Structure-based design of kinetic stabilizers that ameliorate the transthyretin amyloidoses

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Small molecules that bind to normally unoccupied thyroxine (T4) binding sites within transthyretin (TTR) in the blood stabilize the tetrameric ground state of TTR relative to the dissociative transition state and dramatically slow tetramer dissociation, the rate-limiting step for the process of amyloid fibril formation linked to neurodegeneration and cell death. These so-called TTR kinetic stabilizers have been designed using structure-based principles and one of these has recently been shown to halt the progression of a human TTR amyloid disease in a clinical trial, providing the first pharmacologic evidence that the process of amyloid fibril formation is causative. Structure-based design has now progressed to the point where highly selective, high affinity TTR kinetic stabilizers that lack undesirable off-target activities can be produced with high frequency.

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Transthyretin aggregation appears to cause the transthyretin amyloidoses

The TTR amyloid diseases (amyloidoses) appear to be caused by extracellular TTR tetramer dissociation, monomer misfolding and misassembly into a variety of aggregate morphologies [1, 2, 3•] (Figure 1). The field has hypothesized that oligomers formed during the process of amyloidogenesis lead to cellular toxicity [4, 5]. The displacement of tissue by extracellular amyloid is also thought to exacerbate pathology [6]. The TTR amyloidoses include senile systemic amyloidosis (SSA), familial amyloid

Transthyretin's thyroxine binding sites

Each T4 binding site is characterized by a series of subsites [15] (Figure 3D), an outer binding subsite, an inner binding subsite, and an intervening interface that are all composed of pairs of symmetric hydrophobic depressions referred to as halogen binding pockets (HBPs), wherein the iodine atoms of T4 reside (Figure 3B) [16]. HBPs 1 and 1′ in the outer binding subsite comprise the side chains of Ala108/108′, Thr106/106′, Met13/13′ and Lys15/15′ with the pocket lined by methyl and methylene

Small molecule TTR ligands and the basis for kinetic stabilization

Over one thousand aromatic small molecules exhibiting structural complementarity to the T4 binding sites within TTR have been synthesized [18, 19, 20, 21, 22, 23, 24, 25, 26••, 27••, 28••, 29, 30] and are typically composed of two aromatic rings occupying the inner and outer T4 binding subsites (Figure 3D). The aromatic rings composing TTR kinetic stabilizers can either be linked directly (e.g. biphenyls) [19, 20] or tethered through short hydrophobic linkers [26••, 27••] (Figure 3D). Most of

Structure-based design of TTR kinetic stabilizers

Kinetic stabilizers for the treatment of the TTR amyloidoses must be both highly potent and selective. The ligands must not interact with the thyroid hormone receptor (THR), a major concern given the similarity of some kinetic stabilizers with triiodothyronine (T3, the primary thyroid hormone) and T4 (the prohormone). Nonsteroidal anti-inflammatory drug (NSAID) activity is also contraindicated for treating TTR amyloidosis patients; thus, TTR kinetic stabilizers should exhibit minimal NSAID

Optimal aryl-X ring substructures

To optimize the aryl-X ring, a library of 2-arylbenzoxazoles was synthesized in which the benzoxazole ring was purposefully unmodified and the 2-aryl ring was systematically substituted (Figure 4A). The library members were evaluated for TTR amyloid inhibition potency, plasma TTR binding selectivity, as well as undesired COX-1 and THR binding activity (Appendix A, Table S4). Substituents at the 3 and 4 positions and, especially, the 3, 4 and 5 positions afforded the most potent and selective

Optimal linker substructures

We next evaluated possible substructures to link the aryls occupying the inner and outer TTR T4 binding subsites, including CHdouble bondCH, –CH2CH2–, polar 1–3 atom linkers and fused five-membered ring heteroaromatic linkers. One aromatic ring is always unsubstituted in analogues made for this purpose, whilst the other ring comprises a 3,5-X2 or a 3,5-X2-4-hydroxyphenyl substructure, where X is Br or Me (Figure 4B and Appendix A, Table S5). Analysis of the numerous TTR•(ligand)2 structures in the Protein

Optimal aryl-Z ring substructures

Evaluation of aryl-Z ring candidates was carried out using a library composed of a fixed aryl-X substructure (N-(3,5-dibromo-4-hydroxyphenyl) as well as a fixed and suboptimal amide linker (Figure 4C) [28••]. Regardless of the diverse range of aryl-Z substituents evaluated, most were capable of increasing inhibitor potency and plasma TTR binding selectivity (Appendix A, Table S7). Independent of the substituent, 2,6-substituents are generally more potent and selective than 2,5-substituents that

Substructure combination strategy is effective

Using SAR data from the aryl-X, aryl-Z and linker-Y substructure optimization studies [26••, 27••, 28••], we envisioned that a library rich in highly potent and selective TTR kinetic stabilizers could be generated by simply combining the most highly ranked substructures. As a demonstration, we utilized the highly ranked 3,5-dibromo-4-hydroxyphenyl aryl-X ring (and isosteric variants replacing the OH functional group with a NH2), a variety of highly ranked aryl-Z rings and two hydrophobic

Conclusions

The systematic optimization of the three substructures comprising a typical TTR kinetic stabilizer has enabled rank ordering of these substructural components and these data now enable us to confidently predict the structures of diverse, potent and highly selective TTR kinetic stabilizers that inhibit TTR amyloidogenesis in vivo. From the high-resolution X-ray crystallographic data, we now know why some aryls prefer the inner binding subsite and others prefer the outer binding subsite. We also

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We are grateful to the NIH (DK46335 to JWK and AI42266 to IAW), as well as the Skaggs Institute for Chemical Biology and the Lita Annenberg Hazen Foundation for long standing financial support. The progress outlined within would not have been possible without the hard work, determination, and creativity of numerous co-workers cited within, including those who co-authored this review.

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