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Brief Article
Design, Synthesis, and Characterization of Fatty Acid Derivatives of a Dimeric Peptide-Based Postsynaptic Density-95 (PSD-95) Inhibitor Klaus B. Nissen, Julie J. Andersen, Linda M. Haugaard-Kedström, Anders Bach, and Kristian Strømgaard J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501755d • Publication Date (Web): 15 Jan 2015 Downloaded from http://pubs.acs.org on January 24, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Medicinal Chemistry
Design, Synthesis, and Characterization of Fatty Acid Derivatives of a Dimeric Peptide-Based Postsynaptic Density-95 (PSD-95) Inhibitor Klaus B. Nissen, Julie J. Andersen, Linda M. Haugaard-Kedström, Anders Bach, and Kristian Strømgaard* Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. ABSTRACT: Dimeric peptide-based inhibitors of postsynaptic density-95 (PSD-95) can reduce ischemic brain damage and inflammatory pain in rodents. To modify the pharmacokinetic profile we designed a series of fatty acid linked dimeric ligands, which potently inhibits PSD-95 and shows improved in vitro blood plasma stability. Subcutaneous administration in rats showed extended stability and sustained release of these ligands. This can facilitate new pharmacological uses of PSD-95 inhibitors and further exploration of PSD-95 as a drug target.
The family of ionotropic glutamate (iGlu) receptors are ligand-gated ion channels that mediate the majority of excitatory synaptic transmission in the vertebrate brain and are crucial for normal brain function.1-3 Dysfunction of iGlu receptors is involved in a range of neurological and psychiatric diseases and inhibition of iGlu receptors is considered a promising strategy for treating brain diseases, such as Alzheimer’s disease, ischemic brain damage and chronic pain.1, 4 Particularly, overstimulation of the Nmethyl-D-aspartate (NMDA) subtype of iGlu receptors is involved in the pathology of ischemia and neuropathic pain and several clinical candidates targeting the NMDA receptor have been in development, but most compounds have failed to reach the market.1, 5-6 This has generally been attributed to a narrow therapeutic window of NMDA inhibition, leading to severe side-effects or low efficacy of the drugs in clinical trials.1, 6 The scaffolding protein postsynaptic density-95 (PSD95) has been shown to simultaneously bind the NMDA receptor and the enzyme neuronal nitric oxide synthase (nNOS)7-9 via its PSD-95/discs large/zonula occludens (PDZ) domains. Upon activation of the NMDA receptor, Ca2+ influx into the neuron stimulates nNOS to generate nitric oxide (NO), which is neurotoxic in high concentrations.10-12 Ligands that bind the first two PDZ domains (PDZ1 and PDZ2) of PSD-95 inhibit formation of the NMDA/PSD-95/nNOS ternary complex, thus uncoupling nNOS and reducing NO formation.13 These ligands have been shown to reduce ischemic brain damage11-16 and reduce the symptoms of chronic pain in rodents.17-20 Importantly, inhibition of PSD-95 does not block synaptic activity or other Ca2+-mediated signal events.13, 21 Therefore, inhibition of PSD-95 is a promising strategy for the treatment of ischemic brain damage and neuropathic pain.
The clinically most advanced PSD-95 inhibitor is TatNR2B9C (NA-1), which is being developed for the treatment of ischemic brain damage after stroke and has recently successfully passed phase II clinical trials.22-23 However, NA-1 suffers from low affinity to PSD-95 (Ki = 4.6 µM),16 and in an attempt to address this, we have previously developed plasma stable, high-affinity dimeric ligands 1 (UCCB01-125) and 2 (UCCB01-144) (Figure 1) that bind both PDZ1 and PDZ2 of PSD-95 simultaneously. These compound have up to 1000-fold increased affinity for PSD-95 compared to NA-1.16, 24 1 was recently shown to reduce the mechanical hypersensitivity associated with chronic inflammatory pain in mice without affecting attention, long-term memory or motor performance, in contrast to conventional inhibitors of NMDA receptors.25 2, containing a cell penetrating peptide (CPP) moiety, was shown to reduce brain damage and improve motor function in mice subjected to focal cerebral ischemia.16 Both 1 and 2 demonstrated improved in vitro plasma stability compared to NA-1,16, 24 and here we were interested in extending this stability further, and create compounds with modified in vivo pharmacokinetic profiles, without compromising biological activity.
