European Journal of Medicinal Chemistry 261 (2023) 115837 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: www.elsevier.com/locate/ejmech Research paper PITB: A high affinity transthyretin aggregation inhibitor with optimal pharmacokinetic properties Francisca Pinheiro a, Nathalia Varejão a, 1, Adrià Sánchez-Morales b, 1, Filipa Bezerra c, d, Susanna Navarro a, Adrián Velázquez-Campoy e, f, Félix Busqué b, Maria Rosário Almeida c, d, Ramon Alibés b, David Reverter a, Irantzu Pallarès a, **, Salvador Ventura a, g, * a Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain b Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain c Molecular Neurobiology Group, i3S – Instituto de Investigação e Inovação em Saúde, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200- 135, Porto, Portugal d Departamento de Biologia Molecular, ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313, Porto, Portugal e Department of Biochemistry and Molecular & Cellular Biology, and Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Unit GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain f Aragon Institute for Health Research, Zaragoza (Spain) and Biomedical Research Network Center in Hepatic and Digestive Diseases (CIBERehd), Madrid, Spain g ICREA, Passeig Lluis Companys 23, E-08010, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: The aggregation of wild-type transthyretin (TTR) and over 130 genetic TTR variants underlies a group of lethal Amyloidosis disorders named TTR amyloidosis (ATTR). TTR chemical chaperones are molecules that hold great promise to Lead optimization modify the course of ATTR progression. In previous studies, we combined rational design and molecular dy­ Pharmacokinetics namics simulations to generate a series of TTR selective kinetic stabilizers displaying exceptionally high affin­ Stabilizers Transthyretin ities. In an effort to endorse the previously developed molecules with optimal pharmacokinetic properties, we V30M conducted structural design optimization, leading to the development of PITB. PITB binds with high affinity to TTR, effectively inhibiting tetramer dissociation and aggregation of both the wild-type protein and the two most prevalent disease-associated TTR variants. Importantly, PITB selectively binds and stabilizes TTR in plasma, outperforming tolcapone, a drug currently undergoing clinical trials for ATTR. Pharmacokinetic studies con­ ducted on mice confirmed that PITB exhibits encouraging pharmacokinetic properties, as originally intended. Furthermore, PITB demonstrates excellent oral bioavailability and lack of toxicity. These combined attributes position PITB as a lead compound for future clinical trials as a disease-modifying therapy for ATTR. Besides its well-known role as a transporter of thyroxine (T4) and retinol 1. Introduction binding protein-vitamin A complex, TTR is increasingly recognized as having a neuroprotective activity in the central nervous system [5]. Amyloid diseases encompass a wide spectrum of disorders charac­ The most common forms of ATTR are related with the aggregation of terized by the accumulation of amyloid fibrils, highly organized struc­ TTR mutants and inherited in an autosomal dominant manner [6]. To tures that arise from the misfolding and subsequent aggregation of date, ATTR onset has been associated with more than 130 genetic var­ proteins [1]. Transthyretin amyloidosis (ATTR) is a group of iants [7], most of them causing peripheral neuropathy [8] (known as life-threatening systemic disorders caused by the extracellular deposi­ familial amyloid polyneuropathy - FAP) or cardiomyopathy [9] (known tion of transthyretin (TTR) amyloid fibrils [2]. as familial amyloid cardiomyopathy - FAC). In some rare cases, TTR TTR is a 55 kDa homotetrameric protein, which is mainly synthe­ mutations can result in leptomeningeal amyloidosis, which is often sized in the liver, choroid plexus and retinal pigment epithelium [3,4]. accompanied by ocular involvement [10]. * Corresponding author. Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bel­ laterra, Barcelona, 08193, Spain. ** Corresponding author. E-mail addresses: irantzu.pallares@uab.cat (I. Pallarès), salvador.ventura@uab.cat (S. Ventura). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ejmech.2023.115837 Received 24 July 2023; Received in revised form 24 September 2023; Accepted 26 September 2023 Available online 6 October 2023 0223-5234/© 2023 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 2. Results and discussion Abbreviations 2.1. Rational design and synthesis of PITB ATTR transthyretin amyloidosis FAP familial amyloid polyneuropathy In this study, we aimed at improving the PK properties of M-23 while FAC familial amyloid cardiomyopathy preserving the primary attributes that enhance its activity relative to FAP familial amyloid polyneuropathy tolcapone. Since the new contacts between the lower phenyl ring of M- IEF isoelectric focusing 23 and TTR were proposed to underlie its increased binding affinity and ITC isothermal titration calorimetry stabilizing activity [27], we kept this ring intact and focused on the 3, TTR transthyretin 4-dihydroxy-5-nitrophenyl moiety. For tolcapone, the in vivo methyl­ ation of the 3-hydroxy substituent has been described to generate a long-lived metabolite displaying TTR kinetic stabilizing activity [28,29]. Owing to the chemical similarity between the two compounds, we hy­ ATTR can also be caused by the wild-type (WT) protein. The depo­ pothesized that the methylation of M-23 could also improve its PK sition of WT-TTR, especially in the heart, underlies the development of profile, while keeping the specific contacts it establishes with TTR senile systemic amyloidosis, a condition that mainly affects elderly man (Fig. 1). [11]. Senile systemic amyloidosis is gaining recognition as a cause of In support of this idea and taking advantage of the plasma samples heart failure, affecting as many as 25% of individuals over 80 years of we collected in a previous M-23 PK study in mice, we confirmed that the age [12,13]. 