Systemic light chain amyloidosis is a disease that occurs when individuals produce too much of an immune protein. The excess protein chains normally exist in the body as individual molecules called “monomers” or in pairs called “dimers,” and they can readily switch between these two forms. However, the monomers are also prone to forming amyloid fibrils, which are difficult to break down. Amyloid fibrils are often deposited in the heart and kidneys and can lead to organ failure and death.

Finding molecules that prevent the formation of amyloid fibrils could help to develop treatments for amyloidosis. Now, Brumshtein, Esswein et al. have screened 27 compounds to identify those that stabilize the dimer form of the protein. This would reduce the number of monomers in the body, and so reduce the number of immune proteins that can form amyloid fibrils.

The experiments identified four compounds that could stabilize the dimers, including one called methylene blue. Comparing the chemical structures of these compounds with the structures of drugs approved for medical use identified thirteen drugs. However, follow-up tests showed that only one, called sulfasalazine, reduced the formation of amyloid fibrils. Neither methylene blue nor sulfasalazine is likely to have a strong enough effect to treat amyloidosis, but they may serve as templates for future drug designs.

Amyloid fibrils are protein deposits often associated with disease. These deposits show common structural and biochemical characteristics, including non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-β X-ray diffraction pattern. The spine of amyloid fibrils, termed a 'steric zipper,' is built by paired β-sheets with interdigitating side chains and serves as the scaffold for fibril extension. The inter-strand and inter-sheet distances give rise to a characteristic cross-β X-ray diffraction pattern of two perpendicular reflections at ~4.8 and ~10 Å (Geddes et al., 1968; Hobbs, 1973; Sipe and Cohen, 2000; Nelson et al., 2005). Formation of a steric zipper requires the presence of a discrete peptide segment with an amino acid sequence capable of forming a self-complimentary propagating structure. Such segments are commonly found as integral parts of globular proteins and trigger aggregation into amyloid fibrils only upon exposure to solvent through full or partial denaturation (Nelson et al., 2005).

Cases of systemic light chain amyloidosis (AL), associated with multiple myeloma, were first described in the mid-19^th century; however, the etiology of the disease was not clear. In 1848, Dr. Bence-Jones discovered the disease-associated substance in urine, though it was only later identified as immunoglobulin light chains (LC) associated with amyloidosis (Kyle, 2001; 2011; Dahlin and Dockerty, 1950; Glenner et al., 1970; 1971; Glenner, 1973; Sipe et al., 2014). Systemic AL amyloidosis is characterized by overexpression of monoclonal LCs, which are able to form homo-dimers. Despite renal clearance of excess protein, the full-length LCs or their fragments still form amyloid fibril deposits in patients’ tissues, most frequently in the heart and kidneys, which result in organ failure (Falk et al., 1997; Buxbaum, 1986). Although it is clear this deposition causes organ dysfunction, much remains to be discovered about the molecular process by which immunoglobulins assemble into amyloid fibrils. It is unknown which peptide segments form steric zippers and how to block their assembly into the amyloid spine.

LCs are produced by plasma cells and their final amino acid sequence is determined by somatic recombination (Sakano et al., 1979; Marchalonis and Schluter, 1989). LCs are classified by their amino acid sequence as either κ or λ. They consist of two domains, the variable (V_L) and constant (C_L), which are connected by a joining segment (J). In patients, amyloid fibrils are found to include either V_Ls or full-length LCs (V_L-J-C_L), yet the ubiquitous presence of V_Ls indicates that this domain may be the minimal and essential unit for fibril assembly (Falk et al., 1997; Buxbaum, 1986; 1992; Olsen et al., 1998; Lavatelli et al., 2008; Vrana et al., 2009; Bodi et al., 2009).

The crystal structures of full-length LCs and structures of just their V_Ls both reveal homo-dimers, which resemble the light chain and heavy chain hetero-dimer of the antigen-binding fragment (Fab) of a full antibody. Fab is a hetero-dimer composed of both light and heavy chains, each with constant (C_L and C_H) and variable (V_L and V_H) immunoglobulin domains. LC homo-dimers, in which the V_L domain is covalently linked to the C_L domain, resemble the quaternary structure of the Fab hetero-dimer, and non-covalently linked dimers of only V_Ls structurally resemble the variable region of the Fab (V_L and V_H) (Edmundson et al., 1969; Colman et al., 1977; Firca et al., 1978). The interfaces between the variable domains of physiological hetero-dimers and pathological homo-dimers are lined with apolar residues encompassing a hydrophobic cavity capable of accommodating small molecules (Edmundson et al., 1984; 1993).

