Structural basis for the stabilization of amyloidogenic immunoglobulin light chains by hydantoins

Abstract

Misfolding and aggregation of immunoglobulin light chains (LCs) leads to the degeneration of post-mitotic tissue in the disease immunoglobulin LC amyloidosis (AL). We previously reported the discovery of small molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which slow or stop the LC aggregation cascade at the outset. A predominant structural category of kinetic stabilizers emerging from the high-throughput screen are coumarins substituted at the 7-position, which bind at the interface between the two variable domains of the light chain dimer. Here, we report the binding mode of another, more polar, LC kinetic stabilizer chemotype, 3,5-substituted hydantoins. Computational docking, solution nuclear magnetic resonance experiments, and x-ray crystallography show that the aromatic substructure emerging from the hydantoin 3-position occupies the same LC binding site as the coumarin ring. Notably, the hydantoin ring extends beyond the binding site mapped out by the coumarin hits. The hydantoin ring makes hydrogen bonds with both LC monomers simultaneously. The alkyl substructure at the hydantoin 5-position partially occupies a novel binding pocket proximal to the pocket occupied by the coumarin substructure. Overall, the hydantoin structural data suggest that a larger area of the LC variable-domain-variable-domain dimer interface is amenable to small molecule binding than previously demonstrated, which should facilitate development of more potent full-length LC kinetic stabilizers.

Figure 1.

The structural characterization of the binding of coumarin 1 to JTO-FL by X-ray crystallography is recapitulated by Autodock Vina. A) Crystal structure of 1 bound to JTO-FL (PDB: 6MG5). The variable domains of different chains are shown in blue and purple, whereas constant domains of both chains are depicted in gray. The small molecule is shown in yellow CPK surface representation. The red arrow indicates the direction of view for the more detailed figures. B) Close-up representation of the crystal structure of coumarin 1 bound to JTO-FLwherein the kinetic stabilizer is shown in yellow stick format and as a surface representation, whereas the FL LC is shown as a ribbon representation. C)The docked structure of 1 (green) is overlaid onto the crystal structure of JTO-FL•1, reflecting docking accuracy.

Figure 2.

Autodock Vina 1.1.2-based models of small molecule kinetic stabilizer screening hits (Table 1) docked into the structure of apo JTO-FL derived from the structure of JTO-FL•1. The position of 1 in the crystal structure of JTO-FL•1 is shown as yellow lines and as a surface representation. Autodock-generated poses of each small molecule are shown as green sticks for: A) Compound 2. B) The E stereoisomer of compound 4a (diethylamino-substituted 4). C) The Z stereoisomer of compound 4a. D) The (5R,6R) stereoisomer of compound 7. E) The S enantiomer of compound 8. F) The R enantiomer of compound 8.

Figure 3.

Selected regions of the ¹⁵N-¹H-HSQC NMR spectra of WIL-FLLC(50μM)in the absence(black contours) and presence (red contours) of small molecule kinetic stabilizers (100 μM). Full spectra are plotted in Figure S2. Note that the reduction in NMR intensities in the presence of compound 7 is smaller than with compounds 1 and 8, most likely because of the more limited solubility of compound 7. (B) Amide groups with resolved resonances that are perturbed upon addition of the ligands are plotted as spheres within the ribbon representation of the crystal structure of 1 bound to JTO-FL (PDB: 6MG5). The same set of resonances were perturbed by all three compounds.

Figure 4.

Sedimentation velocity analytical ultracentrifugation profiles of C214S WIL-FL (10 μM) in the presence or absence of the indicated small molecule kinetic stabilizers (100 μM). Vehicle is 1% DMSO. The monomer-dimer equilibrium is shifted towards the dimer in the presence of either kinetic stabilizer.

Figure 5:

The crystal structure of the JTO-FL•8 complex. A) A comparison of the bound kinetic stabilizers in the JTO-FL•1 (yellow) and JTO-FL•8 (blue) complex structures, wherein the FL LC is depicted in ribbon diagram format, colored as indicated. B)Potentialhydrogenbondinginteractions(reddashed lines) between 8 and JTO-FL, some mediated through ordered water molecules (red spheres). Hydrogen atoms are modeled into the protein and ligand for clarity. C) Interactions between 8 and the labeled side chain residues of JTO-FL. D) Electron density map (2Fo-Fc contoured at ± 1.0 σ) showing the presence of the R enantiomer of 8 bound to JTO-FL. E) Comparison of the crystal structure of 8 in the bound conformation (blue) with the docked models of (S)-8 (green) and (R)-8 (aqua), indicating that they likely bind similarly.

Figure 6.

Differential kinetic stabilizer potencies of isolated enantiomers of 8. WIL-FL (5 μM) was incubated in the presence of 1% DMSO vehicle (Veh) control or the indicated small molecule kinetic stabilizers (100 μM) at a final concentration of 1% DMSO for 2 h at 37 °C with proteinase K (50 nM). The remaining WIL-FL upon treatment with kinetic stabilizer 1, 8E1, or 8E2, shown as the fraction of the no proteinase K control, was measured using analytical-size exclusion chromatography. Each proteinase K sensitivity assay was performed in triplicate. An ideal kinetic stabilizer would exhibit a fraction of WIL-FL remaining of 1.