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. 2020 Mar 12;11(10):1973–1979. doi: 10.1021/acsmedchemlett.9b00688

Brain Permeable Tafamidis Amide Analogs for Stabilizing TTR and Reducing APP Cleavage

Anjana Sinha †, Jerry C Chang †,‡, Peng Xu †, Katherina Gindinova †, Younhee Cho §, Weilin Sun †, Xianzhong Wu ‡, Yue Ming Li ‡, Paul Greengard †, Jeffery W Kelly §, Subhash C Sinha †,*
PMCID: PMC7549266  PMID: 33062181

Abstract

graphic file with name ml9b00688_0006.jpg

Tafamidis, 1, a potent transthyretin kinetic stabilizer, weakly inhibits the γ-secretase enzyme in vitro. We have synthesized four amide derivatives of 1. These compounds reduce production of the Aβ peptide in N2a695 cells but do not inhibit the γ-secretase enzyme in cell-free assays. By performing fluorescence correlation spectroscopy, we have shown that TTR inhibits Aβ oligomerization and that addition of tafamidis or its amide derivative does not affect TTR’s ability to inhibit Aβ oligomerization. The piperazine amide derivative of tafamidis (1a) efficiently penetrates and accumulates in mouse brain and undergoes proteolysis under physiological conditions in mice to produce tafamidis.

Keywords: Alzheimer’s disease, Aβ peptide, γ-secretase, prodrug, tafamidis, transthyretin

Transthyretin (TTR) is a tetrameric carrier protein, transporting thyroid hormone and holo retinol-binding protein.1 It is secreted by liver into the blood at a steady state concentration of about 3–6 μM and by the choroid plexus (CP) into the cerebrospinal fluid (CSF) at a steady state concentration of ≈300 nM.2 In brain, TTR counteracts the neurotoxic effects of Aβ peptides by reducing their aggregation and enhancing clearance of the oligomers and plaques from brain.3−5 TTR may also degrade Aβ peptides.6 Neurotoxic Aβ species seem to initiate Tau-hyperphosphorylation (pTau) and tangle formation that together mediate neuronal damage and memory loss, leading to Alzheimer’s disease (AD).7 There are multiple studies supporting the protective effects of TTR.8 For example, overexpression of the wild-type (WT) human TTR transgene was beneficial, while silencing the endogenous TTR gene or neutralizing TTR protein function by using an anti-TTR antibody accelerated Aβ aggregation in AD mice.4,9

TTR itself is an amyloidogenic protein. Tetramer dissociation is rate-limiting for TTR aggregation, a process also requiring partial monomer denaturation.10,11 There is very strong genetic and pharmacologic evidence that TTR aggregation causes the degenerative phenotypes in the TTR amyloidoses.12−14 The TTR amyloidoses can present as a cardiomyopathy or a peripheral neuropathy,15 whereas some mutations lead to neurodegeneration of the central nervous system or the eye initially.16 If untreated, multiple organ systems become affected upon progression. There is persuasive evidence that pharmacologic kinetic stabilization of the native tetrameric quaternary structure of TTR slows or stops the progression of the TTR amyloidoses.13,17 A small molecule kinetic stabilizer could potentially benefit AD patients provided that it does not adversely affect the TTR–Aβ interaction. Indeed, it has been shown that the TTR effect on Aβ clearance increases when TTR is pharmacologically stabilized.18 There are conflicting reports though that TTR stabilization via binding at the T4-site affects the TTR–Aβ interaction adversely.8,19

Herein, we explore the benefits of using the Food and Drug Administration (FDA) approved TTR kinetic stabilizer tafamidis (1, Figure 1)17 for lowering Aβ oligomerization and production in the Alzheimer’s disease context.20 Toward this end, we have prepared amide derivatives of 1 as potential inhibitors of Aβ oligomerization and production. We have found that tafamidis weakly inhibits γ-secretase activity in a cell-free assay, whereas several amide derivatives of 1 and analogs thereof reduce Aβ production in cells independent of γ-secretase inhibition. Notably, compound 1 and its amide derivatives do not compromise TTR’s ability to reduce Aβ oligomerization. We have also shown that compound 1a, the piperazine amide of 1, accumulates in brain and is partially hydrolyzed to 1, providing proof-of-principle for a tafamidis prodrug strategy for stabilizing TTR in the brain and in the periphery.

Figure 1.