3-O-methylation of M-23 occurs in vivo and that, remarkably, the Pathogenic mutations in TTR decrease its thermodynamic stability resulting derivative exhibited up to 3-fold longer half-life (t1/2) in and/or lower the kinetic barrier for tetramer dissociation [14,15], which plasma than M-23 (Table 1 and Fig. 2). Of note, M-23 methylation is is considered to be the rate-limiting step in amyloid formation. Occu­ anticipated to be a minor metabolic route [28], thus being unlikely to pancy of the T4-binding sites by specific small molecules is known to contribute to the pharmacological action of M-23. With this in mind, we stabilize TTR, inhibiting its dissociation and, thus, preventing ATTR proceeded to synthesize 3-O-methylM23, which we designated PITB, onset [16,17]. The so-called kinetic stabilization strategy is the mode of and to analyze its biological activity. action of the drug tafamidis [18], currently the only pharmacological PITB was prepared according to Scheme 1 following an optimized treatment in the market for both TTR-related polyneuropathy and car­ synthetic sequence developed by us [27]. The synthesis leading to PITB diomyopathy [19,20]. However, the evidence that 30% of patients do involves the coupling reaction of the freshly prepared Grignard reagent not respond to tafamidis [21], together with its high cost [22], has of iodoarene compound 1 to the methyl diprotected nitro-catechol 2 prompted the development of alternative molecules to treat ATTR. followed by a Dess-Martin periodinane oxidation reaction to furnish Using a drug repurposing approach, our group identified tolcapone, ketone 3 in 58% yield for the two steps. Selective removal of two methyl an FDA-approved drug for Parkinson’s disease, as a potent and selective protecting groups on 3 using BBr3 in DCM with controlled equivalents of TTR stabilizer, presenting a higher ex vivo tetramer stabilizing activity BBr3 and reaction time delivered the expected monoprotected catechol than tafamidis [23]. The results of a Phase IIa clinical trial demonstrated PITB in excellent yield. The structural identity and purity of all com­ that tolcapone strongly stabilizes TTR in patients with FAP, supporting pounds were ascertained by NMR spectroscopy and HRMS. The syn­ its further development as a drug to treat ATTR [24,25]. Noteworthy, thesis procedure is detailed in the Experimental Section. tolcapone can cross the blood-brain barrier, unlike tafamidis, and it was shown to bind and inhibit the aggregation of the highly destabilized TTR variants associated with leptomeningeal amyloidosis [26]. 2.2. PITB kinetically stabilizes TTR in vitro The higher stabilizing activity of tolcapone compared to tafamidis was attributed to its similar high affinity for both T4-binding sites, which We tested whether PITB could protect TTR from urea-induced contrasts with the strong negative cooperativity displayed by tafamidis tetramer dissociation. In urea, TTR subunits do not unfold until the [27]. Despite tolcapone binds significantly stronger than tafamidis to the tetramer dissociates [30]. At high urea concentrations, tetramer disso­ second binding cavity, tafamidis affinity for the first cavity is > 3-fold ciation and monomer unfolding are irreversibly linked, making it higher. The high-resolution structure of TTR in complex with tolcapone possible to evaluate the rate of tetramer dissociation [14,31]. Accord­ revealed that its 4-methyl-phenyl ring does not establish any enthalpic ingly, WT-TTR and the two most clinically relevant TTR variants, interaction with the binding pocket, suggesting that there was room for V30M- and V122I-TTR (1.8 μM) were incubated in the absence or improvement [23]. Building upon this evidence, we have recently syn­ presence of PITB (3.6 μM) and TTR denaturation was initiated by adding thesized a set of tolcapone-inspired kinetic stabilizers and identified M-23, a molecule that exhibits one of the highest affinities reported for TTR thus far [27]. Yet, a pharmacokinetic (PK) study in mice revealed that M-23 has suboptimal bioavailability (5.5%) and short plasma half-life (0.9 h), which might compromise its therapeutic use. In the present study, we introduce PITB (Pharmacokinetically Improved TTR Binder), a compound designed to address the PK limita­ tions of M-23. PK studies conducted in mice confirmed that, as intended, PITB has an excellent PK profile. TTR/PITB crystal structures demon­ strated the establishment of protein-ligand contacts at the dimer-dimer interface that are absent in tolcapone. These interactions underlie the higher binding selectivity and stabilizing activity showed by PITB in serum from both control individuals and TTR V30M carriers, when compared to tolcapone. Overall, the obtained results underscore the potential of PITB as a novel molecule for therapeutic intervention in ATTR. Fig. 1. Chemical structures of M-23, 3-O-methylM23 and tolcapone. The hy­ droxy group and its methylated form are highlighted in red, and the corre­ sponding phenyl ring is drawn in bold. 2 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 Table 1 PK parameters for M-23 and 3-O-methylM23 following intravenous (IV) (1 mg/ kg) and oral (PO) (10 mg/kg) administration of M-23 in mice. IV (1 mg/kg) PO (10 mg/kg) M-23 3-O-methylM23 M-23 3-O-methylM23 Tmax (h) — — < 0.25 < 0.25 Cmax (ng/mL) 1363.0 555.3 743.7 96.7 AUC0–24h (h⋅ng/mL) 761.0 462.1 866.4 209.1 t1/2 (h) 1.5 2.7 0.9 2.8 Fig. 3. PITB effect over the