Stabilization of amyloidogenic immunoglobulin light chains by small molecules

Abstract

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.kinetic stabilizer | high-throughput screen | dimerization | structural biology | proteotoxicity S ecretion of an Ig light chain (LC) by a clonally expanded plasma cell population can lead to the disease LC amyloidosis (AL)-both a cancer and a proteinopathy (1, 2). "Free" LCs secreted without an associated antibody heavy chain (HC) initially adopt a well-defined homodimeric structure, wherein the monomers may be covalently linked by an interchain disulfide bond ( Fig. 1A) (3). LC monomers comprise an N-terminal variable (V) domain attached to a C-terminal constant (C) domain (SI Appendix, Fig. S1). Each patient's clonal plasma cells secrete a single, unique LC sequence. Most LCs are rapidly removed by the kidney.However, since amyloidogenic full-length (FL) LCs are generally less stable than nonamyloidogenic FL LCs, they can misfold, or misfold and misassemble, into nonnative species including cross-β-sheet amyloid fibrils, which are a hallmark of AL (4-8). Sequence also seems to play a role, as not all destabilized FL LCs aggregate in patients (4-8). How aggregation occurs in patients is not known, but several processes have been described in vitro, including destabilization-dependent endoproteolysis that releases amyloidogenic LC fragments (4,9,10). LC fragments including V domains are observed in patient deposits alongside FL LCs (11-13).Since we do not understand the structure-proteotoxicity relationships driving AL, a conservative strategy is to block FL LC misfolding at its origin by stabilizing the FL LC native state. Such a strategy has been effective at ameliorating the transthyretin amyloidoses (14-19). A small molecule that stabilizes FL LC dimers should prevent any misfolding and/or endoproteolysis that lead to LC aggregation and organ toxicity. We refer to such molecules as kinetic stabilizers, since they reduce the rate at which LCs transiently visit nonnative, aggregation-prone, and protease-sensitive conformations (20). The interfaces between the domains of the LC dimer are an important...

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis

“…Mapping of the cleavage sites on the available LC structures. The cleavage sites identified on AL-55 were mapped on the already available native structure of JTO full length LC dimer (PDB entry: 6MG4) (24), which, as AL-55, belongs to λ6 isotype and displays 90% sequence identity with AL-55, and on the AL-55 fibrillar structure recently reported (PDB entry: 6HUD) (17) ( Figure 5A and C). AL-H7 cleavage sites were instead mapped on the native structure of AL-H7 full length dimer (PDB entry: 5MUH) (26) and on the fibrillar structure of another λ1 LC belonging to the same IGLV1 family VL and with a sequence identity of 70% (PDB entry: 6IC3) (18) with AL-H7 ( Figure 5 B and D).…”

“…Defining the site and timing of the proteolytic events in AL in vivo has two important implications: a better understanding of amyloid formation in the natural environment, and a therapeutic perspective, to prevent destabilizing proteolysis (24) or to potentiate the tissue defenses that degrade harmful aggregates (22). Identification of the N-and C-termini of the LC fragments is instrumental to understand the enzymes and processes involved in cleavage.…”

Systemic amyloidosis: moving into the spotlight

“…Exciting gene-silencing therapies (patisaran 88 AL amyloidosis has trailed ATTR in these crucial therapeutic aspects. Recently, highthroughput screening and characterization identified several small molecules that kinetically stabilize free light chains by binding at the V-domain-V-domain interface in both kappa and lambda light chains providing the first step to a potential FLC stabilising approach 91 . Whilst pre-clinical work suggests potential in RNA inhibitors in reducing free light chains production 92 , this remains challenging to translate into in vivo models.…”

A kinetic coupling between protein unfolding and aggregation controls time‐dependent solubility of the human myeloma antibody light chain

“…The gene sequence of full-length human λ-6A light chain hLC () was chosen from a study of stabilization of amyloidogenic immunoglobulin light chain by small molecules, 17 and the gene was synthesized by GenScript.…”