Figure 1

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Comparison of the FDA approved TTR stabilizer tafamidis, 1, with DV2-103, 2, and 2a reveals 1 as a suitable pharmacophore for developing compounds with inhibitory effects on Aβ production. Shown in blue and/or red color are the fragments in 1 and 1a identical to some fragments in 2 and 2a.

Chemistry

The synthesis of amide derivative 1a was inspired by the structures of compounds 2(21) and 2a(22) (Figure 1), which are kinase inactive analogs of PD17395523 and reduce production of Aβ peptides with micromolar EC50, similar to Gleevec and analogs.24,25 Compounds 1 and 1a have some substructures in common with 2 and 2a, Figure 1. We prepared compound 1a by forming an amide bond between acid 1(26) and the Boc-protected amine a, followed by Boc-deprotection using 4 M HCl in dioxane (Figure 2A, upper row). We similarly synthesized additional amides of 1 (Figure 2A, upper row), as well as amide analogs 3–9 (Figure 2B), wherein substituents on the core tafamidis structure were varied.

Figure 2.

Figure 2

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Structure and synthesis of various amide derivatives of tafamidis (A) and of the analogous acids (B). Note: Step “i” was used with all compounds, and step “ii” for the Boc-protected intermediates that were obtained in step “i” using the Boc-protected amines a′, c′, or h′.

Screening and Evaluation

We performed screening of 1 and the amide derivatives 1a–i, 3a–b, 3e–g, 4b, 5a–b, 6a, 7a, 8a, and 9a (Figure 2) and determined their effects on Aβ production in N2a695 cells. The latter express human WT amyloid precursor protein 695 (APP695), which undergoes two successive cleavages to produce human Aβ peptides.27,28 First β-secretase or β-cleavage shreds the large extracellular portion of APP as a soluble APP fragment (sAPPβ) leaving the membrane-bound C-terminal fragment (βCTF or C99). Subsequent γ-secretase or γ-cleavage of βCTF leads to multiple Aβ isoforms and the APP intracellular domain (AICD). For the Aβ production assay, we incubated N2a695 cells with candidate compounds (10 μM) for 6 h at 37 °C and collected the culture media and cell lysates, as described.22,25 We quantified the levels of Aβ40 and Aβ42, which are the most prominent and relevant Aβ isoforms in AD, in culture media using commercially available human Aβ ELISA kits. We found that four compounds, 1a, 1h, 3a, and 3e, reduced production of both Aβ40 and Aβ42 peptides by 30–60% compared to the DMSO (vehicle) control (See: SI Figure S-1 and Figure 3A). To confirm that the effect of compounds 1a, 1h, 3a, and 3e on Aβ40 and Aβ42 production is not due to any toxicity, we incubated N2a cells with each compound (10 μM) in a 96-well plate for 24 h and determined the number of viable cells using MTT assay.29 Although, we observed some cellular toxicity with compounds 1h and 3a after 24 h of treatment (SI Figure S-2), it should not have affected the Aβ production, because cells were incubated with the compounds for only 6 h in the Aβ production assay.22,25

Figure 3.

Figure 3

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Tafamidis derivatives 1a and 1h and analogous compounds 3a and 3e reduce Aβ production in N2a695 cells by affecting APP cleavages. Bar graphs are the measure of Aβ40 and Aβ42 peptides in cell supernatants post treatment with these compounds and determined using human Aβ ELISA kits (A), Western blots (lower) and quantitiation of the APP-CTF levels (upper) of N2a695 cell lysates post compound treatment (B), and effect of compound 1 on γ-secretase activity measured in vitro (C). All experiments were performed at least twice in duplicate or triplicate, except for compound 3a, for which Aβ42 shown in part A was measured only once. Data shown in part C are from one experiment in duplicate. Statistical significance (P): *, <0.05%; **, <0.01%; ***, <0.001%.

Subsequently, we determined the effects of 1, 1a, 1h, 3a, and 3e on APP cleavage in comparison to DMSO controls, in N2a695 cells by performing Western blotting (WB) of cell lysates. We probed SDS-PAGE gels with antibody RU369, which recognizes the C-terminus of APP fragments. The results, shown in Figure 3B, clearly revealed an increase in APP-CTF in samples treated with compounds 1a, 1h, 3a, and 3e, but not in the vehicle control (DMSO) or upon treatment with 1. These results suggest γ-secretase inhibition or α-secretase activation. The α-secretases are a group of metalloenzymes known to mediate nonamyloidogenic cleavage of APP. However, by performing γ-secretase activity assay, in vitro,30 we found that compounds 1a, 1h, 3a, and 3e did not inhibit γ-secretase activity appreciably at 30 μM or lower concentration (Figure 3C). In contrast, compound 1, which did not show activity in N2a695 cells, inhibited γ-secretase in a dose dependent manner, with an apparent IC50 of <30 μM. We further studied tafamidis amide 1a. Compound 1a is nontoxic to cells for extended incubation periods (SI Figure S-2).

