Multi-Ligand Binding Mode of Tramiprosate and Effects on Aβ42 Monomer Conformation

To address the high conformational flexibility of Aβ42 and characterize its interaction with tramiprosate, we used IMS with a Q-TOF MS with traveling wave ion mobility. IMS is a powerful technique capable of separating molecular ions based on their size and conformation and can also be used to characterize the stoichiometry of ligand–protein complexes [23].

This IMS–MS analysis (Fig. 3) illustrated both the stoichiometry of the drug–protein complex and the shape of Aβ42 and also showed that multiple molecules of tramiprosate bind to a single molecule of Aβ42, in agreement with the previous studies [24]. These results indicate that tramiprosate formed a dynamic solvation envelope surrounding Aβ42 that interacted with the peptide in a dynamic manner. Figure 3 also shows that Aβ42 alone adopted many different conformations, as indicated by a long yellow zone and how the multitude of those conformations changed with each additional bound molecule of tramiprosate. Analysis of the arrival time distribution clearly showed this conformational shift. As additional tramiprosate molecules interacted with Aβ42 monomer, many conformations of Aβ42 transitioned into a more compact, presumably semi-cyclic, conformation (Sect. 4.4). The most extended conformations of Aβ42 on the right part of the yellow zone gradually disappeared with each additional bound molecule of tramiprosate, indicating the formation of more compact and stabilized conformations. With three or more bound tramiprosate molecules, only the most compact conformer populations, and none of the extended populations, were detected. This suggests that the binding of the drug to the peptide has a significant effect on the generation of more defined and stabilized populations of Aβ42 conformers. Additional bound molecules of the drug further stabilized the narrow peak of the population of Aβ42 conformers.

Fig. 3 Ion mobility spectrometry–mass spectrometry (IMS–MS) driftscope plot of the IMS drift time versus mass/charge (m/z) of amyloid beta Aβ42-tramiprosate stoichiometry. Aβ42 alone shows long time drifts (yellow zone), indicating many different populations of conformers. With an increasing number of bound tramiprosate molecules, the drift time of the Aβ42 conformers changes, indicating the presence of fewer and more stabilized conformations. Some of the extended conformers on the right completely disappear Full size image

Tramiprosate Prevents Formation of Aβ42 Oligomers

We next evaluated whether the Aβ42 conformation-stabilizing activity of tramiprosate affects aggregation, specifically the oligomer aggregation stages from monomers through soluble decamer species. To this end, we examined the formation of soluble Aβ42 oligomers in the absence or presence of tramiprosate by IMS–MS (Figs. 4, 5; Table 1). As expected, the critical neurotoxic oligomers (i.e. dimer, trimer, tetramer, pentamer, hexamer, and decamer) [25, 26] formed following an incubation of Aβ42 monomers; the identities of the oligomer species were further characterized at multiple charge states. However, in the presence of 1000-fold molar excess tramiprosate, the formation of the corresponding oligomers was inhibited. To explore a concentration–response relationship, we incubated monomeric Aβ42 with a 100-fold or 1000-fold molar excess of tramiprosate for 24 h. At a 100-fold molar excess, tramiprosate partially reduced the number of detectable oligomers. Strikingly, at 1000-fold molar excess, tramiprosate completely abrogated the full range of Aβ42 oligomer species (Table 2). Together, the results showed a concentration-dependent effect for tramiprosate in preventing the formation of Aβ42 oligomers, with complete inhibition achieved at the highest concentration tested (i.e., 1000-fold molar excess). Importantly, these findings suggest that tramiprosate stabilizes Aβ42 in its monomeric form and prevents the initiation stage of Aβ42 aggregation.

Fig. 4 Ion mobility spectrometry–mass spectrometry (IMS–MS) 2D arrival time data showing annotated detection of oligomers of amyloid beta Aβ42 after 24-h incubation in the absence of tramiprosate. m/z mass/charge Full size image

Fig. 5 Ion mobility spectrometry–mass spectrometry (IMS–MS) 2D arrival time data. a Detection of no oligomers in the amyloid beta Aβ42 + 1000 × tramiprosate sample after 24-h incubation. b The corresponding mass spectrum detecting only Aβ42 monomers in different charge states. m/z mass/charge Full size image

Table 1 Detection of amyloid beta Aβ42 oligomers by ion mobility spectrometry–mass spectrometry in the absence and presence of tramiprosate Full size table

Table 2 Detection of amyloid beta Aβ42 oligomers by ion mobility spectrometry–mass spectrometry in the absence vs. presence of tramiprosate Full size table

Together, these data show that the tramiprosate-enveloping mechanism, wherein Aβ42 peptide is enveloped by a cloud of tramiprosate reminiscent of a solvation effect (Sect. 4.4), has implications for clinical activity, especially because high molar excess of the tramiprosate was required in the clinical trials [11].

