International Journal of Molecular Sciences Article Diphenyl-Methane Based Thyromimetic Inhibitors for Transthyretin Amyloidosis Bokyung Kim 1 , Young Ho Ko 2 , Massimiliano Runfola 3 , Simona Rapposelli 3 , Gabriella Ortore 3, *, Grazia Chiellini 4, * and Jin Hae Kim 1, *   Citation: Kim, B.; Ko, Y.H.; Runfola, M.; Rapposelli, S.; Ortore, G.; Chiellini, G.; Kim, J.H. Diphenyl-Methane Based Thyromimetic Inhibitors for Transthyretin Amyloidosis. Int. J. Mol. Sci. 2021, 22, 3488. https:// doi.org/10.3390/ijms22073488 Academic Editor: Holger Wille Received: 4 March 2021 Accepted: 25 March 2021 Published: 28 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of New Biology, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, Korea; bkkim@dgist.ac.kr 2 Center for Self-Assembly and Complexity, Institute for Basic Science, Pohang 37673, Korea; yhko@ibs.re.kr 3 Department of Pharmacy, University of Pisa, 56100 Pisa, Italy; massimiliano.runfola@farm.unipi.it (M.R.); simona.rapposelli@unipi.it (S.R.) 4 Department of Pathology, University of Pisa, 56100 Pisa, Italy * Correspondence: gabriella.ortore@farm.unipi.it (G.O.); grazia.chiellini@unipi.it (G.C.); jinhaekim@dgist.ac.kr (J.H.K.) Abstract: Thyromimetics, whose physicochemical characteristics are analog to thyroid hormones (THs) and their derivatives, are promising candidates as novel therapeutics for neurodegenerative and metabolic pathologies. In particular, sobetirome (GC-1), one of the initial halogen-free thy- romimetics, and newly synthesized IS25 and TG68, with optimized ADME-Tox profile, have recently attracted attention owing to their superior therapeutic benefits, selectivity, and enhanced permeability. Here, we further explored the functional capabilities of these thyromimetics to inhibit transthyretin (TTR) amyloidosis. TTR is a homotetrameric transporter protein for THs, yet it is also responsible for severe amyloid fibril formation, which is facilitated by tetramer dissociation into non-native monomers. By combining nuclear magnetic resonance (NMR) spectroscopy, computational simula- tion, and biochemical assays, we found that GC-1 and newly designed diphenyl-methane-based thy- romimetics, namely IS25 and TG68, are TTR stabilizers and efficient suppressors of TTR aggregation. Based on these observations, we propose the novel potential of thyromimetics as a multi-functional therapeutic molecule for TTR-related pathologies, including neurodegenerative diseases. Keywords: transthyretin; thyromimetics; sobetirome; TTR amyloidosis; TTR stabilizers; protein ag- gregation 1. Introduction Transthyretin (TTR) is an essential transporter of the thyroid hormone (TH) and a holo-retinol-binding protein. Under physiological conditions, TTR forms a ~55 kDa homote- trameric complex, on which two hydrophobic pockets are constructed as binding sites for thyroxine (T 4 )[1,2]. Notably, the quaternary stability of this protein is important not only for its physiological function but also to suppress its amyloidogenic propensity [3]. In its native tetrameric state, TTR is a non-amyloidogenic protein, whereas its dissociation into non-native monomers facilitates aggregation and formation of amyloid fibrils [4]. Due to the relative predominance of TTR in human serum and cerebrospinal fluid, TTR amyloido- sis results in detrimental amyloidogenic diseases. For example, senile systemic amyloidosis is caused by the spontaneous aggregation of wild-type (WT) TTR, while familial amyloid polyneuropathy or familial amyloid cardiomyopathy is attributed to genetic modifications of TTR [5]. Indeed, more than 100 mutations have been reported to date, and it was shown that most pathogenic mutations facilitate fibril formation by reducing the stability of the TTR tetramer [6]. Recent investigations in the USA estimated that over three percent of the African American population had a specific TTR mutation that significantly increases the risk of TTR amyloidosis [7], while approximately eight percent of patients with suspected cardiac amyloidosis having pathogenic TTR mutations [8]. Int. J. Mol. Sci. 2021, 22, 3488. https://doi.org/10.3390/ijms22073488 https://www.mdpi.com/journal/ijms