Figure 1. Structures of plasma stable, high affinity dimeric ligands binding simultaneously to PDZ1 and PDZ2 of PSD-95. 1 consists of two PSD-95 binding peptides linked by a PEG(4) moiety. 2 furthermore contains the cell penetrating Tat sequence (YGRKKRRQRRR), attached via the nitrogen atom on the PEG-based ‘NPEG’ linker, to facilitate brain delivery. Letters indicate amino acids except for N (nitrogen) and O (oxygen).
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Journal of Medicinal Chemistry Scheme 1. Synthesis of FA-Linked Dimeric Ligands 7-18 and 22-24. O
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
O IETXV R1
O
a
H N O
R1
O IETXV
3: X=A 19: X=D
O IETXV
O
O
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a
Fmoc
H2N
O
O O
b,c
n N O
O
N OH n H n = 2-3 R 1 = H or COOtBu
IETXV
O R2
O
O IETXV O 4: X=A n=2, R1=H 5: X=A n=2, R1=COOtBu 6: X=A n=3, R1=H 20: X=D n=2, R 1=H 21: X=D n=2, R 1=COOtBu
R1
N m H
O
O
O n
N O O
R2 m
OH
m = 10/16 R 2 = CH3 or COOCH 3
IETXV O 15: X=A, n=3, 16: X=A, n=3, 17: X=A, n=3, 18: X=A, n=3, 22: X=D, n=2, 23: X=D, n=2, 24: X=D, n=2,
7: X=A,n=2, m=10, R1 =H, R 2=CH3 8: X=A, n=2, m=10, R1=H, R2=COOH 9: X=A, n=2, m=16, R1=H, R2=CH3 10: X=A, n=2, m=16, R 1=H, R 2=COOH 11: X=A, n=2, m=10, R 1=COOH, R 2 =CH3 12: X=A, n=2, m=10, R 1=COOH, R 2 =COOH 13: X=A, n=2, m=16, R 1=COOH, R 2 =CH3 14: X=A, n=2, m=16, R 1=COOH, R 2 =COOH
m=10, R1 =H, R 2=CH3 m=10, R1 =H, R 2=COOH m=16 R 1=H, R 2=CH3 m=16, R1 =H, R 2=COOH m=16, R1 =H, R2 =CH3 m=16, R1 =H, R2 =COOH m=16, R1 =COOH, R 2=CH3
a
Reagents and conditions: (a) HATU, collidine, DMF (1 h), repeat coupling with HBTU, DIEA, DMF, then 20 % piperidine in DMF. (b) HBTU, DIPEA, DMF/DCM, 45 min, then TFA/TIPS/H2O (90/5/5); (c) 0.5 M LiOH, H2O/ACN (75/25), 30 min, then TFA to pH95%, Table S1). Table 1. Affinity of Dimeric Ligands 1−2 and FALinked Dimeric Ligands 7−18 for PSD-95 PDZ1-2 as determined by FP.
Cmpd 1
Linker n
R1
m
R2
Ki,PSD95 (nM)a
Ki,PSD-95 +HSA (nM)a
-
-
-
-
14.3 ± 1.1
9.7 ± 1.0
-
-
10.5 ± 0.9
-
-
4.3 ± 0.1
7
10
CH3
13.6 ± 0.6
27.2 ± 2.9
8
10
COOH
15.4 ± 1.2
27.9 ± 2.7
2
2
H
16
CH3
11.1 ± 0.6
1889 ± 155
10
16
COOH
11.4 ± 0.6
5717 ± 298
11
10
CH3
30.2 ± 4.2
24.0 ± 0.6
10
COOH
33.7 ± 1.6
26.4 ± 2.6
16
CH3
17.0 ± 0.6
1391 ± 184
14
16
COOH
9.2 ± 1.6
3594 ± 132
15
10
CH3
20.5 ± 1.1
13.4 ± 1.4
16
10
COOH
13.9 ± 2.1
18.5 ± 0.6
16
CH3
26.5 ± 0.2
1889 ± 100
16
COOH
11.5 ± 1.2
2926 ± 335
9
12 13
17
2
3
18 a
FA
COOH
H
Data shown as mean ± SEM, n≥3.
