Inhibition strategies

130

by the higher propensity S52P-TTR mutant to misfold before dissociation, which exposes Lys48 in a less bulky area ready to be caught up by proteases^42,43.

Because of these diseases, amyloid aggregates were found in brain (FAP), heart (FAC), eye and kidney, which generally leads to several symptoms such as neuropathy, cardiac and gastrointestinal problems and muscles hypotonicity, which over times can lead to death^44–47. Considering the dramatic impact of this life-threatening disease on human’s life, a lot of research on this topic has been carried out. The first-ever gene therapy done in this context was liver transplantation^48,49, which represents nowadays a potent tool to slow or, in the luckiest cases, to halt the progression of FAP. Since TTR is mostly produced in liver, the transplantation indeed replaces effectively mutant gene with a WT gene resulting in a reduction of mutant TTR levels in body < 5% in respect to pretransplant levels⁵⁰. Even if it is considered a useful strategy, this might not eliminate the problem since amyloid fibrils can grow on pre-existing deposits. In addition, some mutations cause CNS amyloidosis derived from production of choroid plexus-related TTR and so do not respond positively to gene therapy by liver transplantation. For these reasons, along with the high invasiveness, risk and cost of liver transplantation, the long-life immunosuppression and the limited organ availability, medicinal chemists are trying to develop new drugs to inhibit these pathological processes.

131

Figure 4.1.3.1.Inotersen (IONIS-TTRRx) chemical structure.

It has been tested on a small number of patients affected by FAP and what turned out, was that over 65 weeks a very good improvement of neuropathy progression was observed.

Unfortunately though, since this conserved region is not generally mutated, these inhibitors, like patisiran and others⁶³, downregulate the mutant TTR expression, as well as WT-TTR expression. Inotersen has recently passed phase 2/3 clinical trial (NCT01737398) as drug for treatment of FAP and so for this reason, it is expected to be approved by FDA for neuropathic applications by the end of 2019^41,64–67.

Figure 4.1.3.2. Schematic representation of protein expression regulation by antisense oligonucleotide and siRNAs. In both pathways mRNA strand is cut off by RISC complex or RNAase H by means of intermolecular pairing with a complementary strand. Figure adapted from reference⁶⁸.

siRNAs⁵¹ are non-codifying short double-stranded oligonucleotides, which, packed in lipid nanoparticles to guarantee delivery to the liver, bind to a conserved regions of TTR mRNA activating their degradation and consequently reducing TTR expression. Their inhibition mechanism passes through endoribonuclease Dicer, a helicase enzyme, which unfolds the dsRNA and gives to RISC complex just one strand. Now RISC complex can recognize the complementary mRNA and degrade it (Figure 4.1.3.2)⁶⁹.

132

The kinetic stabilizers of TTR tetramer, differently from anti-translation drugs, play a stabilizing role in the dissociation of the tetramer. They, in fact, go into both thyroxine cavities acting essentially as a “supramolecular glue” (chaperones), which takes together the two dimers of dimers generally involved in the outbreak of the disease. Wide research has been doing in this field, since these synthetic molecules, smaller and more apolar than oligonucleotides, can be more easily internalized in cells. According to the sharp and very preorganized cavities, all the inhibitors synthesized so far are pretty rigid and flat. Most of them are polyaromatic molecules, such as diflunisal^70,71, tafamidis⁷², tolcapone⁷³, and Thyrphorstin AG10 (Figure 4.1.3.3).

Figure 4.1.3.3. Chemical structure of most common drugs used as kinetic stabilizers in the treatment of ATTR.

Diflunisal is a nonacetylated salicylate nonsteroidal anti-inflammatory drug used for decades in the treatment of arthritis and musculoskeletal pain. In 2003 a randomized experimental study demonstrated that it is able to slow significantly the polyneuropathy progression^74–76. In relative cocrystal two different binding modes have been observed: reverse and forward binding mode (Figure 4.1.3.4 A,B). The molecule can enter the channel in two opposite orientations because of the similar intermolecular interactions at stake. The reverse binding is stabilized by an electrostatic interaction between main chain oxygen atom of Ala 108 and its carboxyl group and the side chain oxygen atom of Thr 119, whereas the forward mode, on the contrary, is dominated by a halogen bond among one fluorine atom and the Thr 119 side chain oxygen atom. Moreover, after computational studies, it has been demonstrated that the binding, in this case, is increased by ionic interaction between Lys 15′ and carboxyl group of diflunisal^70,72. Recently, diflunisal has been taken as lead compound for the development of biphenyl-based inhibitors. According to Adamski-Werner⁷⁰, only small substitutions can be made on biphenyl scaffold like halogen groups. A large library was made and some derivatives have been shown as better inhibitors than diflunisal because of the better positions of groups involved in the binding. As well as Diflunisal, Tafamidis can enter TTR thyroxine-binding pocket and bind to tetramer (Kd1 = 5.7 nM and Kd2 = 260 nM towards WT-TTR). It stabilizes the TTR tetramer by hydrophobic interactions between 3,5-chloro groups of the inhibitor and HBPs. The whole binding is enhanced by water-mediated hydrogen bonds between the carboxylate of Tafamidis and Lys15/15′ and Glu54/54′ residues (Figure 4.1.3.4C).

