Enthalpic Forces Correlate with the Selectivity of Transthyretin

Article pubs.acs.org/jmc

Enthalpic Forces Correlate with the Selectivity of TransthyretinStabilizing Ligands in Human Plasma Irina Iakovleva,† Kristoffer Bran̈ nström,† Lina Nilsson,‡ Anna L. Gharibyan,§ Afshan Begum,‡ Intissar Anan,∥ Malin Walfridsson,† A. Elisabeth Sauer-Eriksson,‡ and Anders Olofsson*,† †

Department Department § Department ∥ Department ‡

of of of of

Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden Chemistry, Umeå University, 901 87 Umeå, Sweden Pharmacology and Clinical Neurosciences, Umeå University, 901 87 Umeå, Sweden Public Health and Clinical Medicine, Umeå University, 901 87 Umeå, Sweden

S Supporting Information *

ABSTRACT: The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design.

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INTRODUCTION

The ability of a ligand to stabilize a complex is directly correlated to its binding affinity (KD). However, the in vivo efficacy is dependent on several other factors. In the present work we have focused on the selectivity of a ligand to its target over other components in plasma, which represents the environment where TTR is predominantly found in vivo. A selection of compounds, illustrated in Figure 1, was identified through screening of a chemical library based on drugs that have previously been approved for other clinical purposes (Prestwick Chemical Library). A total of nine different TTR tetramer-stabilizing ligands were identified, including diflunisal (DIF), diclofenac (DIC), niflumic acid (NIF), aceclofenac (ACL), gemfibrozil (GEM), luteolin (LUT), apigenin (API), meclofenamic acid (MEA), and tolfenamic acid (TOA). All ligands apart from GEM, ACL, and TOA have previously been shown to have a TTR tetramer stabilizing effect.23,31,32 The binding constant (KD) to TTR was determined for all ligands using isothermal titration calorimetry (ITC). In parallel, their efficacy in stabilizing TTR in human plasma was evaluated. The results showed that the level of nonspecific binding to the vast excess of other plasma components can strongly affect the efficacy of certain ligands. Surprisingly, we found that the KD between the ligand and TTR does not

Transthyretin (TTR) is a homotetrameric β-strand protein found in serum and cerebrospinal fluid and is involved in the transport of thyroxin T4 hormone and retinol binding protein. The misfolding and aggregation of TTR are associated with a group of degenerative conditions, including senile systemic amyloidosis,1 familial amyloidotic polyneuropathy,2 familial amyloidotic cardiomyopathy,3 and central nervous system amyloidosis.4 Recently, correlative observations between aggregated TTR and the development of preeclampsia5 and lumbar spinal stenosis6 have also been reported. It is well-known that formation of TTR amyloid requires dissociation of its tetrameric integrity,7−9 and this feature presents an avenue for intervention where binding of ligands to the thyroxine hormone T4 binding site (TBS) on TTR decreases the rate of dissociation and lowers the rate of amyloid formation.10 However, the concentration of T4 in human plasma is too low to be effective, and external administration of the hormone to reach full saturation of all TTR molecules in vivo cannot be done without adverse side effects.11 The TBS is, however, amenable to binding by alternative ligands, and several candidates have been identified.12−26 Some of these ligands are already in clinical use, and promising findings have recently been reported for the nonsteroidal anti-inflammatory ligands diflunisal27 and Vyndaqel28−30 for treatment of familial amyloidotic polyneuropathy (FAP). © XXXX American Chemical Society

Received: April 6, 2015

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DOI: 10.1021/acs.jmedchem.5b00544 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Chemical structures of the nine analyzed drugs within present investigation: LUT, luteolin; API, apigenin; DIF, diflunisal; DIC, diclofenac; aceclofenac, ACL; meclofenamic acid, MEA; NIF, niflumic acid; GEM, gemfibrozil; tolfenamic acid, TOA.