NMR Experiments Identify Aβ42 Residues that Interact with Tramiprosate

Next, we used 2D heteronuclear multiple quantum correlation NMR spectroscopy (2D 1H-15N HMQC NMR) of uniformly 15N-labeled Aβ42 peptide (in 90% H 2 O/10% D 2 O sodium phosphate buffer, pH 7.4 at 37 °C) to determine how tramiprosate binds to the peptide. Based on the peak dispersion of the spectrum (Fig. 6), monomeric Aβ42 adopted a random conformation, as expected [15]. The 2D 1H-15N HMQC NMR experiments were conducted on samples containing 75 μM Aβ42 titrated with tramiprosate to produce tramiprosate to Aβ42 ratios of 10:1, 100:1, 500:1, 1000:1, 3000:1, and 5000:1. Peak assignments of Aβ42 titrated with tramiprosate were then compared with 2D 1H-15N HMQC spectra of Aβ42 alone. When a 1000-fold excess of tramiprosate was added to the peptide solution, significant chemical shift perturbations were observed. No change was observed in the Aβ42 1H-15N HMQC spectrum at a ratio of 10:1 tramiprosate to Aβ42, but minor changes were observed at a ratio of 100:1. Significant changes in the 2D 1H-15N HMQC peaks began to arise at the ratio of 500:1, which plateaued (i.e., reached a steady state) at a ratio of 1000:1 tramiprosate to Aβ42. Further increases of the ratio to 3000:1 and 5000:1 had no effect on the chemical shift perturbation.

Fig. 6 2D 1H-15N heteronuclear multiple quantum correlation (HMQC) nuclear magnetic resonance (NMR) spectrum showing interactions of tramiprosate with amyloid beta Aβ42. a 2D 1H-15N HMQC NMR spectrum with assignments. Aβ42 alone is shown in blue, and Aβ42 with tramiprosate at a ratio of 1:1000 is overlaid in red. b An expanded view of part of a. Assignments in red indicate a significant observed chemical shift perturbation. c Example of a chemical shift perturbation of R5 residue observed when Aβ42 was incubated with tramiprosate at a ratio of 1:1000. Residue R5 is an isolated peak that clearly shows a chemical shift as the tramiprosate concentration is increased. The dotted lines illustrate the center of each peak to gauge the change in chemical shift at each concentration level. The color coding represents no tramiprosate (blue), 100:1 (gold), 500:1 (green) and 1000:1 (red) tramiprosate to Aβ42 Full size image

At a 1000-fold excess of tramiprosate over Aβ42, 22 Aβ42 residues showed significant chemical shift perturbations. The most dramatic changes were observed for R5, H6, S8, G9, Y10, K16, L17, V18, F19, N27, K28, and M35. The 2D 1H-15N HMQC peaks from these residues exhibited at least a 10 Hz chemical shift change in the 1H dimension, with K16 and K28 having chemical shift perturbations of 13.5 and 16.1 Hz, respectively, indicating a substantial interaction with tramiprosate. E3, V12, H13, H14, D23, S26, G25, G33, V36, and V39 showed smaller, yet still significant, chemical shift perturbations, indicating that they also interact with tramiprosate. Taken together, these results show that tramiprosate interacts with residues that span the length of Aβ42 in a concentration-dependent mode, which supports the IMS–MS data. Importantly, the strong tramiprosate binding to K16 and K28 supports tramiprosate-mediated disruption of the Lys28-Asp23 and/or Lys28-Glu22 salt bridges and suppression of neurotoxicity and misfolding [7, 27–29], given that these two lysine residues have been previously demonstrated to play a key role in mediation of these activities [8].

Molecular and Conformational Dynamics

Given the intrinsically disordered nature of Aβ42 and a high conformational dynamics, the interaction with tramiprosate is unlikely to be described by a static structural model with a single tramiprosate molecule bound. Hence, commonly applied structure-based drug-discovery approaches such as molecular docking are unlikely to provide a complete understanding of the MOA of tramiprosate. This represents a challenge to the characterization of the secondary structures of Aβ42 peptides because of their disordered nature and high aggregation propensity.

The characterization of free energy landscapes has been successful in rationalizing the conformational and folding behavior of such disordered proteins, and it provides a concrete representation of the conformational states of such proteins. A previous study [30] described changes in conformation from an α-helical structure to a disordered state, with portions of the peptide adopting a β-sheet structure described by a molecular dynamics simulation. To characterize the structure of Aβ42 alone and with different levels of excess tramiprosate, we performed a series of all atom molecular dynamics simulations. In the absence of tramiprosate, in water alone, Aβ42 adopted a number of very different conformations and was characterized by a disordered structure (Fig. 7a), which is in agreement with other published findings [30]. However, in the presence of increasing concentrations of tramiprosate, the peptide assumed a more conformationally stable form. The observed increase in conformational stability was concentration dependent. A two orders of magnitude molar excess of tramiprosate forced the protein to adopt a semi-cyclic conformation that was stabilized further by a salt bridge formed by the Asp1 N-terminal amino group and the C-terminal carboxylate of Ala42. This semi-cyclic conformation remained stable in the presence of tramiprosate. The Aβ42 was enveloped by multiple molecules of the drug, which interacted with many transient binding sites in a very dynamic manner. Figure 7b shows a molecular dynamics screenshot with six molecules of tramiprosate binding to a semi-cyclic Aβ42 conformer.