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Figure 2. Affinity for PSD-95 PDZ1-2 of dimeric ligands 1−2 and 7−18 as determined by FP. A: FP assay without HSA. B: FP assay with 1 % HSA. Data shown as mean in nM ± SEM, n ≥ 3.
In addition, three FA linked dimeric ligands containing the peptide sequence IETDV instead of IETAV were synthesized, similarly to the procedure described above: Resin-bound, protected IETDV (19) was coupled with Fmoc-GABA-OH or Fmoc-γ-Glu-OtBu to give 20 and 21, which were coupled with C18 acid and C18 diacid methyl ester to give 22−24 (Scheme 1). The affinity of the FA linked dimeric ligands for the tandem PDZ1-2 domain of PSD-95 was evaluated in a fluorescence polarization (FP) assay, as previously described.16 In addition, we examined the influence of HSA on affinity to PDZ1-2 as an indirect measurement of HSA affinity. Without the presence of HSA, the measured affinities of 1 and 2 were comparable Table 2. FA-linked Dimeric Ligands with an IETDV Peptide Ligand. Cmpd
Linker
22
GABA
C18:COOH
23
GABA
C18(COOH)2
24
γ-Glu
C18:COOH
a
FA
Peptide
Ki,PSDa 95 (nM)
Ki,PSD-95 a +HSA (nM)
8.0 ± 0.8
2514 ± 149
IETDV
29.3 ± 0.7
770 ± 27
6.7 ± 0.6
3806 ± 287
with previously published results,16, 24 with Ki values of 14.3 ± 1.1 and 4.3 ± 0.1 nM, respectively. Noticeable, all FA derived dimeric ligands showed affinities in the same range with Ki values between 9–34 nM (Table 1 and Figure 2A). Thus, clearly the strategy of introducing FAs in the NPEG linker did not compromise PSD-95 affinity regardless of the type of linker or FA that was used. Next, we investigated whether HSA influenced the ability of the FA linked dimers to bind PSD-95. The FP assay was therefore performed with buffer containing 1% HSA (~150 µM), which is slightly lower than physiological blood concentrations. At higher HSA concentrations binding of the fluorescent dimeric probe to HSA compromised the FP assay. Here, the affinity of the FA derivatives for PSD-95 PDZ1-2 showed a significant and systematic change: Derivatives with shorter FAs, such as C12 acid (7, 11 and 15) or C12 diacid (8, 12 and 16) were largely unaffected by the presence of HSA, while the affinities of the FA derivatives comprising the longer C18 acid (9, 13 and 17) or C18 diacid (10, 14 and 18) were severely reduced (Figure 2B). In particular, C18 diacid containing analogues showed 250-520 fold increases in Ki values, while the C18 acid derivatives had 70- to 170-fold reduced affinities. FA derivatives 10 and 14 were the most potent derivatives when HSA was not present with Ki values of 11.4 ± 0.6 and 9.2 ± 1.6 nM, respectively, but also the least potent in the modified FP assay with HSA, where Ki values were measured to 5717 ± 298 and 3594 ± 132 nM, respectively. For the three different libkers, we did not observe any systematic difference when HSA was present in the FP assay (Table 1). Compounds 9 and 13 were among the most hydrophobic compounds, as assessed by the retention time (Rt) in analytical HPLC (Table S2). Also, their affinities were greatly affected by HSA and the water solubility of these ligands was reduced. In an attempt to increase the hydrophilicity, we synthesized compounds 22−24 (Scheme 1) corresponding to 9, 10 and 13, but where the peptide sequence IETAV is replaced with IETDV, which should increase hydrophilicity, but not affect the affinity towards PDZ1-2 of PSD-95.16, 24 However, when incorporated into the dimeric structure and attaching the FA, the overall hydrophobicity of 22−24 was not affected according to the HPLC retention times (Table S2). When tested in the FP assay compounds 22 and 24 showed similar affinities to the corresponding compounds, 9 and 13, respectively, both with and without HAS in the FP assay, while 23 were less affected by HSA relative to 10 (Tables 2 and 3). Next, we wanted to determine if FA derivatization of the dimeric ligands influenced the in vitro plasma stability, and four analogues, 7, 10, 13 and 22, were selected for further analysis. However, when subjected to a standard in vitro plasma stability assay24 