Table 1. Tabulation of the ITC Data from Probing TTR and the Evaluated Ligands at 25 °Ca binding affinity to TTR (KD), nM IC50 plasma, μM enthalpy (ΔH), kcal entropy (ΔS), kcal 25 °C free energy (ΔG), kcal/mol (25 °C) molecular weight, g/mol XLogP3 hydrogen bond donors hydrogen bond acceptors rotatable bonds surface area, Å2 charge

LUT

API

DIF

TOA

MEA

NIF

DIC

ACL

GEM

70 7±3 −11.2 −1.4 −9.8 286 1.4 4 6 1 107 0

250 18 ± 3 −9.6 −0.6 −9.0 270 1.7 3 5 1 87 0

580 25 ± 12 −7.8 0.7 −8.5 250 4.4 2 5 2 57.5 −1

150 40 ± 11 −9.4 −0.1 −9.3 261 5.2 2 3 3 49.3 −1

480 50 ± 4 −6.8 1.8 −8.6 295 5.2 3 3 3 49.3 −1

260 130 ± 40 −7.0 2 −9.0 282 3.7 2 7 3 62.2 −1

1000 140 ± 38 −3.8 4.3 −8.1 296 na 2 3 4 49.3 −1

1200 170 ± 39 −6.6 1.4 −8.0 354 4.3 2 5 7 75.6 −1

100 180 ± 27 −1.0 8.6 −9.6 250 3.8 1 3 6 46.5 −1

Data include ΔS, ΔH, ΔG, and the corresponding KD. The table also lists the IC50 in plasma for each compound as well as chemical properties of the corresponding ligands with respect to molecular weight, number of hydrogen-bond donors/acceptors, XLogP3, number of rotatable bonds, topological polar surface area, and the specific charge in water at pH 7. Ligands are luteolin (LUT), diflunisal (DIF), apigenine (API), niflumic acid (NIF), aceclofenac (ACL), meclofenamic acid (MEA), diclofenac (DIC), gemfibrozil (GEM), and tolfenamic acid (TOA). a

drugs with a high selectivity in a complex environment, which is an almost universally desired feature within drug design.

correlate well with the selectivity. Unspecific binding of drugs is a common problem in drug design, and the identification of properties that determine selectivity in a complex environment is of significant interest. Determination of the KD using ITC allows the thermodynamic signature to be assessed with respect to the relative contributions of enthalpic (ΔH) and entropic (ΔS) forces. The ΔH component represents changes in chemical bonds, while the ΔS component corresponds to changes in the free motion of the system. A favorable change in ΔS upon binding of a ligand to its receptor is frequently dominated by the release of bound water molecules from a hydrophobic surface. Interestingly, we found that ΔH correlates better with plasma specificity than KD and that ligands with larger negative ΔH have a proportionally higher selectivity compared to ligands with a lower influence of ΔH, despite having a similar KD. This result is of interest in the quest to identify more efficient TTR stabilizers but might also indicate that screening for a high enthalpic component could be a general parameter to identify

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RESULTS Determination of Binding Affinities between Ligand and TTR As Measured by ITC. The binding affinities of the evaluated ligands were determined with ITC. TTR has two identical ligand-binding sites. As a result of a comparatively strong negative cooperativity, the binding of the first ligand will dominate the total binding energy as well as the stabilizing effect. Although some differences in cooperativity can be noted between different ligands, this will only have a minor influence on the stabilizing effect. Therefore, the results in all cases were fitted to an independent model where two ligand molecules bind to one tetramer with equal affinities. The KD values ranged from 70 to 1200 nM, and the results are summarized in Table 1. Presence of Plasma Strongly Influences Drug Selectivity. TTR is present at a serum concentration of B