133

Figure 4.1.3.4. X-ray crystal structures of Diflunisal and Tafamidis bound to TTR. Diflunisal is shown bound to TTR in the forward binding mode (A) and in the reverse binding mode (B), meanwhile Tafamidis is just observed in the forward binding (C). In figure C it is shown that with Tafamidis the binding is enhanced by two water molecules involved in bridging hydrogen bonds with Glu54, Glu54’and Lys15. Figure adapted from references^70,72.

Tafamidis, for its high efficiency as chaperone and for the good results obtained in phase 3 clinical trial (NCT01994889), has been approved by European Medicinal Agency (EMA) for the treatment of FAP⁷².

A B

C

134

Figure 4.1.3.5. On the left-hand side WT-TTR/tolcapone complex is shown in ribbon representation. Dashed line indicates the twofold symmetry axis of the dimer–dimer interface. A magnification of one of the WT-TTR T4-binding sites is shown on the right-hand side (PDB code: 4D7B). Figure adapted from reference⁷³. Recently, the Parkinson drug Tolcapone has been discovered to be a potent kinetic stabilizer for TTR tetramer through a simultaneous binding in both thyroxine-binding pockets (Kd1 = 21 nM and Kd2 = 58 nM towards WT-TTR)⁷³. In cocrystal structure it has been seen that 4-methylphenyl ring of tolcapone makes hydrophobic interactions in HBP2–2′ and HBP3–3′ with Ala108, Leu110, Ser117 and Thr119, whereas a stabilizing hydrogen bond is established between the hydroxyl side chain of Thr119 and the central carbonyl group of the inhibitor.

Contrary, the 3,4-dihydroxy-5-nitrophenyl ring is engaged into two different interactions:

hydrophobic interactions with Lys15, Leu17, Thr106 and Ala108 in HBP2-2′ and HBP1 -1′ and electrostatic interactions between phenolic OHs and ɛ-amino group of Lys15, which, in turn, establishes polar contacts with the carboxylate group of Glu54. The polar interactions mediated by Lys15 represent a smart idea employed by Tolcapone to exclude solvent and preserve the protein/inhibitor complex. The slightly higher efficiency of Tafamidis over Tolcapone, beside the nature of interactions, can be explained by the entropic effect of a minor number of displaced water molecules upon binding. By a pharmacokinetic point of view, Tolcapone is characterized by rapid absorption properties (85% in gut) and good bioavailability (65%), because of its quantitative complexation in plasma protein, mainly by albumin.

A

Time (h) Plasma concentration (mg ml-1 )

135

Figure 4.1.3.6. A) Plasma concentration of Tolcapone and 3-O-MethylTolcapone (3-OMT) over time. It is clear from the profile that Tolcapone reaches its maximum peak after 2-3 hours followed by a fast clearance. Figure adapted from reference⁷⁷; B) Schematic representation of all reactions involved Tolcapone metabolism. From left to right: glucuronidation reaction, methyl oxidation to carboxylic acid, reduction of nitro group to amine and subsequent acyl capping, 3-O-methylation.

Tolcapone reaches its highest plasmatic concentration after 2 h, but unfortunately at the third hour its concentration is approximately equal to zero (Figure 4.1.3.6). This dramatic inactivation is due mainly to glucuronidation, after which the resulting molecule is rapidly excreted through biles via urine or via faeces. These reactions along with those reported in Figure 4.1.3.6B, are responsible for hepatic overstimulation, which limits tolcapone dosage⁷⁷. For this reason, a lot of ongoing research is synthesizing and testing analogues, in which moieties responsible for pharmacokinetic clearance are combinatorially deleted. In parallel to these type of inhibitors, some researchers have employed some natural flavonoids as TTR tetramer stabilizers. Some of these, such luteolin, genistein and Biochanin A have been demonstrated good stabilizers^78,79. The challenge now is to modify them to increase the binding and pharmacological properties.

Figure 4.1.3.7: Chemical structure of natural molecules used as kinetic stabilizers in the treatment of ATTR.

Obviously, these inhibitors do not remove the pre-existing amyloid deposit, but they just prevent further deposition. With this need in mind, several are the molecules designed as sequestrants for disassembly. For example, Doxycycline (tetracycline antibiotic) and Tauroursodeoxycholic acid (bile acid) have been shown up as efficient tool for disruption of premature amyloid fibrils⁸⁰. Also Epigallocatechin-3-gallate⁸¹, a polyphenol extracted by green tea, is able to either stabilize TTR tetramer or disrupt fibrils, but binding in this case in a different site from that used by tafamidis and diflunisal. Also curcumin^82,83, binding to thyroxine-binding pocket, is able to act as tetramer stabilizer and fibril disruptor.

Besides this “natural approach”, recently, some examples of immunotherapy have been coming B

136

up. One strategy consists in the synthesis of antibody specific to an amino acid sequence in TTR, which is buried in the tetramer, but exposed in the monomer^84,85. This guarantees the possibility to target the dangerous monomer and trigger an immune response finalized to its elimination. Opposite to this, the second strategy exploits the heterogeneous composition of amyloid deposits. Among them, Serum amyloid P component (SAP) is found. Since this protein is a glycoprotein responsible for the resistance of amyloid deposits to proteases, SAP is a good target for amyloid disassembly. A few years ago, a monoclonal G1 anti-SAP antibody was used in this context with very good results⁸⁴. 16 FAP-affected patients within a 6-weeks treatment period were subjected to significant reversal of the disease. By the way, these last systems are getting very interesting devices for the treatment of ATTR, but even if some have passed phase II of clinical trials, they still need to be studied more.