sample recoveries were very low (80%. Thus, the dimeric ligands were incubated in human plasma, analyzed at different time points by HPLC and peak areas were normalized to the amount at T0 and fitted to a first order decay model to determine the halflife (T½). The half-life of the original dimeric ligand 1 was determined to 1.7 h, which was in the same range as previously determined,24 whereas all the FA linked dimeric ligands showed significantly improved half-lives (Figure 3): The dimeric ligand linked to the shorter C12 acid FA, 7, had a T½ = 23.6 h, whereas the dimeric ligands linked to the longer C18 FAs, 10, 13 and 22, all showed T½ > 24 h. In this assay, using human plasma, it is expected that the HSA concentration is in the range of 0.5-0.8 mM, as in normal blood samples.37-39 Thus, the dramatically increased half-lives observed is most likely attributed to the increased HSA affinity of the FA linked dimeric ligands, which lowers the free concentration of ligand available for enzymatic digestion. Additionally, steric hindrance mediated by FA could also prevent enzymatic degradation. To explore if the increased in vitro plasma stability of lipidated compounds translates to in vivo conditions, we established the plasma pharmacokinetic profiles of 1 and analogue 7, 10, 13 and 22 in rats. We measured the concentration of compound in blood following a single subcutaneous (s.c.) bolus injection of male Wistar rats. Clearly, FA linked dimeric ligands have higher T½ and it takes longer before maximal blood concentration is reached (Tmax) compared to dimeric ligand without FA (1)
IETAV
a
a
Dose
T½ (h)
Tmax (h)
3
0.6 ± 0.1
0.5 ± 0.0
30
0.5 ± 0.1
0.5 ± 0.0
30
0.8 ± 0.05
0.8 ± 0.2
15
8.1 ± 0.5
4.7 ± 0.7
13
γ-Glu
C18:0
IETAV
15
10.7 ± 0.6
6.0 ± 1.2
22
GABA
C18:0
IETDV
10
16.3 ± 2.8
8.0 ± 0.0
Data shown as mean ± SEM, n≥3.
(Figure 4). The effect was smallest for 7, but noticeable for 10, 13, and 22, which showed T1/2 between 8-16 hours, corresponding to a 16-to 32-fold increase relative to 1 without FA (Table 3). The increased Tmax is likely due to a prolonged absorption from the injection site, illustrating that FA linked dimeric PSD-95 inhibitors, especially 10, 13, and 22, can be administered by a s.c. depot injection where the compound is released slowly and consistently into the blood. Thereby relevant blood concentrations may be achieved with fewer administrations. In conclusion, we have designed and synthesized a range of dimeric ligands conjugated to four different FAs via three different linkers. The FA linked dimeric ligands were prepared from a common NPEG dimeric derivative providing a systematic investigation of PSD-95 affinity with and without the presence of HSA. PSD-95 affinities of FA-derived dimeric ligands were not compromised, as all ligands showed affinities in the low nanomolar range. However, when adding HSA to the assay affinities were reduced significantly for ligands comprising a long C18 FA, whereas C12 FAs did not affect affinity. Compounds 1 and 2 (without FA) were not influenced by HSA, corroborating that indeed FA leads to interaction with HSA. Selected FA-linked dimeric ligands were investigated in an in vitro plasma stability assay, and especially the C18 FAcontaining ligands showed a dramatic increase in stability compared to 1. Importantly, this property translates into greatly increased in vivo half-lives for up to 16 hours and sustained release into the blood in rats given a s.c. depot injection. Overall, dimeric PSD-95 inhibitors modified with C18 FAs bind strongly to both PSD-95 and HSA, are highly stable in blood plasma, and demonstrate very long in vivo half-lives. Thus these compounds comprise a novel class of PSD-95 inhibitors with unique in vivo pharmacokinetic properties. Future studies are required to examine if this profile translates into improved efficacy in animal disease models, and if these compounds can facilitate the development of new pharmacological strategies and examinations of PSD-95 as a drug target, where a long-term and consistent PSD-95 inhibition is desirable.