kinetic stability and aggregation of WT-, V30M- and V122I-TTR. (A) Unfolding time course at 6 M urea in the absence or presence of 2 molar equivalents of PITB and tolcapone, as monitored by Trp fluorescence. The values correspond to mean ± SEM (n = 3). (B) Percentage of Fig. 2. Plasma concentration-time profiles for M-23 and 3-O-methylM23 after TTR aggregation as a function of PITB and tolcapone concentration as IV (A) and PO (B) administration of M-23 in CD-1 mice. The right panels show measured by turbidity at 340 nm. The values were normalized to 100%, which the concentration of M-23 and 3-O-methylM23 in plasma up to 6 h after was assigned to the turbidity of samples aggregated in the absence of any administration of M-23. The y-axis was stretched to a logarithmic scale (without compound. Data represents mean ± SEM (n = 3). altering the values) to facilitate the comparison between the two compounds. Of note, 3-O-methylM23 was detected after 24 h, in both cases, contrarily to M- exerted by tolcapone for all analyzed TTR variants. 23. Data represents mean ± SEM (n = 3). 2.3. PITB protects TTR from aggregation in vitro Having established the ability of PITB to prevent tetramer dissocia­ tion, we proceeded to investigate whether this effect translated into the inhibition of TTR aggregation. For this purpose, TTR samples were mixed with increasing concentrations of PITB and aggregation was induced by acidification [31,32]. After an incubation of 72 h at 37 ◦ C, the degree of aggregation was quantified by recording turbidity at 340 nm and normalized to the maximum turbidity value, which was assigned to the TTR samples incubated in the absence of compound (Fig. 3B). The data demonstrate that PITB is highly effective at inhibiting the aggre­ gation of WT-TTR and, most importantly, of V30M- and V122I-TTR. In all cases, there was a decrease in TTR aggregation >60% at a 1:1 TTR/PITB ratio and ≥87.5% when PITB concentration is higher than or equal to the one of T4-binding sites. Notably, PITB outperformed tol­ capone, inhibiting up to 89.2% and 92% the aggregation of WT-TTR and Scheme 1. Synthesis of PITB. V30M-TTR, respectively. In the case of V122I-TTR, PITB equaled the potency of tolcapone, reaching up to 90% inhibition at 20 μM. urea to a final concentration of 6 M. Tryptophan (Trp) intrinsic fluo­ rescence was measured every 24 h and used to calculate the fraction of 2.4. PITB binds with high affinity to TTR unfolded protein (Fig. 3A). The effect of PITB was compared with the one of tolcapone. The high stabilization and anti-aggregation effect of PITB on WT-, In the presence of equimolar levels of PITB relative to T4-binding V30M- and V122I-TTR suggests that it binds with high affinity to these sites, 94.1 ± 0.5%, 79.2 ± 0.2% and 92.5 ± 1.6% of WT-, V30M- and proteins. To determine the binding parameters of PITB to TTR, we used V122I-TTR tetramers, were protected from urea-induced dissociation, isothermal titration calorimetry (ITC). Most ligands bind to TTR with respectively. Importantly, PITB stabilizing activity was on par with that negative cooperativity [18,33,34], which implies that occupancy of the 3 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 first binding site results in a reduced affinity for the second site. To structure of the kinetically stabilized TTR variant T119M [39] and could account for this possibility, ITC data was analyzed using two models: one further contribute to the high stabilizing effect of PITB. considering independent binding to both sites, and the other assuming cooperative binding [35,36]. 2.6. PITB selectively binds and stabilizes WT-TTR and V30M-TTR in Our data revealed that PITB binds with high affinity and no coop­ human plasma erativity to both WT and TTR variants V30M and V112I (Table 2 and Fig. 4). Moreover, PITB binds to WT- and V122I-TTR with an affinity ≥ To effectively stabilize the native tetrameric TTR and thus prevent its 3-fold higher than that of tolcapone, and to V30M-TTR with an affinity aggregation in patients with ATTR, PITB must selectively bind to TTR in >12-fold higher. Remarkably, PITB presents a significantly higher af­ plasma over all other plasmatic proteins. To test this, we examined the finity for V30M- and V122I-TTR than tafamidis [33,37], the only capacity of PITB to compete with T4 for TTR binding in human plasma approved kinetic stabilizer for FAP and FAC. Independently of the tested from control individuals (WT-TTR) and individuals carrying the V30M protein, the binding of PITB was entirely enthalpically driven (ΔH < 0; mutation. Tolcapone was included as a control. In this assay, plasma -TΔS > 0), suggesting the establishment of favorable enthalpic non­ samples were incubated with [125I]-T4, in the absence or presence of covalent interactions (e.g., hydrogen bonds or ionic interactions) at the compounds, and subjected to native gel electrophoresis [40]. T4-binding binding interface. proteins were detected by autoradiography, as shown in Fig. S3A. In the absence of any compound, three main plasma proteins bound to T4: the 2.5. PITB stabilizes the weaker TTR dimer-dimer interface main binding protein was T4-binding globulin (TBG), followed by al­ bumin (ALB) and TTR. The samples incubated with tolcapone and, To elucidate the underlaying interactions that drive the binding of especially, PITB, presented weaker T4-TTR bands, indicating that these PITB to WT-TTR and TTR variants V30M and V122I, we determined the molecules can compete the binding of T4 to TTR in human plasma. X-ray crystal structures of TTR:PITB complexes at 1.85 (WT-TTR:PITB), Densitometry analysis of the bands revealed that PITB is a more potent 1.20 (V30M-TTR:PITB) and 1.42 (V122I-TTR:PITB) Å resolution competitor than tolcapone, presenting a