Fig. 7 Analysis of molecular dynamics simulations with and without 1% tramiprosate. a Representative disordered structure of amyloid beta Aβ42. b Representative Aβ42 semicyclic ordered structure with six tramiprosate molecules bound. c Principle component analysis of simulation of Aβ42 folding alone in water. d Principle component analysis of simulation of Aβ42 folding in the presence of 1% tramiprosate Full size image

To describe the large conformational changes observed in these simulations, we performed a PC analysis of the free energy surface. This analysis distills the complex motions of a flexible protein into the largest uncorrelated motions, or PCs. The first major motion (PC1) of Aβ42 can be described as a bending of the two helices towards each other like a hinge, and the second motion (PC2) can be described as a twisting of the two helices. Without tramiprosate, Aβ42 exhibited a typical trait of intrinsically disordered proteins: it lacked a narrow, well-defined energy minimum for any single folded structure (Fig. 7c). When PC1 and PC2 were mapped according to their free energy, a number of energy wells were observed (Fig. 7c), which correspond to the multiple Aβ42 conformations detected experimentally via IMS–MS. The 1% tramiprosate solution, corresponding to an Aβ42 : tramiprosate molar ratio of 1:250, stabilized the peptide in the semi-cyclic conformation; the energy surface as described by PC analysis showed stabilization of the semi-cyclic conformation as a well-defined energy well (Fig. 7d). This correlates well with the conformer stabilization detected by IMS–MS arrival time distribution (Fig. 3). The stabilization of a single conformation prevents Aβ42 from changing form and aggregating into pathogenic oligomers. Both in the stabilization of a single conformation and in the characterization of multiple transient tramiprosate binding sites, these results correlate with the IMS–MS experiments, where we detected up to 13 molecules of tramiprosate bound to Aβ42, in agreement with previous MS data [24, 31]. Interestingly, tramiprosate above 3 mM concentrations did not bind to plasma proteins from human, dogs, and rats in a standard plasma protein-binding study using an ultrafiltration technique [32], suggesting an absence of non-specific binding to plasma proteins such as albumin (data not shown).

Molecular dynamics simulations with free energy landscape analysis predicted a strong effect of tramiprosate on the intrinsically disordered conformations of Aβ42; the effect leads to a defined population of semi-cyclic conformers characterized as a stabilized energy well in the PC plot (Fig. 7d). Structurally, this conformation has a cyclic nature and retains more order than Aβ42 alone. Visual inspection of the simulations demonstrated the transient binding and unbinding of numerous tramiprosate molecules simultaneously along the Aβ42 surface. All of these interactions form a dynamic equilibrium, leading to a very tight conformer population. Taken together, these results suggest that tramiprosate stabilizes the semi-cyclic conformation of Aβ42 and prevents the formation of an initiation seed, thus preventing aggregation of the peptide (Table 3).

Table 3 Summary of the ion mobility spectrometry–mass spectrometry, nuclear magnetic resonance, and molecular dynamics data Full size table

Translational Analyses of Human Brain Drug Exposure vs. the Target

Tramiprosate was measured in the CSF specimens of patients with AD at week 78 from the phase III study [10, 22, 33], and its average concentration at the top tramiprosate dose of 150 mg twice a day (bid) was 60.4 nM (n = 11). Furthermore, based on the brain tissue/plasma exposure ratio derived from rodents and human plasma drug exposure at week 78 after tramiprosate 150 mg bid, we projected the steady-state tramiprosate concentration in brain parenchyma to be approximately 130 nM (Table 4).

Table 4 Steady-state plasma, cerebrospinal fluid, and brain drug exposures following oral administration of tramiprosate 150 mg twice daily in the phase III study Full size table

In an earlier phase II trial in patients with AD, tramiprosate produced a dose-related CSF Aβ42 reduction, suggesting target engagement [22]. In this study, the basal mean CSF concentration of Aβ42 was 179 ± 101 pg/ml (i.e., 0.04 nM, n = 46; Table 5); this concentration aligns with the reported CSF Aβ42 levels that ranged from 144 to 500 pg/ml [4, 34–36] corresponding to 0.035–0.1 nM, in patients with AD (n = 100) [34] or prodromal/early-stage AD (n = 100) [35, 36], as measured by ELISA or MS. Furthermore, brain Aβ42 measures vary in the AD literature, but the reported microdialysis studies in humans have shown that brain interstitial soluble Aβ42 are approximately equivalent to CSF Aβ42 levels [37, 38], and, therefore, the latter can be used as a suitable surrogate for brain pharmacokinetic–pharmacodynamic analyses. Thus, when comparing the ratio of brain tramiprosate : Aβ42, there is an approximately 1300- to 3700-fold excess of tramiprosate over soluble Aβ42 at the steady state based on tramiprosate measured in the brain from patients with AD (Table 4), sufficient to exert a full therapeutic effect of tramiprosate. This analysis fully aligns with the molecular stoichiometry as characterized by the the IMS–MS, NMR, and molecular dynamics approaches.