Poor clearance of the aggregation-prone Aβ peptide leads to oligomers and plaque build-up in the brain. In vivo studies with an anti-TTR antibody in a mouse model have earlier shown that neutralizing the TTR protein increases Aβ load.9 Conversely, increasing TTR expression reduces Aβ load, likely through reducing Aβ oligomerization and enhancing Aβ clearance.31 To test that compounds 1 and 1a do not interfere with the Aβ/TTR interaction, we used fluorescence correlation spectroscopy (FCS), which conveniently distinguishes and predicts the levels (particle number) of smaller vs the larger Aβ particles.32 In this study, the Aβ peptide, mixed with a minute amount of a fluorophore-labeled Aβ peptide, is used and temporal fluctuations of fluorescence caused by diffusion of fluorophore-labeled Aβ peptide passing through a small confocal volume (<1 fL) are recorded. Samples containing monomeric Aβ are expected to show lower autocorrelation functions in comparison to oligomeric Aβ. To perform FCS, 10 μM Aβ peptide (Aβ40 and Aβ42 in 1:1 ratio), containing 2 nM of tetramethylrhodamine (TMR)-labeled Aβ40, and 2 nM cholesterol were distributed in a spectroscopy 8-well plate. Fluorescence intensity and frequency of the intensity fluctuations in each well were recorded at 0, 2, and 4-h time point.

It became evident that significant Aβ aggregation occurred over a 4-h period, as indicated by the large amplitude increase in the autocorrelation curve between 2 and 4 h (Figure 4A, red dashed arrow) in the absence of TTR. This did not occur in the presence of TTR (Figure 4B), indicating a strong inhibition effect of TTR on Aβ aggregation.33 To test whether tafamidis or the amide derivative 1a might interfere with the ability of TTR to inhibit Aβ aggregation, addition of compound 1 or 1a to Aβ and TTR results in only fractional changes in diffusion times over 4 h, indicating that under our experimental conditions transition from monomer to aggregated Aβ is minimal (Figure 4C–D). Notably, when Aβ/TTR solution was mixed with 1a the autocorrelation curves displayed much lower diffusion times in comparison to Aβ/TTR solution alone or upon adding 1 to Aβ and TTR (Figure 4D vs 4B–C). This indicated a slower phase of Aβ aggregation with 1a, as the concentration of Aβ, which could be reflected by the autocorrelation curves in Figure 4D, remains high over a 4 h period. However, in the absence of TTR, compound 1 or 1a was unable to inhibit Aβ aggregation (SI Figure S-4). In summary, these results suggest that usage of 1 or 1a has no discernible adverse effect on the beneficial effect of TTR on inhibiting Aβ oligomerization.

Figure 4.

Figure 4

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TTR inhibits Aβ polymerization in both the presence or absence of 1 and 1a, as determined using FCS. Shown are fitted autocorrelation curves for diffusion of decreasing Aβ (Aβ40:Aβ42, 1:1) particle concentrations as a function of time. (A) The amplitude of the autocorrelation curve at a given diffusion time (τ = 10–4 s, red dashed-line arrow) increases along the reaction time point 0, 2, and 4 h representing the decreasing particle numbers as a result of Aβ polymerization. In addition, the autocorrelation functions show increasing contribution from long delay times, indicating the increasing size of Aβ particles. A minimum change in autocorrelation curves was found when Aβ polymerization was followed in the presence of: (B) TTR, (C) TTR + 1, or (D) TTR + 1a, over a 4-h period, indicating that TTR inhibited Aβ polymerization in both the presence or absence of 1 and 1a. Lower autocorrelation curves found with TTR + 1a indicate a strong beneficial effect of compound 1a on TTR-mediated Aβ aggregation inhibition.

