to mimic a pair of solvent-exposed loops important for the binding and activation of the BDNF receptor, trkB. The monomer components that make up the dimers were based on a monocyclic monomeric peptide mimic of a single loop of BDNF (loop 2) that we had previously shown to be an inhibitor of BDNF-mediated neuronal survival (O'Leary, P. D., and Hughes, R. A. (1998) J. Neurochem. 70, 1712–1721). Bicyclic dimeric peptides behaved as partial agonists with respect to BDNF, promoting the survival of embryonic chick sensory neurons in culture. We reasoned that the potency and/or efficacy of these compounds might be improved by reducing the conformational flexibility about their dimerizing linker. Thus, we designed a highly conformationally constrained tricyclic dimeric peptide and synthesized it using an efficient, quasi-one-pot approach. Although still a partial BDNF-like agonist, the tricyclic dimer was particularly potent in promoting neuronal survival in vitro (EC50 11 pm). The peptides described here, which are greatly reduced in size compared with the parent protein, could serve as useful lead compounds for the development of true neurotrophic drugs and indicate that the structure-based design approach could be used to obtain potent mimetics of other growth factors that dimerize their receptors. BDNF 1The abbreviations used are: BDNF, brain derived neurotrophic factor; NGF, nerve growth factor; neurotrophin 3, NT-3; NT-4/5, neurotrophin 4/5; r.m.s., root mean square; Acm, acetamidomethyl; TFA, trifluoroacetic acid; Z, benzyloxycarbonyl; Bzl, benzyl.1The abbreviations used are: BDNF, brain derived neurotrophic factor; NGF, nerve growth factor; neurotrophin 3, NT-3; NT-4/5, neurotrophin 4/5; r.m.s., root mean square; Acm, acetamidomethyl; TFA, trifluoroacetic acid; Z, benzyloxycarbonyl; Bzl, benzyl. is a member of the neurotrophin family of neurotrophic factors, which includes nerve growth factor (NGF), neurotrophin (NT)-3, and NT-4/5 (1Hallböök F. Curr. Opin. Neurobiol. 1999; 9: 616-621Crossref PubMed Scopus (142) Google Scholar). In addition to its critical role in helping shape the vertebrate nervous system during development, BDNF is of particular therapeutic interest because of its neurotrophic actions on neuronal populations involved in several neurodegenerative diseases (2Froestl W. Pharm. Acta Helv. 2000; 74: 247-251Crossref PubMed Scopus (7) Google Scholar), including: sensory neurons, implicated in peripheral sensory neuropathies (3Lindsay R.M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1996; 351: 365-373Crossref PubMed Scopus (194) Google Scholar); motor neurons, which degenerate in amyotrophic lateral sclerosis (4Askansas V. Adv. Neurol. 1995; 68: 241-244PubMed Google Scholar); dopaminergic neurons of the substantia nigra, lost in Parkinson's disease; and cholinergic neurons of the basal forebrain, involved in Alzheimer's disease (5Siegel G.J. Chauhan N.B. Brain Res. Brain Res. Rev. 2000; 33: 199-227Crossref PubMed Scopus (445) Google Scholar). BDNF, like the other neurotrophins, produces its effects on neurons through two transmembrane receptors. Binding of a neurotrophin to a specific member of the trk family of receptor tyrosine kinases (NGF binds to trkA, BDNF and NT-4/5 bind to trkB, and NT-3 preferentially binds to trkC) results in the step-wise homodimerization of the receptor, leading to receptor autophosphorylation and the initiation of multiple signal transduction pathways, including those leading to neuronal survival (6Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1644) Google Scholar). In contrast, the glycoprotein p75 acts as a common low-affinity receptor (K D ≈ 10–9m) for all the neurotrophins (7Chao M.V. J. Neurobiol. 1994; 25: 1373-1385Crossref PubMed Scopus (399) Google Scholar). Unlike the trk family, p75 signals apoptosis via a unique intracellular death domain, Chopper (8Coulson E.J. Reid K. Baca M. Shipham K.A. Hulett S.M. Kilpatrick T.J. Bartlett P.F. J. Biol. Chem. 2000; 275: 30537-30545Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), although its precise biological function remains controversial. The high affinity binding sites for BDNF and the other neurotrophins on neurons (K D ≈ 10–11m) probably consist of a combination of the appropriate trk member and p75. The overall response to a neurotrophin therefore depends on the balance of signaling through a trk family member and p75, with the opportunity for modulation and cross-talk at multiple levels of the signaling process. The neurotrophins are homodimers consisting of two monomers, each of ∼120 residues. X-ray crystal structures of NGF (9McDonald N.Q. Lapatto R. Murray-Rust J. Gunning J. Wlodawer A. Blundell T.L. Nature. 1991; 354: 411-414Crossref PubMed Scopus (429) Google Scholar), NT-3 (10Butte M.J. Hwang P.K. Mobley W.C. Fletterick R.J. Biochemistry. 