displacement of T4 binding to (Fig. 5). Additionally, we determined the cocrystal structure of tolca­ plasmatic TTR of 97.3 ± 1.2% and 94.5 ± 2.8% for normal plasma or pone with V30M-TTR, at a resolution of 1.57 Å. As expected, PITB binds plasma from TTR V30M carriers, respectively (Fig. 6A). to TTR T4-binding sites at the weaker AB/CD dimer-dimer interface. Once we confirmed that PITB selectively binds to TTR in human Because of the two-fold symmetry axis that runs through the binding plasma, we investigated its effect on the stability of TTR in the plasma of pockets, two symmetry-related binding conformations appear for PITB. normal individuals and TTR V30M carriers. The ability of PITB to pre­ In all structures, PITB was found in the forward binding mode, with the vent tetramer dissociation was monitored ex vivo by isoelectric focusing 3-methoxy-4-hydroxy-5-nitrophenyl pointing to the outer binding cav­ (IEF) under partially dissociating conditions (4 M urea) [41]. Tolcapone ity, where it makes hydrophobic interactions with the residues from was also used as a control. Briefly, plasma samples were incubated with halogen binding pockets (HBPs) 2/2′ and 1/1’ (Fig. 5). In this orienta­ or without compound overnight at 4 ◦ C and, after IEF, the bands were tion, the 4-hydroxy group of PITB forms hydrogen bonds with the K15/ compared. Under the conditions used, it is possible to distinguish two K15′ residues of TTR, which in turn stabilize their electrostatic in­ main sets of bands, one corresponding to monomers (normal and teractions with E54/E54’. These interactions by K15 are also observed in oxidized forms), and one including several bands of lower pI that TTR:tolcapone complexes and were proposed to close the binding cav­ represent different forms of tetramers (Fig. S3B). The tetramer/mo­ ity, protecting the protein-ligand complex from the solvent [23]. In the nomer ratios were used to calculate the degree of tetramer stabilization WT-TTR:PITB structure, the hydroxyl side chain of T119 is not at a in each sample. The results showed that PITB has a higher stabilizing hydrogen bonding distance from the central carbonyl group, contrarily activity than tolcapone, especially in plasma from TTR V30M carriers, to what is observed for tolcapone (Fig. 5A). This fact can be explained by with an increase in tetramer stability of 17.4 ± 5.1% (versus 4.9 ± 2.3% the different conformation adopted by the side chain of T119 in the first for tolcapone) (Fig. 6B). The increased binding selectivity and stabili­ structure (Fig. S1A). Interestingly, for the same reason, no hydrogen zation effect of PITB in human plasma, when compared to tolcapone, is bond is seen between the carbonyl group of both PITB and tolcapone, likely due to the higher number of interactions it establishes with the and the side chain of T119, when bound to V30M- and V122I-TTR protein, as observed in the crystal structure. Additionally, differences in (Fig. 5B,C and Fig. S1B). On the other hand, PITB establishes impor­ their respective binding to plasma proteins might also contribute to this tant interactions in the inner binding cavity, which are not observed in heightened efficacy. the TTR:tolcapone structures. In particular, the fluorine atom of the 3-fluoro-5-hydroxyphenyl ring interacts with A108, while the hydroxy 2.7. PITB displays no toxicity for human cells group forms hydrogen bonds with the S117 residues from adjacent subunits. These additional contacts made by PITB likely account for its The assessment of compound toxicity is a crucial aspect in the higher affinity towards WT-, V30M- and V122I-TTR in comparison to development of new drugs. Therefore, we conducted experiments to tolcapone. evaluate the potential cytotoxicity of PITB using human cervical carci­ Remarkably, the hydrogen bonds between PITB and S117 help to noma (HeLa) and human hepatoblastoma (HepG2) cells. These cell lines bridge the dimers at the weaker dimer-dimer interface, increasing the have been extensively studied and used in in vitro cytotoxicity studies kinetic barrier for tetramer dissociation [33,38]. Additionally, in one of [42–44]. The HepG2 cell line, in particular, is the most commonly used the S117 side-chain conformations, a short hydrogen bond can be for detecting drug-induced hepatotoxicity [45,46]. Liver injury is one of formed between the S117 residues from adjacent monomers in the same the primary causes of drug withdrawal [47] and many medications dimer (Fig. S2). This inter-subunit interaction was also observed in the require monitoring liver function during treatment, including tolcapone Table 2 Thermodynamic parameters for the interaction of PITB with WT-, V30M- and V122I-TTR determined by ITC. PITB Tolcapone Kd (nM) ΔG (kcal/mol) ΔH (kcal/mol) -TΔS (kcal/mol) Kd (nM) ΔG (kcal/mol) ΔH (kcal/mol) -TΔS (kcal/mol) WT-TTR 16 − 10.6 − 13.4 2.8 48 − 10.2 − 13.6 3.6 V30M-TTR 36 − 10.2 − 11.0 0.8 440 − 8.7 − 13.8 5.1 V122I-TTR 14 − 10.7 − 16.1 5.4 50 − 10.0 − 12.2 2.2 4 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 Fig. 4. Interaction of PITB with WT-, V30M- and V122I-TTR. The top panels correspond to the raw data after baseline correction, while the lower panels represent the integrated data as a function of the molar ratio between the concentration of PITB and protein. The solid lines in the bottom panels represent the best fit to the data to acquire the binding parameters. [48]. In both procedures, the concentration of PITB in plasma was still In this study, HeLa and HepG2 cells were incubated with increasing quantifiable 24 h post-dosing. The area under the plasma concentration- concentrations of PITB or tolcapone and cell viability was assessed after time curve (AUC) values were 3162.3 h⋅ng/mL and 26895.9 h•ng/mL 72 h using