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Figure 2. Determination of IC50 of TTR-stabilizing ligands in human plasma. The potencies of the TTR-stabilizing ligands were evaluated using a urea-mediated denaturation assay. Dissociation of the TTR-tetramer, in human plasma, is observed as presence of a monomeric TTR band on a Western blot. The fraction of denaturation is quantified through densitometric analysis, and representative examples for each ligand are shown where also the IC50 is indicated: (A) LUT, (B) API, (C) DIF, (D) DIC, (E) MEA, (F) ACL, (G) NIF, (H) GEM, (I) TOA.

around 5 μM.33 This implies that the effective concentration of a ligand must be equal to or greater than 5 μM in the plasma to be effective, i.e., to reach a stoichiometric ratio corresponding to at least one bound ligand molecule per tetramer. We used a urea-mediated denaturation assay similar to Sekijima et al. (2006),34 with slight modifications as described in the materials and methods section, to determine the half maximal inhibitory concentration (IC50) values of the evaluated ligands in the presence of human plasma (Figure 2). The transition from a native tetrameric form to a monomeric state formed under denaturing conditions in urea is monitored. Detection of dissociated TTR is performed using a western approach where appearance of a monomeric band is monitored. Under the given conditions TTR can be stabilized and prevented to dissociate in the presence of stabilizing ligands, and the ligand concentrations where 50% is dissociated (IC50) are indicated in each panel (Figure 2) which shows representative examples of single experiments, while the summarized results with standard deviation are given in Table 1. The results show a large variation with regard to the required ratio between ligand and tetramer to reach the IC50 in plasma. For example, the flavonoid LUT shows an extremely high selectivity and displays an IC50 corresponding to only 7 μM

which is very close to a 1:1 stoichiometric ratio between one ligand and a TTR-tetramer in plasma. On the contrary, GEM requires nearly a 40-fold excess of the ligand relative to the TTR concentration in plasma to reach IC50. A plot of the KD value of the ligands in a pure system versus the IC50 value in plasma is shown in Figure 3 and shows a great variation between different drugs and their relative specificity in plasma as a function of their KD. Enthalpic Force Predicts the Selectivity. Stabilization of the tetramer is directly proportional to the affinity of the bound ligand within the TBS. Because of the large differences between the ability of the ligands to prevent dissociation of the TTR in the presence of plasma and because of the strong deviation from a linear correlation between KD and IC50 for GEM, DIF, and NIF, it is of interest to elucidate the mechanism and binding properties that determine the selectivity. Because the binding properties might vary depending on the relative contribution of enthalpic and entropic components despite a similar binding constant, it is also of interest to determine if the thermodynamic properties of the binding affect the selectivity of the ligand in the complex matrix represented by human plasma. The relative contributions of ΔH and ΔS are simultaneously acquired in ITC upon determination of binding C



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affinity, also shows a correlation where a high contribution of ΔH is associated with a high selectivity in plasma (Figure 4B). The Crystal Structure of the TTR-GEM Complex Shows the 2,5-Dimethylphenoxy Group Buried within the TBS. The crystal structure of the TTR-GEM complex, which corresponds to the ligand-TTR complex having the lowest enthalpic contribution, was determined at 1.75 Å resolution (Table 2). The TTR-GEM complex was cocrystallized with a 5 Table 2. Data Collection and Refinement Statistics for the TTR-GEM Complexa

Figure 3. Plot between the KD in PBS and IC50 in plasma of the evaluated TTR ligands LUT (filled circle), API (open square), DIF (filled square), DIC (filled down-pointing triangle), MEA (open down-pointing triangle), ACL (filled diamond), NIF (filled triangle), GEM (open circles), and TOA (open triangle). The IC50 results are reported as the mean ± standard deviation (SD) of three independent experiments.

