Novel transthyretin amyloid fibril formation inhibitors: synthesis, biological evaluation, and X-ray structural analysis.

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of beta-aminoxypropionic acids (compounds 5-21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR:inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp(2)-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22-32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding.

Structural insight into pH-induced conformational changes within the native human transthyretin tetramer.

Acidification of the transthyretin (TTR) tetramer facilitates dissociation and conformational changes in the protein, allowing alternatively folded monomers to self-assemble into insoluble amyloid fibers by a downhill polymerization mechanism in vitro. To investigate the influence of acidification on the quaternary and tertiary structures of TTR, crystal structures of wild-type human TTR at pH 4.0 and pH 3.5 have been determined to 1.7 A resolution. The acidic pH crystals are isomorphous to most of the previously reported TTR structures, containing two subunits in the asymmetric unit (the so-called A and B subunits) but forming a tetramer through crystallographic symmetry. The pH 4.0 crystal structure reveals that the native fold of the tetramer remains mostly undisturbed. In particular, subunit A of the TTR pH 4.0 structure is very similar to the wild-type TTR pH 7.4 structure with an r.m.s.d. of 0.38 A. In contrast, subunit B of the TTR pH 4.0 structure exhibits several significant changes. The EF-helix (residues 75-81) and the adjacent EF-loop (residues 82-90) show an r.m.s.d. greater than 2.0 A. The acidic residues within this region (Glu72, Asp74, Glu89, and Glu92) undergo significant conformational changes that instigate movement of the EF helix-loop region and make residues Lys70, Lys76, His88, and His90 orient their side chains toward these acidic residues. In particular, Glu89 undergoes a maximum deviation of 5.6 A, occupying Phe87's initial position in the wild-type TTR pH 7.4 structure, and points its side chain into a hydrophobic pocket of the neighboring subunit. In the pH 3.5 structure, the EF helix-loop region is completely disordered. These results demonstrate that acidic conditions increase the susceptibility of the EF helix-loop region of the TTR B subunit to undergo conformational changes and unfold, likely destabilizing the tetramer and identifying at least the initial conformational changes likely occurring within the tetramer that leads to the amyloidogenic monomer.

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis.

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.

Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation.

Amyloid fibril formation by the plasma protein transthyretin (TTR), requiring rate-limiting tetramer dissociation and monomer misfolding, is implicated in several human diseases. Amyloidogenesis can be inhibited through native state stabilization, mediated by small molecule binding to TTR's primarily unoccupied thyroid hormone binding sites. New native state stabilizers have been discovered herein by the facile condensation of arylaldehydes with aryloxyamines affording a bisarylaldoxime ether library. Of the library's 95 compounds, 31 were active inhibitors of TTR amyloid formation in vitro. The bisaryloxime ethers selectively stabilize the native tetrameric state of TTR over the dissociative transition state under amyloidogenic conditions, leading to an increase in the dissociation activation barrier. Several bisaryloxime ethers bind selectively to TTR in human blood plasma over the plethora of other plasma proteins, a necessary attribute for efficacy in vivo. While bisarylaldoxime ethers are susceptible to degradation by N-O bond cleavage, this process is slowed by their binding to TTR. Furthermore, the degradation rate of many of the bisarylaldoxime ethers is slow relative to the half-life of plasma TTR. The bisaryloxime ether library provides valuable structure-activity relationship insight for the development of structurally analogous inhibitors with superior stability profiles, should that prove necessary.