Our work focuses on a λ patient-derived V_L homo-dimer named Mcg whose structure and properties were detailed in pioneering studies by Edmundson and coworkers (Edmundson et al., 1969; 1984; 1993; Firca et al., 1978). The cavity between the two V_L domains in Mcg is capable of binding a wide array of ligands, including synthetic organic molecules and peptides, which form contacts with the side chains of the dimer cavity. The cavity spans most of the 24 Å-long dimer interface; it is cylindrically shaped and can be considered to constitute three sites: A, B and C (Edmundson et al., 1984). Based on the geometry of the dimer with some deviation from the original nomenclature by Edmundson et al., we designate side chains of residues Y34, Y93, D97 and F99 on one side of the cylindrical cavity as the 'A-site,' S36, Y51, E52, S91 and F101 as the 'B-site,' and Y38, Q40, V48 and Y89 as the 'C-site' (Figure 1). Some ligands bind the cavity in a sequential manner without a distinct preference for one of the sites. For example, 1-anilinonaphthalene-8-sulfonic acid binds in either the A or C-sites and can migrate between them, whereas fluorescein binds to both sites with partial occupancy. Other molecules, such as menadione, preferentially bind to only one of the sites (Edmundson et al., 1984). Regardless of binding location, this demonstrates the dimer cavity is capable of accommodating various hydrophobic and aromatic ligands.

Figure 1

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V_Ls exist in equilibrium between homo-dimers and amyloid-prone monomers. Experiments conducted in denaturing conditions indicate that reducing the stability of the monomeric state promotes amyloid fibril formation, and mutations that induce dimer disassociation or promote monomer unfolding increase the propensity to form amyloid fibrils (Bernier and Putnam, 1963; Kishida et al., 1975; Qin et al., 2007; Wetzel, 1994; Hurle et al., 1994; Brumshtein et al., 2014; Baden et al., 2008). Likewise, mutations that stabilize the structure of V_Ls or covalently fix V_L dimers inhibit formation of amyloid fibrils. These results indicate that formation of amyloid fibrils involves two steps: V_L dimer disassociation into monomers followed by partial or full unfolding. The mechanism of amyloid formation also suggests that shifting the equilibrium away from the amyloid-prone monomer by stabilizing the dimer would hinder formation of amyloid fibrils (Figure 2) (Bulawa et al., 2012; Bellotti et al., 2000).

Figure 2

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The monomer-dimer equilibrium of V_Ls suggests that systemic AL amyloidosis may be mitigated by binding ligands to the cavity at the V_L dimer interface (Figure 2). This approach proved effective for transthyretin-related amyloidosis, another type of systemic amyloidosis for which stabilizing the quaternary state led to the development of therapeutics (Miroy et al., 1996). Upon transthyretin tetramer disassociation into amyloid-prone monomers, it forms amyloid fibrils in an acidic environment. The binding of thyroxine inhibits disassociation and subsequent amyloid formation (Baures et al., 1998). Following the same principle, a modified ligand with a disassociation constant in the nano-molar range prevents transthyretin from forming amyloid fibrils and is effective in vivo.

Here we apply structural and biochemical methods to investigate ligands that hinder amyloid formation by stabilizing the V_L homo-dimer. We identify ligands that may serve as prototypes for therapies for treating LC amyloidosis and our results are consistent with a mechanism for amyloidosis that proceeds via dimer disassociation to amyloid-prone monomers (Qin et al., 2007; Brumshtein et al., 2014).

Identification of ligands that inhibit formation of amyloid fibrils. Based on the previous work of Edmundson et al., we screened a panel of 27 hydrophobic and aromatic ligands for an inhibitory effect on the formation of amyloid fibrils (Figure 3) (Edmundson et al., 1976; 1984; 1993). Such ligands may stabilize V_L dimers and decrease disassociation into amyloid-prone monomers. Varying concentrations of ligands up to 1 mM were used to evaluate the effect of complexes on formation of amyloid fibrils. Formation of amyloid fibrils was monitored using both ThT fluorescence assays and EM micrographs. Molecules that showed an inhibitory effect with concentrations under 1 mM were 8-anilino-1-naphthalene sulfonic acid [5], methylene blue [10], Chicago Sky Blue 6B [14] and Phenol Red [20] (Figure 3B).

Figure 3

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Methylene blue, Chicago Sky Blue 6B, and Phenol Red (10, 14 and 20) were used to search DrugBank for approved biomedical compounds resembling their chemical structures and properties with a similarity threshold value of 0.3 (Law et al., 2014). The search identified 13 compounds that were tested for an amyloid inhibitory effect by means of ThT assays and EM analysis (Figure 3C). Of these 13 molecules from DrugBank, only sulfasalazine [34] showed an inhibitory effect on formation of amyloid fibrils. Among all the molecules tested, including the ligands initially screened and the subsequent approved biomedical compounds, the two that show the most potent effect are methylene blue [10] and sulfasalazine [34]; both hinder amyloid fibril formation at effective concentrations of 0.1 and 0.5 mM respectively (Figure 4).