parameter

TTR-GEM

Data-Collection Statistics wavelength (Å) 1.5418 space group P21212 unit-cell parameters (Å) a = 42.69, b = 85.41, c = 63.62 resolution limits (Å) 35.0−1.75 (1.80−1.75) total no. of reflections 192453 no. of unique reflections 23805 (2064) multiplicity 10.0 (4.9) completeness (%) 98.55 (87.31) Rsym 0.063 (0.135) ⟨I/σ(I)⟩ 28.95 (3.65) Refinement and Model Building Statistics resolution range (Å) 35.0−1.75 R factor (%) 17.0 (25.7) Rfree (%) 21.2 (31.1) no. of atoms protein 2217 water 244 ligand atoms/sodium ion 36/1 average B-factor (Å2) protein 19.00 water 31.90 ligands 20.30 Root-Mean-Squared Deviations from Ideal Geometry bond length (Å) 0.012 bond angle (deg) 1.37 Ramachandran Plot residues in most favored regions (%) 98.5 residues allowed regions (%) 1.5 residues in disallowed regions (%) 0.0 Protein Data Bank PDB code 5BOJ

affinities, and the result for each ligand is given in Table 1. The results show a rather high variation, and the enthalpic contribution ranges between −1 to −11.2 kcal/mol. Interestingly, plotting the IC50 against the enthalpic component of the binding shows good correlation with the selectivity in plasma, in contrast to KD (Figure 4A). A plot of the relative ratio between ΔH/ΔG versus IC50, which then omits the factor of

a

Values in parentheses refer to the highest resolution shell.

M excess of inhibitor in the space group P21212 and contains one dimer in the asymmetric unit. By application of the crystallographic 2-fold symmetry along the c-axis, the biological tetramer can be obtained (Figure 5A). The inner β-sheets of the dimer−dimer (AB−A′B′) interface form two ligand-binding site cavities (TBSs) referred to as sites AA′ and BB′, respectively. High-affinity binding of GEM to TTR was confirmed from difference Fourier electron density maps (Figure 5B). Structural Differences between TTR-LUT and TTRGEM. The crystal structure of the TTR-LUT complex has recently been determined at 1.1 Å resolution.35 Superimposing the two structures shows differences in protein−ligand interactions with the TBS. Both compounds interact with two residues, Ser117 and Thr119, deep within the TBS channel. However, to accommodate both the hydrophobic 2,5-

Figure 4. (A) A correlation plot between the enthalpic component of the binding (−ΔH) and the IC50 for TTR-tetramer stabilization in human plasma: LUT (filled circle), API (open square), DIF (filled square), DIC (filled down-pointing triangle), MEA (open downpointing triangle), ACL (filled diamond), NIF (filled triangle), GEM (open circles), and TOA (open triangle). (B) Correlation plot between the relative contribution of the enthalpic component and the total Gibbs free energy of binding (ΔH/ΔG) and the IC50 for TTRtetramer stabilization: LUT (filled circle), API (open square), DIF (filled square), DIC (filled down-pointing triangle), MEA (open down-pointing triangle), ACL (filled diamond), NIF (filled triangle), GEM (open circles), and TOA (open triangle). The IC50 results are reported as the mean ± standard deviation (SD) of three independent experiments. D



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Figure 5. Crystal structure of the TTR-GEM complex. (A) The TTR monomers in the dimer structure are shown as ribbons and are labeled A and B. The symmetry-related monomers are labeled A′ and B′. Two GEM molecules bind in the thyroxin-binding channels and are shown as sticks. For clarity, only one of the symmetry-related GEM orientations is shown. (B) Quality of the electron density map at the BB′ dimer−dimer interface. The σA-weighted (m|Fo| − D|Fc|) electron density contoured at 3 times the root-mean-square value of the map is shown in orange. To reduce model bias, the GEM molecule was excluded from the coordinate file that was subjected to one round of simulated annealing refinement before calculation of the map. (C) Detailed view of the LUT and GEM binding site in the deep end of the TBS. Residues and the LUT molecule are shown in yellow, whereas residues and the GEM molecule are shown in orange. Hydrogen bonds are shown as dotted lines. The Oγ1 atoms of both B-Ser117 and BThr119 form extensive hydrogen bonds with luteolin and a water molecule. Both Ser117 and Thr119 change their side chain rotamer in the TTRGEM complex, and the two symmetry-related Ser117 side chains now form a hydrogen bond over the dimer interface.