Next, we determined brain permeability of compound 1a in 3-month old 5xFAD mice.34 These mice exhibit Aβ pathology within 3 to 6 months and are suitable for performing this efficacy study, in vivo. Mice were dosed with compound 1a in the drinking water for 2 weeks, and the concentration of 1a in plasma and in brain tissue was determined using LC-MS/MS.35 Because amide 1a could undergo proteolysis to afford tafamidis in the stomach, and/or the plasma, and/or in brain, we also determined the concentration of 1 in both blood and brain tissue extracts. As shown in Figure 5A, the piperazine amide derivative of tafamidis (1a) appears to penetrate mouse brain (Figure 5A), although the blood brain barrier (BBB) in 5xFAD mice is known to be compromised. In future studies, it is important to rule out blood contamination of brain extracts. Amide 1a underwent proteolysis in mice to produce tafamidis, detected both in murine brain and in plasma when mice consumed 1a in the drinking water over a period of 15 days.

Figure 5.

Figure 5

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Brain penetration and activity of tafamidis and the amide derivative 1a. (A) Brain and serum concentration of 1a and product 1. Compound 1a (50 mg/kg) was given to 3 months old 5xFAD mice (n = 3) for 15 days. Blood and brain tissues were collected on the 15th day and analyzed to determine the concentration of 1a and 1 using LC-MS/MS. (B) Microsomal stability of 1a, measured by incubating it with mouse and human liver microsomes and determining the remaining 1a periodically using LC-MS/MS. (C) Effects of compound 1a on TTR stabilization as compared to tafamidis, 1, and determined using TTR subunit exchange assay.

To estimate the pharmacokinetics of compound 1a in humans, we determined its metabolic stability using human and mouse liver microsomes and determined amounts of the nonmetabolized 1a using methodology, as described.25,36 We incubated 1a at two concentrations (1 and 10 μM) with mouse and human liver microsomes for 30 and 60 min at 25 °C, before the remaining amounts of 1a were quantified. The results, tabulated in Figure 5B, established that compound 1a is likely more stable in humans than in mice. The stability of 1a in human or murine plasma was not established.

Compound 1a appears to penetrate the BBB and undergo hydrolysis in the brain and in the plasma to produce tafamidis, providing proof-of-principle that analogous compounds could become compound 1 prodrugs. Since there are both 1 and 1a in plasma and brain samples, we probed the ability of both compounds to stabilize TTR in plasma using the TTR subunit exchange assay.17,37,38 Prior to the initiation of subunit exchange, compound 1 or 1a was added to healthy donor plasma at concentrations ranging from 0 to 60 μM. Following a 30 min incubation period at 25 °C, subunit exchange assays were initiated by adding 1.8 μM recombinant dual-FLAG-tagged WT (FT2-WT) TTR to the plasma samples. The results, shown in Figure 5C, clearly demonstrate that compound 1a stabilizes TTR, but not as well as tafamidis does. WT TTR subunit exchange, quantified by kex (subunit exchange rate or the rate of tetramer dissociation), proceeds faster with 1a than with 1 below a concentration of 30 μM.

In conclusion, amide derivatives of tafamidis and of compound 3 significantly reduced production of Aβ peptides in N2a695 cells. There was an increase in the levels of CTFs suggesting that the test compounds inhibited the γ-secretase enzyme. By using a sensitive FCS detection method, we provide direct evidence that the inhibitory effects of compound 1 and 1a on Aβ oligomerization are TTR dependent. Further investigations are required to characterize the interaction of compound 1a and TTR on a molecular level. Compound 1a also provides proof-of-principle that with further refinement, it may be possible to make a brain permeable prodrug of 1, which has implications for treatment of TTR-associated amyloidosis in the brain and the eye.39

Experimental Section

Synthesis and Characterization of Compounds

All amide derivatives of acids 1 and 3–9 were prepared by usual HATU coupling of these compounds with various amines, and those possessing N-Boc protection were deprotected using 4 M HCl in dioxane, as described in the SI.

Aβ Production and APP Metabolism Studies

N2a695 cells (4.0 × 105–4.5 × 105 cells/mL, 2 mL/well, 90–95% confluent) were treated with compounds (10 μM final concentration) at 37 °C under 5% CO2 atmosphere for 6 h. Subsequently, cell media and cell lysates were collected for determining Aβ levels and APP and CTF levels, respectively. Aβ concentration was determined by transferring cell culture media (50–100 μL, 5–10× dilution for Aβ40 and no dilution for Aβ42 measurement) to strips of 96-well ELISA plates precoated with the human Aβ40 and Aβ42 peptide capture antibodies. Further processing was carried out per manufacturer instructions, and signals for Aβ were measured using PerkinElmer Envision ELISA reader. For determining APP metabolites in cell lysates, the latter (30 μL) were loaded and separated on a 16.5% Tris-Tricine gel (Criterion) and electrotransferred to PVDF membrane (EMD Millipore). The membrane was then treated with glutaraldehyde (Sigma) solution (0.25% in PBS) before it was blocked using milk PBST and probed using antibody RU369, HRP-linked secondary antibody, and enhanced chemiluminescence ECL reagent for detection.