EXPERIMENTAL SECTION Figure 4. Plasma profiles of dimeric ligands 1 (two doses) and FAderived dimeric ligands 7, 10, 13 and 22 after s.c. administration in rats.
General synthetic procedure. See Supporting Information.
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Synthesis of 4-6, 20-21. The protected linkers (FmocGlu-OtBu, Fmoc-GABA-OH and Fmoc-5-Ava-OH were coupled to the nitrogen of the NPEG linker of 3 and 19 by two consecutive couplings. For the first coupling, FmocGlu-OtBu, Fmoc-GABA or Fmoc-5-Ava-OH (2 eq.) was activated by O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HATU, 3 eq, 0.5M) and collidine (4 eq) in DMF) before addition to the drained resin. After 45 min of shaking and a DMF flowwash, the coupling was repeated with HBTU/DIEA as coupling reagent and the reaction was continued overnight. The Fmoc group was then removed with 2 x 20% piperidine in DMF (2 x 5 min shaking, DMF wash in between). 4: MS (ESI+) calcd. for C65H112N12O27 [M + H]+, 1493.8; found m/z 1493.9, calcd. For [M + 2H]+, 747.4; found m/z 747.5. 5: MS (ESI+) calcd. for C64H112N12O25 [M + H]+, 1449.8; found m/z 1449.8, calcd. For [M + 2H]+ 725.4; found m/z 725.6. 6: MS (ESI+) calcd. for C65H114N12O25 [M + H]+, 1463.8; found m/z 1464.1, calcd. For [M + 2H]+, 731.9 found m/z 732.3. 20: MS (ESI+) calcd. for C67H112N12O31 [M + H]+, 1581.8; found m/z 1581.8, calcd. For [M + 2H]+, 791.4; found m/z 791.5. 21: MS (ESI+) calcd. for C66H112N12O29 [M + H]+, 1537.8; found m/z 1537.8, calcd. For [M + 2H]+ 769.4; found m/z 769.6. Synthesis of 7‒18 and 22‒24. The fatty acids (C12, C12 diacid monoester, C18 and C18 monoester, 4 eq.) were coupled to the linker-dimer conjugates (4‒6 and 20‒21) using HBTU (4 eq., 0.5M) and DIEA (8 eq.) in dry DMF for the shorter-chain acids and dry DCM:DMF (75:25) for the longer-chain acids. After the coupling was complete, the FA-linked dimeric ligands were cleaved from the resin with concomitant side-chain deprotection using TFA/TiPS/H2O (90:5:5). The methyl protection group of the mono-protected FAs was then removed by stirring the cleaved products in 0.5M LiOH in H20/ACN 75/25 (100 mL) for 30 minutes followed by acidification with TFA. The resulting mixture was lyophilized. Purification of the crude FA-linked dimeric ligands was conducted by dissolving the ligand in the smallest possible volume of 100% DMSO (95% purity (Scheme 1). Characterization of 7‒18 and 22‒24 is shown in table S1.
ASSOCIATED CONTENT Supporting Information. General information on chemistry and peptide synthesis, synthetic procedure for C18 diacid methyl ester, procedures for protein expression, fluorescence polarization assay, plasma stability assay, and in vivo pharmacokinetic studies; and Table S1-S2 and Scheme S1. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author
[email protected], +45 35336114
ACKNOWLEDGMENTS We are grateful for financial support from the Lundbeck Foundation (K.S.).
ABBREVIATIONS USED 5-Ava, 5-amino valeric acid; CPP, cell penetrating peptide; FA, fatty acid; FP, fluorescence polarization; GABA, γ-butyric acid; γ−Glu, γ−glutamic acid; HSA, human serum albumin; iGlu; ionotropic glutamate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NMDA, N-methyl-D-aspartate; PDZ, PSD-95, discs large, zonula occludens-1; PEG, polyethylene glycol; PPIs, protein-protein interactions; PSD-95, postsynaptic density-95. SAR, structure-activity relationship.
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Journal of Medicinal Chemistry
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Journal of Medicinal Chemistry
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