dimethylphenoxy group of GEM and the hydrophilic A-ring of LUT with its two hydroxyl groups, the orientation (rotamer) of the Ser117 and Thr119 side chains changes (Figure 5C).

unspecific binding to other components, which then requires an excess of the ligand versus TTR to accomplish a stabilizing effect. Although we show that there is some correlation between KD and IC50 in plasma, some ligands display a strong deviation from linearity. This is well illustrated by comparing the two ligands LUT and GEM that both bind to TTR with a KD within the lower nanomolar range (70 and 100 nM, respectively) but differ dramatically with regard to their ability to stabilize TTR in plasma. LUT has an IC50 value in plasma of 7 μM, while the corresponding figure for GEM is around 180 μM. Comparing NIF and API, which have essentially the same binding affinity in PBS, shows a large difference in their selectivity in serum where NIF has an IC50 in plasma of around 130 μM while API has an IC50 value of 18 μM. DIF also deviates from linearity but has a comparatively high selectivity where the KD is around 580 nM while the IC50 in plasma is only 25 μM. These results strongly suggest that KD on its own cannot fully predict the selectivity of a ligand in a complex matrix, and this points to the need for a more predictive parameter. The KD between a ligand and its target can, as for all interactions, be described by the Gibbs free energy (ΔG). The ΔG relates to the equilibrium given in eq 1 where R is the gas constant and T the temperature in kelvin.

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DISCUSSION AND CONCLUSIONS The tissue-damaging mechanisms of TTR have not yet been fully elucidated. Although massive amyloid depositions can be detrimental to specific organs,36 biopsies of peripheral nerves from FAP patients suggest that oligomeric TTR assemblies produce tissue damage before amyloid can be observed.37 In support of this notion, a cytotoxic effect from soluble TTR has also been shown in cell-viability experiments.38 Interestingly this effect is also dependent on the dissociation of the tetrameric integrity of TTR, and the highly stable TTRT119M variant cannot adopt a cytotoxic conformation.39 The notion that dissociation of the tetramer is a ubiquitous prerequisite for a toxic effect is further supported by the finding that tetramer-stabilizing ligands such as DIF and DIC can inhibit a toxic response from TTR.40 Thus, stabilization of the TTR tetramer provides an attractive therapeutic approach irrespective of the downstream events. In the present study, we have focused on the selectivity of TTR-stabilizing ligands in human plasma with the aim of elucidating properties that influence their efficacy. In humans, the physiological concentration of tetrameric TTR in plasma is around 5 μM, which represents the lower concentration limit for a ligand to occupy at least one of the two binding sites.33 It is important to point out here that TTR displays a negative cooperativity between the two binding sites. Regarding the T4 hormone, the affinity between the two sites differs by a factor of 100.41,42 This means that the second ligand will only contribute 1% to the stabilizing energy. Although similar values can be expected for most of the ligands, even a very low ratio corresponding to, for example, 1:10 or 1:5 would only affect the affinity by 10−20% and consequently have a comparatively small impact on the results. As a consequence, binding of the first ligand will give the dominating stabilizing energy. In the presence of plasma, we show that the ability of a ligand to bind TTR can be significantly decreased because of

ΔG = −RT ln K

(1)

The ΔG is in turn made up of an enthalpic and entropic component according to eq 2. ΔG = ΔH − T ΔS

(2)

Because the relative contribution between enthalpic and entropic forces can vary between different ligands, despite identical KD values, the properties of the binding might also vary. The potential change in ΔH upon binding of a ligand to its receptor reflects the change in energy as a result of formation of chemical bonds. All chemical bonds have a distance dependency, and hydrogen bonds have additional orientation requirements, and this implies that the ΔH component of the E