1998; 37: 16846-16852Crossref PubMed Scopus (56) Google Scholar), NT-4/5, and a BDNF/NT-4/5 heterodimer (11Robinson R.C. Radziejewski C. Spraggon G. Greenwald J. Kostura M.R. Burtnick L.D. Stuart D.I. Choe S. Jones E.Y. Protein Sci. 1999; 8: 2589-2597Crossref PubMed Scopus (70) Google Scholar) reveal a common fold for the neurotrophins. Each monomer consists of seven β-strands (contributing to three longitudinal antiparallel β-sheets) connected by three solvent-exposed hairpin loops (loops 1, 2, and 4) and a longer loop (loop 3) and contains three disulfide bridges between six fully conserved cysteine residues arranged in a cystine-knot motif, characteristic of this growth factor superfamily. However, in contrast to most of the other members of the superfamily, the neurotrophin monomers are arranged in the dimer in a parallel fashion, and are held together solely by non-covalent (largely hydrophobic) interactions. A number of studies implicate the solvent-exposed loops of BDNF and the other neurotrophins in mediating their biological effects. Site-directed mutagenesis analyses have revealed that the ability to bind and activate trkB can be conferred to NGF by replacing residues in loop 2 with the corresponding residues from BDNF (12Ibáñez C.F. Ilag L.L. Murray-Rust J. Persson H. EMBO J. 1993; 12: 2281-2293Crossref PubMed Scopus (132) Google Scholar). The crystal structure of NGF in complex with one of the Ig domains of trkA shows direct contact between residues in the receptor and loop 1 of NGF (13Wiesmann C. Ultsch M.H. Bass S.H. de Vos A.M. Nature. 1999; 401: 184-188Crossref PubMed Scopus (311) Google Scholar), although the authors of this article have since conceded that additional regions of the neurotrophins may well interact with other trk domains (14Wiesmann C. de Vos A.M. Cell Mol. Life Sci. 2001; 58: 748-759Crossref PubMed Scopus (150) Google Scholar). In support of the functional role for loop 2, we have reported that conformationally constrained peptides (of which the monocyclic monomeric peptide 1 (Table I) was identified as the most effective) designed to mimic a single loop 2 of BDNF are inhibitors of BDNF-mediated survival of sensory neurons in culture, probably by acting as competitive trkB antagonists (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar).Table IStructures, mass spectral data, and summary of in vitro neuronal survival effect of compounds described in this study Despite promising preclinical data, clinical trials with recombinant BDNF in patients with amyotrophic lateral sclerosis have proven unsuccessful (16BDNF Study Group: Phase III Neurology. 1999; 52: 1427-1433Crossref PubMed Google Scholar). This failure is likely to have been caused, at least in part, by the unfavorable pharmacokinetics of BDNF; for example, the plasma half-life of recombinant BDNF in rats is less than 1 min (17Poduslo J.F. Curran G.L. Brain Res. Mol. Brain Res. 1996; 36: 280-286Crossref PubMed Scopus (474) Google Scholar). These and similar data from other neurotrophic factors have led to the view that low molecular weight drugs, with more appropriate pharmacokinetic properties than the parent proteins, might prove a more fruitful means of harnessing neurotrophic action for therapeutic use (18Skaper S.D. Walsh F.S. Mol. Cell Neurosci. 1998; 12: 179-193Crossref PubMed Scopus (88) Google Scholar). As a step toward the development of such neurotrophic drugs, we describe here a structure-based approach for the discovery of potent mimetics of BDNF. Given our previous data with monocyclic monomeric BDNF inhibitors (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar), we reasoned that appropriately designed dimeric loop 2 mimetics should be able to bring about trkB homodimerization and thus mimic the actions of BDNF through this receptor. Using this approach, three classes of peptides were designed and synthesized: bicyclic dimeric peptides linked by a disulfide bridge internal to the sequence of monomeric monocyclic peptide 1; a bicyclic dimer linked by an amide bond external to the peptide 1 sequence; and a highly constrained tricyclic dimeric peptide containing both disulfide and amide dimerizing linkages. The latter compound exhibited extremely high potency, comparable with BDNF, in promoting the survival of chick sensory neurons in culture. The results suggest that this compound is worthy of further preclinical development and that our structure-based approach may be of general utility in developing potent mimetics of other growth factors and cytokines that dimerize their receptors. Molecular Modeling—Bicyclic dimeric peptides were designed using Sybyl molecular modeling software (version 6.4; Tripos, St. Louis, on an The loop 2 from a of the BDNF dimer by modeling (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google corresponding to two of the sequence was to the loops were one between of were used to the of dimerizing between the which were as A disulfide bond was each of the linked loops at and as described previously (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar). The residues the residues were the the and the of each compound The low of each bicyclic dimeric peptide was the BDNF loop 2 and the of was as the root mean of the between common and of peptide were synthesized from using with the of peptides for the of bicyclic dimers were on with residues involved in were the for the dimerizing was for the of the bicyclic dimer and tricyclic dimer were synthesized on amide which the peptide The and residues were as trifluoroacetic and the were of and and and were on the and the additional were as the and the tricyclic dimer the for the was as the The of peptides and were the of the was to all peptides as the R.C. P. J. Chem. Soc. Trans. Scopus Google Scholar). peptides were from the with The peptides were to the corresponding monocyclic monomers by the peptide in a of in W. J. Chem. Soc. 1991; Scopus Google Scholar). of Bicyclic dimers were using a of the of A. K. H. P. W. Helv. Scopus Google Scholar) by the appropriate monocyclic monomeric peptide in containing 1 in and the at the was with 1 and the bicyclic dimer was by of Bicyclic bicyclic dimer was by the two monocyclic monomers in the addition of and and the was with and the bicyclic dimer was by of tricyclic dimer was using a combination of the described for and dimers the two monocyclic monomers were in the of The bicyclic dimer was to the tricyclic dimer by treatment with and were from bicyclic dimer and tricyclic dimer by the peptides with in the of using and of was and peptides as appropriate by using on peptides were as single of all peptide and was by mass using a mass with an at the of peptides and molecular with a of were in of root sensory neurons from embryonic as described previously (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar). to were to in 1 with recombinant BDNF BDNF BDNF were by in at in of neurons with were for were by neuronal survival in the of the number of neurons to and survival in to survival was as the mean from and were from to survival in the of peptides was compared with by of by multiple of Bicyclic of the loop 2 of a of the structure of the BDNF revealed two sites in 1 sequence of the monocyclic monomeric peptide 1, and 2 the sequence of peptide 1 The between of and the of as by conformational of a dimerizing at these two the bicyclic dimers and (Table which low and the loop 2 The between the of the that longer might be more at this However, of peptides that the BDNF of and the was in well because of In 2, to the of the monocyclic monomer 1, was as a for a dimerizing because the other sites and were to to peptides with the in this the was better to this than longer of Bicyclic bicyclic dimers were using appropriate to the of and dimerizing disulfide The dimers were synthesized using for for the disulfide and for involved in the dimerizing disulfide The success of the of and to the dimer was to be on the of the results were with of other in multiple The dimer was synthesized using a combination of and derived of the and monomers, was with the amide bond agents and to multiple and/or The step in the of was the of the on and by treatment with spectral data to the bicyclic dimers can be in of Bicyclic on in effects of the bicyclic dimers on neuronal survival were in of embryonic chick root sensory neurons, an used in the to the survival effects of BDNF and the other neurotrophins. to of embryonic chick sensory neurons, the bicyclic dimeric peptides 2 and and the bicyclic dimeric peptide and in neuronal survival Although peptides 2, and a similar survival effect of the survival effect of peptide was the most potent of the bicyclic dimers of more than more potent than 2 peptides 2, and the survival effect a their a In contrast to the survival promoting effects of 2, and the bicyclic dimer the monocyclic monomeric to the bicyclic dimers neuronal survival compared with the the partial of the bicyclic dimeric the bicyclic dimeric peptide was to chick sensory in with BDNF. to its loop monocyclic monomeric 1 (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar), dimeric bicyclic peptide and of BDNF-mediated neuronal survival of at 1 of these data suggest that bicyclic dimer acts as a partial on the of neurons that to BDNF, than a on a of BDNF of reasoned that the low potency and partial of the bicyclic dimers may be caused, at least in part, by conformational flexibility about the dimerizing that the two monomeric the peptides are likely to be of to one The bicyclic dimers probably as trkB agonists in a able to the of trkB in a parallel the of the two monomeric is to receptor homodimerization the are the bicyclic dimers probably as leading to a in in and of the as previously shown for monocyclic monomeric peptides acting as competitive trkB antagonists (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar). this and to to obtain a BDNF with potency and we designed tricyclic