Bifunctional crosslinking ligands for transthyretin

Abstract

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR-ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.

Keywords: amyloidosis; crosslinking; plasma clearance; transthyretin.

Figure 1.

Bifunctional ligands for crosslinking TTR tetramers based on the dichlorophenylaminobenzoic acid head group with either polyproline (group I) or polypiperidine (group II) linkers. Ligand IIe has a modified ether linkage in place of the aminopropoxy linkage.

Figure 2.

(a) A₂₈₀ UV absorbance gel filtration profiles of native TTR (dotted line) and EDC crosslinked TTR (E-TTR, solid line). Native untreated TTR chromatographs as a tetramer on gel filtration eluting at 14.2 ml while EDC crosslinked TTR elutes mainly as high molecular weight oligomers (Ve 7.5 9.0 ml). Both native TTR (T) and EDC-treated TTR (E-TTR) can bind ¹²⁵I-thyroxine as shown in the native gel autoradiogram (inset). Native TTR migrates towards the anode while EDC-TTR remains mostly at the site of sample deposition (arrowed). As some of the aggregates may have reduced thyroxine-binding capacity, the relative intensities in the autoradiogram may not reflect the absolute amounts of material present on the gel. The gel filtration A₂₈₀ UV absorbance profile gives a more accurate representation of relative amounts. (b) Clearance of EDC-treated ¹²⁵I-TTR (closed circles) in wild-type mice is faster than for TTR control (open circles); data are expressed as mean and s.d. (n = 3). (c) ¹²⁵I-TTR is localized in the spleen of animals treated with oligomerized EDC-TTR. Kid, kidney; spl, spleen; liv, liver.

Figure 3.

(a) The A₂₈₀ UV absorbance gel filtration profiles of native TTR (dotted line) and TTR incubated with a fourfold excess of ligand Ie (Pro9, solid line). The ligand has caused an increase in peak volume and a concomitant decrease in retention volume consistent with the formation of a ligand octamer complex followed by dissociation on the column. (b) The effect of ligands on peak width is dose-dependent for all ligands except Ia (Pro5) where no octamer formation has occurred.

Figure 4.

(a) Treatment of TTR with the polypiperidine ligand IIc generates an octameric complex which elutes on gel filtration with a retention volume of 12.8 ml and is separated from native tetrameric TTR at 14.6 ml. (b) The octamer (fraction 12) is stable to a second step of gel filtration. (c) Mass spectrometric examination of the complex of TTR with ligand IIc is consistent with the formation of a bis ligand octamer. Native apo TTR appears as the tetramer (T, charge states 11+ to 14+, 56 500 ± 50 Da). Addition of increasing levels of the ligand results in the dose dependent formation of an octameric species containing two ligands (O₂, charge states 17+ to 20+, 115 715 ± 50 Da). (d) The TTR/IIc complex dissociates as the desolvation energy is increased (80, 100 and 120 V, top to bottom panels). The holo octameric TTR complex with two ligands bound (17+ to 20+, O₂) dissociates to the octamer with one ligand (O₁) and apo octamer (O) confirming stoichiometry of the TTR ligand complex formed. Release of monomers (M) also occurs under increasing desolvation energy. Mass spectra for the TTR/IId complex are shown in the electronic supplementary material, S1.

Figure 5.

(a) There is no difference in the clearance of TTR and the stable TTR/IIc octamer in TTR knockout mice. (b) Analysis of the 60 min serum samples showed that radiolabelled octamer was still present in the circulation.

Figure 6.

(a) The complex formed between ligand IIe and TTR contained an appreciable amount of a larger molecular mass species chromatographing on gel filtration as a dodecamer at approximately 11.0 ml (solid line) and separated from native TTR (dotted line). The complex was unstable, regenerating the tetramer (at 14.7 ml) together with a small amount of octamer (12.8 ml) on a second step of gel filtration (inset). (b) Nanoflow electrospray mass spectrum of TTR in the presence of 1 molar equivalent of ligand IIe. Under soft desolvation conditions, several charge state series are observed corresponding to apo tetrameric TTR (11+ to 13+, T), holo octameric TTR with one ligand bound (17+ to 19+, O₁) and holo dodecameric TTR with two or three ligands bound (22+ to 24+, D_2/3). Higher oligomeric species are also observed above 8000 m/z. (c) Tandem mass spectrum of the 19+ charge state of O₁ shows the release of individual monomeric TTR (M) and the formation of ‘stripped complex’ H₁ corresponding to heptameric TTR with one ligand bound. (d) Tandem mass spectrum of the 24+ charge state of D_2/3 shows the release of individual monomeric TTR (M) and the formation of ‘stripped complex’ U₂ corresponding to undecameric TTR with two ligands bound. Ion charges are shown for the most intense species in each ion series.

Figure 7.

Models for the proposed TTR/Ie octameric complex (a) and the bracelet structure for TTR/IIc (b). The pictures were made using PyMOL Molecular Graphics System, version 0.99, Schrödinger, LLC.