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beneficial effects in a clinical trial on FAP.27 It is interesting to note that DIF, despite its comparatively low affinity, displays a high selectivity in plasma that is also likely to be a contributing factor to its clinical efficacy. The present approach to correlate thermodynamic data with the properties of selectivity in a complex matrix does not require any structural information. It is nevertheless of interest to investigate if the different thermodynamic properties can be supported by structural properties. Through cocrystallization, we could confirm that all compounds bind within the TBS (data not shown). Although a detailed structural analysis is outside the scope of this investigation, we have chosen to include the structures of the two TTR-compound complexes with the highest enthalpic component (TTR-LUT) and the lowest enthalpic component (TTR-GEM). The results suggest that residues Ser117 and Thr119 play a crucial role in providing binding selectivity. The structure of TTR-LUT35 compared with the 1.75 Å structure of TTR-GEM reported here shows how the side chain orientations of Ser117 and Thr119 change the shape and hydrophobicity of the deep end of the TBS channel. These rotamer changes allow both hydrophilic (e.g., LUT) and hydrophobic (e.g., GEM) ligands to bind; however, as indicated by the ITC result, the hydrophobic properties of GEM make it a suitable partner for many other components in plasma. Taken together, we show how the selectivity of TTRstabilizing ligands can be significantly compromised in the presence of other plasma components. We show that the ΔH of the binding correlates with selectivity and that it might be a better predictor of selectivity than the KD. This information is valuable for the design of novel and more specific TTRstabilizing drugs, which is of high therapeutic importance. An interesting question is whether a high enthalpic component can be used as a general approach to screen for drugs having a high selectivity in a complex matrix, which is an almost universally desired feature in drug design.

interaction between a molecule and its protein target is largely orientation dependent. The change in ΔS upon binding, which represents both the decreased freedom of motion and the release of bound water, is less dependent on the molecular orientation.43 We therefore hypothesized that ligands with a high ΔH in the binding to its receptor also would be more specific in a complex environment than a ligand with similar affinity but with a higher ΔS component. The results show that LUT, which by far is the most selective ligand within the analyzed group of ligands, has the largest negative ΔH value, which supports our hypothesis. Plotting the ΔH versus IC50 for all ligands shows that, in contrast to the affinity, the contribution of the enthalpic component describes the selectivity well. Interestingly, only considering the relative contribution of ΔH within the total ΔG of the binding, and thus normalizing the affinity for each ligand, also correlates well with selectivity (Figure 4B). Another interesting question is whether selectivity can be correlated to properties available from de novo analysis of the ligand’s structure. Some of the chemical properties that can be derived from a structural analysis are listed in Table 1. As shown in the table, all investigated ligands had approximately the same molecular weight and neither their size nor their overall topological polar surface area correlated with selectivity. Also, the number of possible chemical bonds (hydrogen bonds and charge interactions) failed to correlate with selectivity, which is somewhat surprising but likely reflects the fact that the formation of chemical bonds requires a strict orientation and that frequently only a subset of the atoms is involved in bond formation, which then abrogates de novo prediction. The entropic component of the interaction between the ligand and TTR displayed a negative correlation with the selectivity in plasma. A favorable entropic component is frequently the result of water exclusion due to hydrophobic interactions. However, the XLogP3, which is a commonly used theoretical figure of the overall hydrophobicity represented by the partition coefficient,44 also failed to show a correlation with selectivity. The most selective ligands (LUT, API, and DIF) all have a comparatively low number of rotatable bonds. It is reasonable to anticipate that a higher number of rotatable bonds would more easily enable structural adaptations to a higher number of different targets and, therefore, a loss in selectivity. From a therapeutic point of view, the extremely high selectivity of LUT is of interest to explore further. We have recently showed that LUT effectively rescues a neuronal cell model as well as a Drosophila melanogaster model of FAP.35 Unfortunately, LUT undergoes rapid modification in vivo through glucuronidation in the intestines and the liver45 that fully inactivates the molecule and renders it less suitable as a drug.35 GEM, also known by the brand name Lopid and used for the treatment of hypercholesterolemia,46 is shown here for the first time to be a stabilizer of TTR. However, GEM also degrades quickly with a plasma half-life in vivo of around 1.5 h, which together with its low selectivity reported here, makes it less suitable as a therapeutic alternative. ACL, API, NIF, MEA, TOA, and DIC unfortunately all fail as suitable drug candidates because of adverse side effects at the required serum concentrations and because of rapid degradation in vivo. DIF, on the contrary, has a low degree of reported side effects and a comparatively long half-life in vivo, and it has recently shown