PrestoBlue®. No significant toxicity was observed in either for the IV (1 mg/kg) and PO (10 mg/kg) administration of PITB, cell line following exposure to PITB, even at the highest concentration of respectively. This indicates an excellent oral bioavailability (%F) of 100 μM (Fig. 7A,B). Importantly, PITB was substantially less toxic than 85.1%, which is significantly higher than the one reported for AG10 tolcapone at concentrations higher than 10 μM, suggesting that PITB (26.8%) in mice following PO administration at 30 mg/kg [33]. Notably, may be a safer alternative than tolcapone when administered at high AG10 is a TTR kinetic stabilizer currently undergoing clinical trials for doses. To ensure the generalizability of these findings beyond tumor ATTR-related cardiomyopathy. cells, we assessed the cytotoxicity of the two compounds on normal For comparative purposes, the PK properties of tolcapone after PO human fibroblast MRC-5 cells (Fig. 7C). The results echoed previous administration (10 mg/kg) in CD-1 mice were also determined (Fig. 8B findings, as there was no reduction in the viability of non-tumor cells and Table 3). Following administration of equimolar doses of PITB and following PITB administration, in stark contrast to the toxic effect of tolcapone, the last was more readily absorbed, achieving a Cmax of tolcapone. 7616.7 ng/mL in less than 15 min (Tmax). In contrast, the half-life of PITB was significantly higher than the one of tolcapone (t1/2 for PITB = 2.8. PK studies in mice confirm the bioavailability and stability of PITB 10.1 h; t1/2 for tolcapone = 1.4 h), which is due to its higher volume of distribution (Vd) and decreased clearance (Cl). Accordingly, no tolca­ Altogether, the previous results support the potential of PITB to pone was detected in plasma after 24 h. As a result, the exposure to PITB become a drug candidate for the treatment of both hereditary and non- was substantially greater than the one to tolcapone (AUC0-24 for PITB = hereditary forms of ATTR. To further explore this option, the PK pa­ 26895.9 h ng/mL; AUC0-24 for tolcapone = 11927.5 h ng/mL). rameters of PITB were determined from studies conducted in CD-1 male Overall, these findings indicate that PITB exhibits more favorable PK mice. The PK profiles for PITB following intravenous (IV) (1 mg/kg) or properties than tolcapone, which may potentially translate into oral (PO) (10 mg/kg) administration of the compound are shown in improved pharmacological activity in vivo. Fig. 8A, and the calculated parameters are summarized in Table 3. Following the IV injection of PITB, a Cmax value of 4043.3 ng/mL and a 3. Conclusion t1/2 of 3.5 h were determined. Additionally, our results demonstrated that PITB is rapidly absorbed after PO administration, reaching a Cmax of In the present study we have developed PITB, a M-23 derivative with 4176.7 ng/mL, at 0.5 h (Tmax). While the concentration achieved by the optimal PK properties. PITB was designed based on the known existence oral route was similar to that observed for the IV infusion, the elimi­ of 3-O-methyl tolcapone, a minor metabolite of tolcapone with an nation of PITB was considerably slower in this case, with a t1/2 of 10.1 h. increased stability in plasma, and that can act as a TTR stabilizer [28, 5 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 Fig. 5. Crystal structures of WT-TTR (A), V30M-TTR (B) and V122I-TTR (C) in complex with PITB. On the left, the global view of TTR bound to PITB (cartoon representation). The dashed line corresponds to the twofold symmetry axis of the dimer-dimer interface. The insets show the detailed view of one of the TTR binding sites for PITB and for tolcapone. PITB, tolcapone and some of the TTR interacting residues are shown as sticks. The dashed blue lines show key interactions between each ligand and TTR. The structural data for Tolcapone:WT and Tolcapone:V122I was obtained from PDB files 4D7B and 5A6I, respectively. 29]. Since methylation is a common pathway in the metabolism of catechol-containing compounds [49], we hypothesized and confirmed that M-23 has a similar fate. Here, we show that PITB binds strongly to the T4-binding sites of WT-TTR and the two most clinically relevant TTR variants V30M and V122I. As a result, PITB greatly increases the sta­ bility of TTR tetramers, inhibiting their aggregation. Remarkably, compared with tolcapone, PITB presents a higher binding selectivity and stabilization potency in plasma. The high-resolution crystal structures of TTR:PITB complexes confirmed that, as intended, PITB keeps the crit­ ical interactions established by M-23, contributing to its improved effi­ cacy in plasma relative to tolcapone. Additionally, our cytotoxicity Fig. 6. PITB binding and stabilizing activity in human plasma from control assays suggest a low risk associated with PITB administration. Most individuals (WT-TTR) or TTR V30M carriers. (A) Fraction of [125I]-T4 bound to importantly, the PK evaluation of PITB in mice revealed a favorable PK TTR determined by densitometry analysis of the native gel electrophoresis, as profile, with a half-life of 10.1 h and an oral bioavailability of 85.1%. explained in the experimental section. The values were normalized to the negative control and correspond to mean ± SEM (n = 3). (B) Percentage of TTR Remarkably, PITB demonstrates superior PK parameters when tetramer stabilization calculated from the densitometry analysis of the IEF gels, compared to tolcapone, an FDA-approved drug, encouraging future in as detailed in the experimental section. Error bars represent SEM of mean vivo studies, including sex-specific toxicity and efficacy evaluations. values (n = 2 for WT plasma; n = 3 for V30M plasma). Interestingly, PITB is prone to undergo 3-O-demethylation