Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies

Abstract

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.

Sporadic inclusion-body myositis: A degenerative muscle disease associated with aging, impaired muscle protein homeostasis and abnormal mitophagy

“…Fluorescent amyloid ligands termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusions in s-IBM muscle fibers [33]. Their use requires fluorescence microscopy, and they are not yet commercially available, to our knowledge.…”

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™stains

“…Specifically, we used Amytracker™ 480 (related to HS163) and 680 (related to HS169) which are fluorescent amyloid ligands, also termed luminescent conjugated oligothiophenes (LCOs) [72,84]. These two fluorescent markers bind rapidly and with high sensitivity to detect protein amyloid formation in fibrin(ogen).…”

“…In particular, Nilsson and colleagues have developed a number of novel fluorescent amyloidogenic markers. Some of these are referred to as luminescent conjugated oligothiophenes (LCOs) [68][69][70][71][72][73][74][75][76] and are marketed as Amytracker™ 480 and 680 (derived, respectively, from HS163 and hFTAA HS169 in the literature [71,74]), but proprietary structures that are not identical to them; (see Figure 1 for the chemical structures of HS163 and HS169). Although they too show strong selectivity for, and enhanced fluorescence when binding to, classical amyloid proteins, their staining properties clearly differ from those of ThT [68,70,[77][78][79].…”

APTES mediated modular modification of regenerated silk fibroin in a water solution

“…It should be noted that hydrophobic interactions with amyloid proteins have been previously reported for oligothiophene compounds. 26 However, when the SF doped samples and SF-APTES-T3 were washed with DMSO in Amicon-ultra centrifugal filter with a cutoff 100 KDa (thus able to retain SF, MW 350KDa) and centrifuged, while for the doped samples the fluorescence was observed on both the filtered SF and in the washing solution, for SF-APTES-T3 sample fluorescence was observed only in the retained SF phase. This evidence suggest the occurrence of strong interactions between SF and oligothiophenes but also unequivocally confirms that only by using the APTES linker the oligothiophene fluorophore can be stably grafted on SF.…”

“…1 H-NMR spectra were recorded at a field of 14.4 T (600 Mhz for 1 H) by dissolving a small portion of the dried film in D 2 O, and using a indirect triple resonance probe for maximum sensitivity. PGSE spectra were recorded using the BPP-STE pulse 26 sequences using an array of gradient strengths (1-65 G/cm) and a 200 ms diffusion delay. 512 to 1024 scans were recorded for each 1 H spectrum.…”