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EXPERIMENTAL PROCEDURES

Protein Expression and Purification. Expression and purification have been described previously.39 Briefly, the transformation of the expression vector into Escherichia coli BL21 was performed according to standard procedures. Induction of protein expression was achieved with 0.4 mM isopropyl thiogalactopyranoside at 37 °C. After 18 h of culturing, cells were harvested and lysed by freeze−thaw and sonication. Cell debris was removed by centrifugation at 20 000g for 30 min. The supernatant was collected and loaded onto an anion exchange column (Q-Aepharose, Amersham Biosciences) and eluted with a NaCl gradient. The fractions containing TTR were concentrated using Centriprep filter units with Ultracel YM-10 membranes (Millipore) and loaded onto a gel filtration column (Sephadex G-75, Superdex 16/60, Amersham Biosciences) Monitoring Drug Efficacy in Human Plasmas. To evaluate the kinetic stability of TTR in a complex matrix, an indirect method of detection is required. The assay is based on denaturation in urea, which causes a monomerization of the tetramer and is performed in a similar manner as previously34,47 but where the cross-linking step is omitted. The addition of stabilizing ligands prior to the addition of urea prevents dissociation in a concentration-dependent manner, and the relative increase in stability can be measured as a function of ligand concentration. In contrast to analysis of pure TTR where a direct visualization of the proteins on the polyacrylamide gel can be made, a Western blot approach has to be applied because of the bulk amount of other proteins in the human plasma. Only the intrinsic TTR present in plasma was analyzed, and only plasma samples from a single healthy F



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ion, and 244 water molecules. Molecular graphics were produced using CCP4mg.52 Structure factors and coordinates of the TTR-GEM complex have been deposited in the Protein Data Bank (accession code 5BOJ).