in vivo [50], originating M-23, which would further sustain its kinetic stabilizing activity. All in all, the findings presented here underscore the strong potential of PITB as a therapeutic agent for ATTR, particularly in cases of FAP 6 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 caused by V30M-TTR - the most prevalent mutation in hereditary ATTR cases. Considering that tolcapone has already demonstrated efficacy in a phase IIa clinical trial in this genetic background and given that PITB outperforms this drug in nearly every analyzed aspect, our results pro­ vide compelling support for further preclinical and clinical development of PITB. 4. Experimental section 4.1. General methods Commercially available reagents were used as received. Solvents were dried by distillation over the appropriate drying agents. All re­ actions were monitored by analytical thin-layer chromatography (TLC) using silica gel 60 precoated aluminum plates (0.20 mm thickness). Flash column chromatography was performed using silica gel Geduran® SI 60 (40–63 μm). 1H NMR, 19F NMR and 13C NMR spectra were recorded at 250, 400 MHz and 90, 100 MHz, respectively. 1H NMR spectra were referenced to the residual proton signals of CDCl3, 7.26 ppm, and acetone-d6 2.05 ppm. 13C NMR spectra were referenced to the residual 13C signal of CDCl3, 77.16 ppm, and acetone-d6, 29.84 ppm. The purity of the PITB was determined to be over 95% by HPLC analysis (see the Supplementary Data). Melting points were determined on hot stage and are uncorrected. HRMS were recorded using electrospray ionization (ESI). 4.2. Synthesis procedure Tolcapone was purchased from Fisher. PITB was prepared as described below. 4.2.1. (3,4-Dimethoxy-5-nitrophenyl) (3-fluoro-5 methoxyphenyl) Fig. 7. Toxicity of PITB to different cell lines. Cell viability of HeLa (A), HepG2 methanone (3) (B) and MRC-5 (C) cells after 72 h exposure to PITB or tolcapone as assessed by To a solution of 1 (2.50 g, 9.93 mmol) in dry THF (25 mL) at − 40 ◦ C, the PrestoBlue assay. Data represents mean ± SEM (n = 3). iPrMgBr (1 M in THF, 15.9 mL, 11.92 mmol) was added dropwise under an Ar atmosphere and the reaction mixture was stirred for 1 h. Then, a solution of 3,4-dimethoxy-5-nitrobenzaldehyde, 2, (2.53 g, 12.00 mmol) in dry THF (20 mL) was added and the mixture was warmed to rt and stirred overnight. The reaction was quenched by the slow addition of brine (20 mL) and the aqueous layer was extracted with EtOAc (3 × 35 mL). The resulting organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to furnish the corresponding alcohol as a yellow oil (1.98 g) which was used in the next step without further purification. To a solution of the alcohol (1.98 g, 5.87 mmol) in dry CH2Cl2 (90 mL) under an Ar atmosphere, a solution of DMPI (5.08 g, 12.03 mmol) in dry CH2Cl2 (30 mL) was added dropwise and the mixture was stirred overnight at rt. The reaction was quenched with the addition of 12 mL of a prepared solution of Na2S2O3 (17.0 g) in a Fig. 8. Plasma concentration-time profiles for PITB and tolcapone after administration in CD-1 mice. (A) PK profiles for PITB following IV (1 mg/kg) saturated aqueous solution of NaHCO3 (80 mL) and the mixture was and PO (10 mg/kg) administration. Data represents mean ± SEM (n = 3). (B) stirred for 15 min. The aqueous phase was extracted with CH2Cl2 (3 × Mean plasmatic levels of tolcapone (±SEM) over 24 h after PO administration 50 mL) and the combined organic extracts were dried over anhydrous (10 mg/kg) (n = 3). Noteworthy, no tolcapone was detected after 24 h. Na2SO4 and concentrated under vacuum. Purification by flash column chromatography (hexanes/EtOAc, 5:2) of the resulting residue provided the ketone 3 as a white solid (1.92 g, 5.72 mmol, 58% yield for the two- Table 3 steps). 3: Mp 96–94 ᵒC (from CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.67 PK parameters of PITB and tolcapone following administration in CD-1 mice. (d, J = 2.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.06 (m, 1H), 7.02 (ddd, J PITB Tolcapone = 8.4 Hz, J = 2.3 Hz, J = 1.4 Hz, 1H), 6.86 (dt, J = 10.2 Hz, J = 2.3 Hz, IV (1 mg/kg) PO (10 mg/kg) PO (10 mg/kg) 1H), 4.06 (s, 3H), 3.97 (s, 3H), 3.84 (s, 3H); 19F NMR (250 MHz, CDCl3) δ − 110.1 (s); 13C NMR (90.5 MHz, CDCl3) δ 192.4 (d, J = 2.8 Hz), 163.3 Tmax (h) – 0.5 < 0.25 Cmax (ng/mL) 4043.3 4176.7 7616.7 (d, J = 248.3 Hz), 161.1 (d, J = 10.8 Hz), 154.4, 146.7, 144.2, 139.0 (d, AUC0–24h (h⋅ng/mL) 3162.3 26895.9 11927.5 J = 8.4 Hz), 132.0, 118.7, 116.3, 111.2 (d, J = 2.8 Hz), 109.1 (d, J = t1/2 (h) 3.5 10.1 1.4 23.1 Hz), 106.4 (d, = 25.0 Hz), 62.4, 56.8, 56.1. HRMS (ESI+) calcd. for ke (h− 1) 0.201 0.069 0.500 [C16H14FNO6+Na]+ 358.0703; found [M+Na]+ 358.0705. Vd (L/kg) 1.572 4.482 1.600 Cl (L/h⋅kg) 0.315 0.308 0.800 F (%) – 85.1 – 7 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 4.2.2. (3-fluoro-5-hydroxyphenyl) (4-hydroxy-3-methoxy-5-nitrophenyl) of the test sample by that of a sample aggregated in the absence of methanone, PITB compound, and multiply by 100. BBr3 (1 M in CH2Cl2, 9.50 mL, 9.50 mmol) was added dropwise to a solution of 3 (400 mg, 1.19 mmol) in CH2Cl2 (6.0 mL) at − 10 ◦ C. The 4.6. Isothermal titration calorimetry reaction was allowed to proceed at rt for 1.5 h (TLC, CH2Cl2/MeOH, 10:1). The reaction was quenched with water (8 mL) and the resulting All ITC measurements were carried out with protein and compounds aqueous layer was extracted with EtOAc (2 × 20 mL). The organic ex­ dissolved in the same buffer (in a PBS buffer pH 7.0 containing 100 mM tracts were dried with anhydrous Na2SO4 and the solvent removed KCl, 1 mM EDTA and 2.5% DMSO), and degassed. The ITC measure­ under vacuum to give the crude product which was purified by flash ments were performed at 25 ◦ C using a Nano ITC calorimeter (TA In­ column chromatography (CH2Cl2/MeOH, 20:1) to afford PITB as a struments). Titrations were performed as a set of 20 injections of 100 μM yellow solid (311 mg, 1.01 mmol, 85% yield). Mp 157 - 155 ◦ C (from compound into protein (5 μM), programmed as a 2.02 μl injection with CHCl3). 