Figure 4

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Equilibrium dialysis binding of methylene blue and sulfasalazine. Equilibrium dialysis was used to assess the binding constants of methylene blue and sulfasalazine to Mcg. Measured concentrations were fit to the corresponding model equations and their curves were represented as binding and Scatchard plots (Figure 5) (Scatchard, 1949; Spitzer and McDonald, 1956). The constants were derived from a least squares fit of equations to data and are given in Table 1. Although both methylene blue and sulfasalazine bind to Mcg, the Scatchard plots indicate that binding proceeds through somewhat different pathways: methylene blue shows positive cooperative binding, signifying at least two sites with different binding constants, while sulfasalazine shows no cooperativity and suggests an additional, non-specific binding site (Figure 5). The best fit for the sulfasalazine-binding data was achieved using a model for two identical, independent binding sites per V_L dimer, followed by non-specific binding.

Figure 5

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Crystal structures of methylene blue and sulfasalazine bound to the V_L dimer. In the crystal structures of Mcg with methylene blue and sulfasalazine, the ligands bind at the cavity between the two V_L domains (Figure 6, Table 2). In the structure of Mcg with methylene blue, one ligand is bound to the A-site of the dimer. This differs from equilibrium dialysis results in solution, which indicate at least two methylene blue binding sites. The structure of Mcg with sulfasalazine indicates two symmetry-related molecules bound in the hydrophobic cavity of the dimer with both ligands simultaneously binding to three sites of the V_L dimer cavity.

Figure 6

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Effect of ligand binding on quaternary state. We used analytical ultracentrifugation to assess whether ligand binding shifts the equilibrium of Mcg towards more dimers or whether ligands bind to monomers as well. The wavelengths used for optical density readings of methylene blue (550 nm) and sulfasalazine (420 nm) did not overlap with protein absorbance. Distributions of molecular weights were fit to the absorbance data, thus permitting detection of only the apparent molecular weights of the protein in complex with ligand (Figure 7, Table 3). The observed molecular weights indicate that ligands bind only to V_L dimers. For methylene blue, the measured molecular weight was slightly larger than expected; therefore, we performed an additional ultracentrifugation meniscus depletion experiment to detect possible monomers. The experiment identified no methylene blue bound to V_L monomers.

Figure 7

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The identification of ligands that inhibit LC amyloid formation by stabilizing the V_L dimer has therapeutic implications for systemic light chain amyloidosis. By utilizing the known ability of ligands to bind the hydrophobic cavity of V_L dimers, we prevent dimer disassociation into monomers and thus hinder amyloid formation (Edmundson et al., 1984; 1993). A similar approach was used to design therapies for transthyretin-related amyloidosis by stabilizing the tetrameric state of the protein (Miroy et al., 1996). Initial screening for ligands that hinder V_L amyloid formation revealed four ligands showing this effect, with methylene blue as the strongest binding ligand. A subsequent search in the DrugBank aimed to identify similarly effective ligands that are already approved for medical use.

Since the emission spectra of ThT partly overlaps with the absorption spectra of the ligands, both ThT assays and EM analysis were necessary to confirm the ability of the ligands to inhibit amyloid formation. Analysis of electron micrographs and ThT assays indicates that increasing the concentration of methylene blue or sulfasalazine hinders the formation of amyloid fibrils. One of the molecules from our screen, epigallocatechin gallate, was recently suggested to hinder the formation of amyloid fibrils, yet in our experiments it did not show any effect (Pelaez-Aguilar et al., 2015).

Though both of the ligands bind Mcg and induce amyloid hindering effects, the mechanism of binding slightly differs. Methylene blue binds through positive cooperativity. This result indicates the presence of two binding sites, one strong and one weak, where binding to the second site begins only after the first site is fully occupied. Scatchard plots for sulfasalazine show that the two specific binding sites on Mcg are identical with the same disassociation constant and that this specific ligand binding is followed by non-specific binding.