donor were used throughout the work so as to eliminate individual variations. The plasma sample was buffered to pH 7.4 prior to analysis by addition of 1 M phosphate buffer to reach a final concentration of 20 mM. The compounds DIF, DIC, NIF, ACL, GEM, LUT, API, MEA, and TOA (all from Sigma-Aldrich, Stockholm, Sweden) were added at the indicated concentrations, and each ligand was incubated in plasma for 1 h at room temperature prior to a denaturation step. To initiate denaturation, an equal volume of an 8 M urea solution was added to the sample, which resulted in a final concentration of 4 M urea. To quantify the relative dissociation, the denaturation step in urea was followed by gel electrophoresis separation using tricine-based SDS− PAGE with 0.025% SDS in the running buffer and 0.2% SDS in the loading buffer. These concentrations of SDS do not denature the tetramer but do prevent reassociation of monomers. TTR was detected using the well-established rabbit anti-TTR antibody (antiprealbumin, DAKO, Glostrup, Denmark) followed by a horseradish peroxidase-labeled antibody (anti-rabbit HRP, GE Healthcare, Uppsala, Sweden). Blocking of nonspecific binding on the membrane and incubation of the antibodies were performed in 5% fat-free powdered milk containing 0.3% Tween-20. All washing steps were performed in 40 mM Tris, 150 mM NaCl, and 0.3% Tween-20. Bound antibodies were visualized on photographic film using enhanced chemiluminescence (ECL-prime, GE Healthcare, Buckinghamshire, U.K.). The employed rabbit anti-transthyretin antibody (antiprealbumin, DAKO, Glostrup, Denmark), however, does not detect the native fraction of TTR very well, and as a consequence, only the change in monomers, which reflect the level of dissociation, is monitored. The presence of monomers on the photographic film was quantified through a densitometric analysis using the ImageJ 1.48 program, which also monitors that all analysis is performed within a linear range. The IC50 results are reported as mean ± standard deviation (SD) of three independent experiments. Isothermal Titration Calorimetry. Binding experiments were performed using an Auto-iTC200 (MicroCal, Malvern, U.K.) at 25 °C. Protein concentrations in the cell were 145 μM, and the ligands were titrated at a 10-fold molar excess. For the control experiment, compounds were titrated into the cell with buffer (PBS with the corresponding DMSO concentration used in the experiment). For each experiment, 19 automated injections of 2 μL each were performed (duration 0.8 s) with 300 s intervals between each injection and with a stirring speed of 1000 rpm. The titrations were done with high feedback and a filter period of 5 s and were repeated twice. Calorimetric data were plotted and fitted using the standard single-site binding model. Crystallization of the TTR-GEM Complex. The protein was crystallized as described previously.48 Briefly, the purified TTR was dialyzed against 10 mM Na phosphate buffer with 100 mM KCl (pH 7.6) and concentrated to 5 mg·mL−1 using an Amicon Ultra centrifugal filter device (Millipore, 3 kDa molecular-weight cutoff) and cocrystallized at room temperature with a 5 M excess of GEM using the vapordiffusion hanging drop method. A drop containing 3 μL of protein solution was mixed with 3 μL of precipitant and equilibrated against 1 mL of reservoir solution containing a range of 1.3−1.6 M sodium citrate and 3.5% v/v glycerol at pH 5.5 in 24-well Linbro plates. Crystals grew to dimensions of 0.1 × 0.1 × 0.4 mm3 after 5 days. The crystals were cryoprotected with 12% v/v glycerol. Data Collection, Integration, and Structure Determination. The X-ray diffraction data were collected on our in-house Bruker MicroStar facility at 1.75 Å resolution and were processed and scaled with SAINT and SADABS. See Table 1 for details of data collection and refinement statistics. The structure of wild-type TTR (PDB code 1F41,49) and X-ray data from 35.0−1.75 Å resolution were used in molecular replacement searches with the program PHASER.50 The GEM molecule is best defined in the BB′ cavity. The TTR-GEM complex was refined against all the diffraction data using phenix.refine from the PHENIX program suite51 resulting in an R factor of 17.0% and an Rfree of 21.2%. The refined structure comprises TTR residues 10−125 from monomer A and B, two GEM molecules, one sodium

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00544. Molecular formual strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46703543301. E-mail: anders.olofsson@medchem. umu.se. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Ole Suhr of the Department of Public Health and Clinical Medicine, Umeå University, for valuable discussions and input on the manuscript. This work was supported by the Swedish Research Council, Parkinsonfonden, Insamlingsstiftelsen vid Umeå Universitet, Alzheimerfonden, Åhlenstiftelsen, J. C. Kempes Stiftelse, the Swedish Research Council, Hjärnfonden, Magnus-Bergvalls Stiftelse, O.E. och Edlas Stiftelse, Torsten Söderbergs Stiftelse, Västerbottens Läns Lasting (ALF-medel), Patientföreningen FAMY/AMYL, and the Medical Faculty of Umeå University.

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ABBREVIATIONS USED TTR, transthyretin; TBS, thyroxine binding site; ΔH, enthalpy; ΔS, entropy; DIF, diflunisal; DIC, diclofenac; NIF, niflumic acid; API, apigenin; ACL, aceclofenac; GEM, gemfibrozil; LUT, luteolin; API, apigenin; TOA, tolfenamic acid; MEA, meclofenamic acid

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REFERENCES

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