1H NMR (300 MHz, acetone-d6) δ 8.06 (br s, 1H), 7.61 (br s, 150 s of equilibration period. The NanoAnalyze software (TA In­ 1H), 7.07 (br s, 1H), 6.97 (br d, J = 9.0 Hz, 1H), 6.87 (dt, J = 10.4 Hz, struments) was used to integrate the peaks and evaluate heat effects per 1H), 4.03 (s, 3H); 19F NMR (250 MHz, CDCl3) δ − 110.01;13C NMR (90.5 injection. The fittings were performed with a general model for a protein MHz, acetone-d6) δ 192.4, 164.0 (d, J = 244.3 Hz), 159.7 (d, J = 11.6 with two-ligand binding sites implemented in Origin 7.0 (OriginLab), Hz), 155.1, 142.1 (d, J = 9.2 Hz), 135.1, 125.5, 120.3, 113.1, 112.3, which allows to establish if the binding is cooperative or non- 107.6 (d, J = 23.2 Hz), 106.4 (d, J = 24.2 Hz), 57.2. HRMS (ESI-) calcd. cooperative [35,36]. for [C14H10FNO6-H]- 306.0414; found [M − H]- 306.0424. 4.7. Crystallization and structure determination 4.3. Recombinant TTR expression and purification Cocrystals of WT-TTR/PITB, V30M-TTR/PITB, V122I-TTR/PITB The cDNA encoding for WT-TTR was cloned into a pET28a vector and V30M-TTR/tolcapone were obtained following previously (Novagen). The vectors encoding for V30M- and V122I-TTR were pre­ described methods [26]. In brief, purified proteins (140 μM) were mixed pared by classical site-directed mutagenesis protocols using the WT-TTR with 1.4 mM of PITB/tolcapone and co-crystallized at 18 ◦ C using the vector as a template. WT-, V30M- and V122I-TTR were expressed in hanging-drop vapor diffusion technique. The reservoir solution con­ Escherichia coli BL21 (DE3) and purified as previously described [26]. sisted of 20–30% PEG 400, 200 mM CaCl2, and 100 mM HEPES, with a The purest fractions eluting from the gel filtration chromatography were pH range of 7.0–8.0. The crystals were rapidly frozen in liquid nitrogen combined and kept at − 20 ◦ C until use. The concentration of protein was (100 K), and diffraction data were collected at the BL13-XALOC beam­ determined spectrophotometrically at 280 nm, using a molar extinction line from the ALBA Synchrotron in Barcelona [52]. Data integration and coefficient of 77 600 M− 1 cm− 1. merging were performed using XDS [53], and further analysis was conducted with CCP4 [54], including scaling and reduction of the data. 4.4. Urea-induced TTR tetramer dissociation measured by Trp The structures of the TTR/PITB and V30M-TTR/tolcapone complexes fluorescence were determined from the X-ray data through molecular replacement with Phenix (version 1.19.2–4158) [55], utilizing a previously solved Samples containing TTR (1.8 μM in PBS) were incubated with 3.6 μM TTR structure (PDB 1F41) as a reference model. Model refinement and PITB or tolcapone for 30 min at RT, and 6 M urea was added to trigger building were carried out using Phenix [55] and Coot [56], respectively. denaturation. Since the stock solutions of both compounds were pre­ Refinement and data statistics can be found in Table S1. The atomic pared in DMSO, a control sample containing the same percentage of coordinates have been deposited in the PDB [PDB accession codes 8PM9 DMSO instead of compound was prepared. Trp fluorescence spectra (PITB:WT), 8PMA (PITB:V30M), 8PMO (PITB:V122I), and 8PM8 (Tol­ were recorded over time (λex = 295 nm; λem = 310–410 nm) using an FP- capone:V30M)]. The structural representations were generated using 8200 Spectrofluorometer (Jasco). Upon denaturation, the Trp residues Pymol (The PyMOL Molecular Graphics System, Version 2.0, become more exposed to the polar solvent, which is accompanied by a Schrödinger, LLC). change in the emission maximum from ~335 to ~355 nm. The fluo­ rescence intensity ratio 355/335 nm was calculated for each time point 4.8. T4 binding competition assays and the values normalized from minimum (folded state) to maximum (unfolded state), with the maximum being the one of the control sample The ability of PITB and tolcapone to displace T4 from plasmatic TTR after 96 h incubation. was evaluated by incubation of whole plasma (5 μl) with 1 μl of [125I]-T4 (specific radioactivity 1250 μCi/μg; concentration 320 μCi/mL; Perki­ 4.5. TTR in vitro aggregation inhibition nElmer) in the presence of the test compounds (5 molar excess relative to TTR tetramer). A negative control containing the same percentage of To assess the effect of PITB and tolcapone in the aggregation of WT-, DMSO than the samples was also prepared. Following 1 h incubation at V30M- and V122I-TTR, a well-established TTR aggregation assay was room temperature, plasma proteins were separated by native PAGE employed [31,51]. Briefly, TTR solutions (7.2 μM in 10 mM sodium [40], the gels were dried, and subjected to phosphor imaging using a phosphate, 100 mM KCl, 1 mM EDTA, 1 mM DTT, pH 7.0) were incu­ Typhoon 8600 variable mode imager (Molecular Diagnostics, Amer­ bated with varying concentrations of test compound for 30 min at 37 ◦ C. sham Biosciences). Then, the autoradiography films were scanned, and The percentage of DMSO was the same in all samples (5%). Following the bands quantified using Image Lab software version 5.2.1 (Bio-Rad). incubation, the pH of the solutions was dropped to 4.2 via addition of an The amount of T4 bound to TTR in comparison to total T4 (T4-TBG + equal volume of acidification buffer (100 mM sodium acetate, 100 mM T4-ALB + T4-TTR) was determined for each sample and normalized to KCl, 1 mM EDTA, 1 mM DTT, pH 4.2) and the samples were kept at 37 ◦ C the maximum value, which corresponds to