Amyloid β-protein assembly: The effect of molecular tweezers CLR01 and CLR03

Abstract

The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer's disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment.

Figure 1

Different binding effects of CLR01 and CLR03 on Aβ42. (a, b) Molecular structures of CLR01 and CLR03 compounds; (c–e) mass spectra of Aβ42 samples: (c) 1:10 mixture of Aβ42 and CLR01; (d) 1:10 mixture of Aβ42 and CLR03; (e) Aβ42 alone. Each species is noted in brackets where the first number is the number of Aβ42 molecules and the second number represents the number of bound small molecules. The charge is noted as a superscript.

Figure 2

Effects of low concentration CLR01 on Aβ42 early oligomerization. ATDs of (a) z/n = −5/2 Aβ42 (m/z = 1805) in an Aβ42 sample without CLR01; (b) z/n = −5/2 Aβ42 and CLR01 complex (m/z = 1950); and (c) z/n = −5/2 Aβ42 and CLR01 complex (m/z = 2096) in a 1:1 mixture of Aβ42 and CLR01. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section, and the oligomer order (n) is noted for each feature. (d) A mass spectrum of the 1:1 mixture of Aβ42 and CLR01 is shown as an example. Each species is noted in brackets where the first number is the number of Aβ42 molecules and the second number represents the number of bound CLR01 molecules. The charge is noted as a superscript. The dashed line represents the theoretical position for the uncomplexed −5/2 peak. Mass spectra of mixtures of Aβ42 and CLR01 with different ratios are shown in Figure S2 (Supporting Information). (e) Cross sections of dimer and tetramer in the uncomplexed or CLR01-complexed −5/2 Aβ42. The error for the cross sections reported here is between 0 and 1%.

Figure 3

CLR01 remodels the early oligomerization of Aβ42. (a) Mass spectrum of Aβ42 alone with ∼4 h of incubation on ice; (b) mass spectrum of the Aβ42 sample immediately after the addition of 1:1 CLR01. Each species is noted in brackets where the first number is the number of Aβ42 and the second number represents the number of bound CLR01 molecules. The charge is noted as a superscript. (c) ATD of the z/n = −5/2 Aβ42 peak for the Aβ42 in the absence of CLR01 after ∼4 h of incubation on ice. (d) ATD of the z/n = −5/2 Aβ42 peak after addition of 1:1 CLR01. (e and f) ATDs of −5/2 Aβ42 oligomer complexes after addition of 1:1 CLR01 to the preaggregated Aβ42 sample. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section, and the oligomer order (n) is noted for each feature.

Figure 4

Effects of low concentration CLR01 on Aβ40 oligomerization. (a) ATD of z/n = −5/2 Aβ40 (m/z = 1731) for Aβ40 alone; (b and c) ATDs of z/n = −5/2 Aβ40 and CLR01 complexes (m/z = 1876 and 2021) in the 1:1 mixture of Aβ40 and CLR01. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section, and the oligomer order (n) is noted for each feature. Note the ATDs with CLR01 bound, panels b and c, are significantly narrower than wild type, panel a.

Figure 5

Time-dependent ion mobility study of the effects of CLR03 on Aβ42 early oligomerization. (a–c) ATDs of the −5/2 Aβ42 peak (m/z = 1805) for the 1:10 mixture of Aβ42 and CLR03 at different time points; (d–f) ATDs of the −5/2 Aβ42 peak (m/z = 1805) for Aβ42 alone at different time points. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section. The oligomer order (n) and cross section are noted for each feature.

Figure 6

CLR03 facilitates Aβ42 oligomer formation: (a) ATD of the z/n = −3 peak for Aβ42 alone, (b and c) ATDs of z/n = −3 Aβ42 without and with CLR03 bound for a 1:10 mixture of Aβ42 and CLR03, (d) ATD of the z/n = −4 peak for Aβ42 alone, (e and f) z/n = −4 Aβ42 without and with CLR03 bound for a 1:10 mixture of Aβ42 and CLR03. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section. M₁ and M₂ represent two conformations of the Aβ42 monomer. The arrows indicate the formation of oligomers (n ≥ 2) in the mixture of Aβ42 and CLR03.

Figure 7

CLR03 facilitates Aβ40 assembly. (a, b) ATDs of the −5/2 Aβ40 peak (m/z = 1731) for the Aβ40 samples in the absence or presence of CLR03. Each ATD is fit with multiple features using the procedure described in the Experimental Methods section, and the oligomer order (n) is noted for each feature. (c) Cross sections of −5/2 Aβ40 oligomers for samples of Aβ40 in the absence or presence of CLR01 or CLR03. The error for the cross sections reported here is between 0 and 1%.

Figure 8

Different effects of CLR01 and CLR03 on Aβ early oligomerization. Oligomerization of (a) Aβ42 wild type, (b) Aβ42 with the presence of CLR01, (c) Aβ40 wild type, and (d) Aβ40 with the presence of CLR03. Aβ42 and Aβ40 are represented with blue and red balls, respectively. CLR01 and CLR03 molecules are noted as X and Y.