dimeric peptides of the and in which two monomeric loop 2 were linked by both an amide and a disulfide Molecular of these peptides that greatly reduced conformational compared with their bicyclic in to the loop 2 of BDNF of these tricyclic was for of of the tricyclic dimeric peptide was using a combination of the used to the bicyclic and to be the of the multiple and dimerizing Using this approach, the two to peptide were from the and and and the monomers as described for the bicyclic dimer The was by the bicyclic peptide with about the step-wise of the residues and the of the to the as described for bicyclic dimers of the tricyclic dimer with the dimeric peptide which was by the of this approach, we a to 4) on the of the two peptide the two of one with the other with were and the peptide on the The peptides were and as a the number both of the and The and as described a to that of Using this quasi-one-pot approach, it is to tricyclic dimeric peptide from in a of on to of embryonic chick sensory neurons, the tricyclic dimeric peptide and in neuronal survival survival at 1 The tricyclic dimer was particularly with of to an of 11 it more potent than the bicyclic dimer and more potent than the bicyclic dimer However, like the bicyclic tricyclic dimer a although the was than In with BDNF, the tricyclic dimer and of BDNF-mediated neuronal survival at 1 These were from those of the bicyclic dimeric peptide the monocyclic monomeric peptide 1 of multiple This article the design of low molecular conformationally constrained dimeric peptide mimetics of the neurotrophic factor BDNF. The peptides were based on the monocyclic monomeric peptide 1 of a single solvent-exposed loop 2 of a compound we had previously shown to as a competitive trkB (15O'Leary P.D. Hughes R.A. J. Neurochem. 1998; 70: 1712-1721Crossref PubMed Scopus (48) Google Scholar). Using the structure of the BDNF dimer as a a of bicyclic dimeric peptides consisting of of of peptide 1 were designed and Unlike their monomeric three of the bicyclic dimeric peptides as BDNF-like promoting the survival of embryonic chick sensory neurons in with potency and efficacy by the and of the dimerizing We reasoned that neuronal survival of these bicyclic dimers might be by conformational flexibility about the disulfide amide dimerizing this we designed and synthesized a tricyclic dimeric peptide in which the monocyclic monomeric were linked by two dimerizing linkages. 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PubMed Scopus Google Scholar). is of interest to that the structure of a peptide to the receptor to the structure of the tricyclic dimer the activate have This the that the molecular design approach we describe in this using two appropriately dimerizing to greatly conformational could be of general utility in the potency of a of dimeric peptide Despite the ability of the bicyclic and tricyclic dimers to sensory survival in are partial agonists with respect to BDNF, in that are able to support about of those neurons that be by BDNF and the neuronal survival effect of BDNF in for this is that the compounds are partial agonists of trkB, because are less than BDNF in about the of trkB because of conformational flexibility of the dimerizing the of partial by the highly constrained tricyclic compound conformational effects on receptor have been for a number of G. Curr. 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Google Scholar), the of multiple a trk member although effects to be through a trk member a A.M. Neurol. PubMed Scopus Google Scholar). modeling studies we have suggest that the dimeric by acting solely as trkB may be to action through p75, to the reduced survival and R. A. Hughes, in the of this partial a of could be the binding of the bicyclic dimeric peptides to trkB and p75 to be A functional lack of p75 toward the action of the bicyclic dimeric peptides could be using p75 such as G. 1991; Full Text PDF PubMed Scopus Google Scholar) the effects of the peptides on such of p75 as and F.D. D.R. Cell Mol. Life Sci. 2001; 58: PubMed Scopus Google Scholar). using trkB such as P. F. M. Google Scholar), could be used to the of trkB. The tricyclic dimeric peptide described here is the most potent member of a of neurotrophin including a trkA from a peptide based on loop of NGF S. K. Mol. 2000; Google Scholar), and a of dimeric mimetics that as agonists of S. K. K. Chem. 2001; Scopus Google Scholar). the neurotrophins have to clinical success in the treatment of neurodegenerative diseases. remains to be the neurotrophin mimetics lead to drugs that neurotrophin actions for therapeutic use. In the structure-based design approach we describe has potent weight peptide mimetics of BDNF with potency comparable with that of the These compounds could serve as a for the development of useful BDNF the between and other growth receptor dimerizing the approach may have general utility a of receptor receptor