the negative control. Plasma for more 72 h. The aggregation was assessed by measuring turbidity at from three control individuals (WT-TTR) and three TTR V30M carriers 340 nm using a Varian Cary Eclipse Fluorescence Spectrophotometer was analyzed. (Agilent Technologies). Since PITB and tolcapone display dose-dependent absorbance at 340 nm, the turbidity of a sample con­ 4.9. TTR stabilization in human plasma assessed by isoelectric focusing taining the same concentration of compound, but lacking TTR, was subtracted to every measurement. The percentage of TTR aggregation To assess the effect of PITB and tolcapone on the stability of TTR in for each inhibitor concentration was calculated by dividing the turbidity plasma, isoelectric focusing (IEF) under semi-denaturing conditions was 8 F. Pinheiro et al. European Journal of Medicinal Chemistry 261 (2023) 115837 performed [41]. To carry out this assay, 30 μl of human plasma from administered at 10 mg/kg. The administered volume was 10 mL/kg. control individuals (n = 2) or TTR V30M carriers (n = 3) were incubated Plasma samples were obtained as described above at 0.25, 0.5, 1, 2, 4, 6 overnight at 4 ◦ C in the presence of 19.1 μM compounds. Additionally, and 24 h post-dosing (three animals for time point). Three animals were control samples containing the same amount of DMSO rather than used as blank with no administration (predose). All plasma samples compound were prepared. After incubation, the samples were subjected were analyzed using the API 3200 LC-MS/MS system (Sciex) coupled to to native PAGE, and the TTR gel band was excised and applied to an IEF an UPLC-Acquity (Waters). A calibration curve was done for each gel. IEF was performed in the presence of 4 M urea (semi-denaturing compound in plasma. The lower limit of quantification (LLOQ) for M-23, conditions) and 5% (v/v) ampholytes (pH 4–6.5; Sigma-Merck), at 1200 PITB and tolcapone were 2.93 ng/mL, 7.68 ng/mL and 2.73 ng/mL, V for 5 h. Proteins were then fixed with 20% trichloroacetic acid and respectively. For PITB quantification in plasma samples from the PK stained with Coomassie blue. The gels were scanned using a GS-900 studies of M-23 (IV and PO), the same UPLC-MS/MS detection system calibrated densitometer (Bio-Rad) and the bands analyzed by densi­ was used. The calibration curve done for PITB PK studies was applied in tometry with the Image Lab software version 5.2.1 (Bio-Rad). The ratio this case. PK parameters were determined with a non-compartmental of the TTR tetramer over total TTR (TTR tetramer + monomer) was model using Phoenix 64 8.3 (WinNolin) software. determined for each sample, and the percentage of tetramer stabiliza­ tion was calculated as ((ratio treated sample − ratio control sam­ Declaration of competing interest ple)/ratio control sample) × 100. Authors have submitted a patent application on the basis of the here 4.10. Cytotoxicity studies presented research. It is protected the use of PITB as a therapy for TTR amyloidosis. Inventors: S.V., I.P, F.P., D.R., N.V., R.A., F.B., A.S Title: The potential chemical toxicity of PITB was evaluated in vitro in Compound for the treatment of transthyretin amyloidosis. Property: HeLa, HepG2 and MRC-5 human cell lines. Tolcapone was tested in Universitat Autònoma de Barcelona. Request number: EP2023/025333. parallel for comparison. HeLa, HepG2 and MRC-5 cells were cultured in All other authors declare no competing interests. MEM ALPHA medium (Gibco) supplemented with 10% fetal bovine serum at 37 ◦ C in a 5% CO2 humidified atmosphere. Cells were seeded in Data availability 96-well plates at 3500 cells/well (HeLa), 4500 cells/well (HepG2) or 1500 cells/well (MRC-5) and incubated with increasing concentrations Data will be made available on request. of compound (2–100 μM) for 72 h at 37 ◦ C. Controls were prepared with the equivalent amount of DMSO relative to each concentration of Acknowledgements compound. Then, 10 μl of PrestoBlue® reagent (Thermo Fisher Scien­ tific) were added to each well, and after incubating for 15 min at 37 ◦ C, This work was funded by the Spanish Ministry of Science and Inno­ the fluorescence emission was recorded using either a 590/20 filter with vation (PDC2021-120914-I00 to S.V. and PID2021-124602OB-I00 to D. an excitation wavelength of 535 nm in a Victor3 Multilabel Reader R), and by the Universitat Autònoma de Barcelona (PROOF OF (PerkinElmer) or a monochromator (λex = 560 nm; λem = 590 nm) in a CONCEPT 2020) and ICREA, ICREA-Academia 2015 and 2020 to S.V. R. Spark® multimode microplate reader (Tecan). The assays were per­ A. acknowledges the financial support of the Spanish Ministry of Econ­ formed in triplicate and the percentage of cell viability for each well was omy and Competitiveness (PID2019-106403RB-I00). A.V.C acknowl­ calculated as (intensity sample − mean intensity blank)/(mean intensity edges the Spanish Ministry of Science and Innovation MCIN/AEI/ control – mean intensity blank) × 100, where ″mean intensity blank″ 10.13039/501100011033/and “ERDF A way of Making Europe” corresponds to the mean intensity of wells with PrestoBlue® alone and (PID2021-127296OB-I00). F.P. and A.S. acknowledge the Universitat ″mean intensity control″ is the mean intensity of wells that contain the Autònoma de Barcelona for their doctoral grant. This work was funded corresponding percentage of DMSO. by FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020 to M.R.A., and through the support of F.B. with a 4.11. In vivo pharmacokinetic studies in mice PhD fellowship (SFRH/BD/123674/2016). The PK studies of M-23 (IV and PO), PITB (IV and PO) and tolcapone Appendix A. Supplementary data (PO) in CD-1 mice were conducted by Draconis Pharma S.L. 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