We addressed the difference in the binding mechanisms of methylene blue and sulfasalazine to Mcg in solution by crystal structures and analytical ultracentrifugation. The crystal structure of Mcg with methylene blue shows binding at the A-site of the V_L dimer cavity and coordination by the side chains of residues Y34, Y93, D97 and F99. The crystal belongs to space group P2₁ with one dimer per asymmetric unit, and the symmetry between the two V_L domains of Mcg is broken by the presence of a sulfate ion bound to only one of the V_L domains (Figure 6A). Analytical ultracentrifugation analysis of the soluble quaternary state indicates the dimer as the most prevalent form of the complex versus monomer without a bound ligand (Brumshtein et al., 2014). We also observed a small fraction of oligomers with molecular weights larger than dimers, as confirmed by the meniscus depletion experiment. The meniscus depletion distributes quaternary states along the path length of the sample cell during analytical centrifugation, thus verifying which state accounts for the deviation in apparent molecular weight. The most significant outcome is the complete absence of soluble monomer in complex with the ligand, which verifies our conjecture that the ligands bind and stabilize dimers. In the case of sulfasalazine, to confirm binding specifically to soluble dimers, the analytical ultracentrifugation experiments identified dimers as the only quaternary state of the ligand-bound complex.

The reconciliation of our three experiments on the binding of methylene blue to Mcg V_Ls suggests blocking of the secondary binding site in the crystals. The three experimental findings are: cooperative binding of methylene blue as measured by equilibrium dialysis, binding of the ligand to the A-site in the crystal structure, and the absence of monomeric V_L in complex with ligand. Taken together, these observations indicate that the secondary binding site of methylene blue may not be available in the crystalline state, but available in solution. Secondary binding to either the B or C-sites may require conformational changes that are less favorable in crystals or induce the higher-order assembly of complexes larger than dimers.

The crystal structure of Mcg with sulfasalazine explains why only one disassociation constant for specific binding was measured. Crystals of the complex belong to space group P2₁2₁2 with two symmetry-related ligands bound per Mcg dimer, and thus one ligand bound per V_L in the asymmetric unit (Figure 6B). Therefore, the equivalence of the two sulfasalazine ligands in the crystal explains why only one disassociation constant was measured by means of equilibrium dialysis. The crystal structure shows that each of the sulfasalazine ligands occupy all three A-B-C-sites simultaneously; each ligand binds to the A and B-sites on one of the V_Ls composing the dimer, while C-site binding is on the symmetry-related V_L. This contrasts with methylene blue, which is bound only to the A-site. Despite differences in the exact orientation of the side chains where the ligands bind, both ligands bind in the cavity of the Mcg dimer (Figure 6C).

Although our experiments were performed using only V_Ls rather than full-length LCs, the ligand-binding site between V_Ls is maintained in full-length LCs. While full-length LCs are disulfide linked at their C_L C-termini, crystal structures indicate that the conformation of V_Ls is independent of connected constant domains (Ely et al., 1989; Terzyan et al., 2003; Makino et al., 2007). In full-length LCs, the joining (J) segment between V_Ls and C_Ls provides the flexibility for V_Ls to maintain the same hydrophobic cavity as that seen in the V_L homo-dimer alone. This indicates the strategy of using ligands to stabilize the V_L dimer and hinder amyloid fibril formation may also be effective for full-length homo-dimers.

The ability of ligands to hinder the aggregation of V_Ls into amyloid fibrils by stabilizing V_L dimers indicates a promising approach for treatment of systemic light chain amyloidosis disease. While the light chain amino acid sequence differs between patients, the characteristic hydrophobic cavity of the homo-dimer formed by overexpressed LCs is conserved (Bodi et al., 2009; Bork et al., 1994; Edmundson et al., 1976; Solomon and Weiss, 1995). Although our experiments were performed using a single V_L variant, LCs of both λ and κ maintain a similar tertiary structure and hydrophobic cavity (Qin et al., 2007; Wetzel, 1994; Brumshtein et al., 2014; Bellotti et al., 2000; Fink, 1998; Khurana et al., 2001). This conserved hydrophobic pocket indicates a possible target for designing medications that inhibit LC amyloidosis. Although methylene blue and sulfasalazine may not bind tightly enough to be directly repurposed as treatments for the disease, these ligands may serve as prototypes for development of effective medications. As indicated by the ability of these molecules to inhibit amyloid fibril formation, this approach may result in alleviated symptoms for systemic light chain amyloidosis patients.

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Author details

1. Boris Brumshtein

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   BB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Shannon R Esswein

   For correspondence

   boris@mbi.ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

2. Shannon R Esswein

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   SRE, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Boris Brumshtein

   For correspondence

   sesswein@mbi.ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5142-0190

3. Lukasz Salwinski

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   LS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Martin L Phillips

   1. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   2. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   MLP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Alan T Ly

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   ATL, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Duilio Cascio

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   DC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Michael R Sawaya

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   MRS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. David S Eisenberg

   1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
   2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
   3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States

   Contribution

   DSE, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   david@mbi.ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

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