Steroid-Quinoline Hybrids for Disruption and Reversion of Protein Aggregation Processes

Abstract

Reversing protein aggregation within cells may be an important tool to fight protein-misfolding disorders such as Alzheimer's, Parkinson's, and cardiovascular diseases. Here we report the design and synthesis of a family of steroid-quinoline hybrid compounds based on the framework combination approach. This set of hybrid compounds effectively inhibited Aβ1-42 self-aggregation in vitro by delaying the exponential growth phase and/or reducing the quantity of fibrils in the steady state. Their disaggregation efficacy was further demonstrated against preaggregated Aβ1-42 peptides in cellular assays upon their endocytosis by neuroblastoma cells, as they reverted both the number and the average area of fibrils back to basal levels. The antiaggregation effect of these hybrids was further tested and demonstrated in a cellular model of general protein aggregation expressing a protein aggregation fluorescent sensor. Together, our results show that the new cholesterol-quinoline hybrids possess wide and marked disaggregation capacities and are therefore promising templates for the development of new drugs to deal with conformational disorders.

Figure 1

Framework combination to prepare “angular” and “linear” quinoline–steroid hybrids.

Scheme 1. Synthesis of Hybrid Compounds 4a–d and 6a–d and (inset) Crystal Structure of 4c

Reagents and conditions: (i) Al(OⁱPr)₃, cyclohexanone, toluene, reflux, 12 h; (ii) 3a–d, p-TsOH, EtOH, MW, 100 °C, 15 min; (iii) PCC, CH₂Cl₂, rt, 24 h.

Figure 2

Influence of compounds 4a–d and 6a–d (20 μM) on fibrillation of Aβ1–42 peptides (10 μM) monitored by ThT fluorescence. Data are presented as the mean of three experiments (n = 3).

Figure 3

Evaluation of compounds’ disaggregation effect in SH-SY5Y cells incubated with preaggregated Aβ1–42. (A) Numbers of protein aggregate foci per cell. (B) Average areas of protein aggregate foci per cell. Both control and Aβ-exposed cells were incubated in the presence of 0.5% EtOH (compound vehicle). Data are presented as mean ± SD (10–20 fields of view, n = 2, average of ∼370 cells analyzed per condition). Significance was determined using the unpaired Student’s t test: (+) for comparison between positive (Aβ) and negative (C) control conditions and (*) for comparison between Aβ and Aβ + compounds. +/*, p < 0.05; ++/**, p < 0.01; +++/***, p < 0.001; ++++/****, p < 0.0001. (C) Micrographs of cells incubated with Aβ1–42 alone (left) or in combination with compound 4c (center) or 6c (right).

Figure 4

Analyses of the disaggregation capacities of compounds 4a–d and 6a–d under conditions of general protein aggregation. (A) Numbers and (B) average areas of green-fluorescing foci of protein aggregates per cell. Control and NTB-exposed cells were grown in the presence of 0.5% EtOH (compound vehicle). Data are presented as mean ± SD (n = 4–10, average of a total of ∼4700 cells analyzed per condition). Significance was determined using the unpaired Student’s t test: (+) for comparison between positive (NTB) and negative (C) control conditions and (*) for comparison between NTB and NTB + compounds. +/*, p < 0.05; ++/**, p < 0.01; +++/***, p < 0.001; ++++/****, p < 0.0001. (C) Micrographs of representative cells of compounds 4c and 6c. Aggregates are in green (HSP27:GFP fusion protein) and cell nuclei in blue (DAPI).