TTR Subunit Exchange Assay

Subunit exchange assays were done as previously described.37,38 Briefly, prior to subunit exchange assays, compounds were added ex vivo to two healthy donor plasma samples at the indicated concentrations. Samples were then incubated at room temperature for 30 min. Subunit exchange was initiated by the addition of 1.8 μM FT2-WT TTR to 40 μL of the plasma samples which were then incubated at 25 °C for 48 h. At the end of the 48 h incubation, a 10 μL aliquot of the exchange mixture was transferred to a UPLC sample glass vial (Waters) to which 1 μL of 3 mM stock solution of A2, a fluorogenic small molecule (S-(4-fluorophenyl) (E)-3-(dimethylamino)-5-(4-hydroxy-3,5-dimethylstyryl)benzothioate),40 was added for a 3 h reaction period, followed by addition of 52 μL of 50 mM phosphate buffer, pH 7.6. Samples were analyzed via ion exchange chromatography and fluorescence from the TTR·A2 conjugate allows detection of TTR tetramers. The rate of subunit exchange was quantified by the rate of appearance of the (WT TTR)3(FLAG-tagged TTR)1 heterotetramer·A2 conjugate peak.

FCS Data Acquisition

All FCS measurements were performed on the Zeiss LSM 880 confocal microscope (Carl Zeiss, Thornwood, NY). A 40× Water immersion 1.2 NA objective from Carl Zeiss was used to perform all measurements. A 561 nm wavelength laser was used to excite the sample, and the detection range was between 570 and 650 nm. Laser power has been kept very low (between 0.1 and 0.3%) in order to minimize triplet state transitions while keeping the CPM (counts per molecule) rate high. Pinhole alignments were made using free dye in solution at the start of the day of each experiment. For each experiment, 10 traces of 20 s each were recorded.

FCS Data Analysis

Individual autocorrelation curves were calculated using Zeiss Zen Black software (Carl Zeiss, Thornwood, NY) and then plotted in MATLAB (The MathWorks, Natick, MA). The autocorrelation function G(τ) is defined as

graphic file with name ml9b00688_m001.jpg 1

where F(τ) is the fluorescence obtained from the volume at delay time τ, brackets denote ensemble averages, and ∂F(τ) = F(τ) – ⟨F(τ)⟩. The fitting formula is based on the multicomponent diffusion model:

graphic file with name ml9b00688_m002.jpg 2

where N is the number of fluorescent particles in the confocal volume, Di is the diffusion coefficient of the i-th species; wxy and wz are the effective lateral and axial dimensions of the Gaussian focal volume, respectively, and τ is the lag time. For Aβ polymerization, we considered the value of i to be 2.41

Statistical Analysis

The data are presented as means ± SEM. Data were analyzed by Student’s t test for single comparison and one-way ANOVA for multiple comparisons, and those showing P value <0.05 were considered significant.

Acknowledgments

We are thankful to Mondana Ghias and Emily Chang for performing some preliminary experiments, to Joseph Fernandez and Uygar Sozer of RU for performing MS analysis of compounds, and to William Netzer, and Victor Bustos for helpful discussions. We are also thankful to RU Proteomics and High Throughput Screening and Spectroscopy Centers for the MS analysis and NMR spectroscopy and Molecular Cytology Core Facility at Memorial Sloan-Kettering Cancer Center for the Fluorescence Correlation Spectroscopy study. This work was supported by The JPB Foundation (Grant # 839 to S.C.S.) and The Fisher Center for Alzheimer’s Research Foundation (P.G.)

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00688.

  • General synthetic procedures and characterization of synthesized compounds including 1H NMR spectra and MS data. Figures showing screening data of all synthetic analogs and evaluation of selected compounds. (PDF)

  • SMILES data for compounds (XLSX)

Author Contributions

AS, JCC, PX, YHC, WS, YML, PG, JWK, and SCS designed the experiments. AS, JCC, PX, KG, YHC, WS, and XZW performed and analyzed the experiments. AS, JCC, PX, YHC, YML, JWK, and SCS wrote the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ml9b00688_si_001.pdf (894.1KB, pdf)
ml9b00688_si_002.xlsx (9.2KB, xlsx)

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