The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry-mass spectrometry (IMS-MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS-MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.

/pdf/screening-and-classifying-small-molecule-inhibitors-of-4ixa2wgpax.pdf

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry

Neurodegenerative disorders and type 2 diabetes are global epidemics compromising the quality of life of millions worldwide, with profound social and economic implications. Despite the significant differences in pathology – much of which are poorly understood – these diseases are commonly characterized by the presence of cross-β amyloid fibrils as well as the loss of neuronal or pancreatic β-cells. In this review, we document research progress on the molecular and mesoscopic self-assembly of amyloid-beta, alpha synuclein, human islet amyloid polypeptide and prions, the peptides and proteins associated with Alzheimer's, Parkinson's, type 2 diabetes and prion diseases. In addition, we discuss the toxicities of these amyloid proteins based on their self-assembly as well as their interactions with membranes, metal ions, small molecules and engineered nanoparticles. Through this presentation we show the remarkable similarities and differences in the structural transitions of the amyloid proteins through primary and secondary nucleation, the common evolution from disordered monomers to alpha-helices and then to β-sheets when the proteins encounter the cell membrane, and, the consensus (with a few exceptions) that off-pathway oligomers, rather than amyloid fibrils, are the toxic species regardless of the pathogenic protein sequence or physicochemical properties. In addition, we highlight the crucial role of molecular self-assembly in eliciting the biological and pathological consequences of the amyloid proteins within the context of their cellular environments and their spreading between cells and organs. Exploiting such structure–function–toxicity relationship may prove pivotal for the detection and mitigation of amyloid diseases.

Implications of peptide assemblies in amyloid diseases

Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases

The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

/pdf/ion-mobility-spectrometry-mass-spectrometry-defines-the-rplr4ei3m2.pdf

Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2013.01.046

Islet amyloid: From fundamental biophysics to mechanisms of cytotoxicity

Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide

The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of type 2 diabetes mellitus (T2DM). Salvia miltiorrhiza (Danshen), one of the most commonly used of traditional Chinese medicines, is often used in Compound Recipes for treating diabetes, however with unclear mechanisms. Since salvianolic acid B (SalB) is the most abundant bioactive ingredient of salvia miltiorrhiza water-extract. In this study, we tested whether SalB has any effect on the amyloidogenicity of hIAPP. Our results clearly suggest that SalB can significantly inhibit the formation of hIAPP amyloid and disaggregate hIAPP fibrils. Furthermore, photo-crosslinking based oligomerization studies suggest SalB significantly suppresses the toxic oligomerization of hIAPP monomers. Cytotoxicity protection effects on pancreatic INS-1 cells by SalB were also observed using MTT-based assays, potentially due to the inhibition on the membrane disruption effects and attenuated mitochondria impairment induced by hIAPP. These results provide evidence that SalB may further be studied on the possible pharmacological treatment for T2DM.

Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptide and protects pancreatic beta-cells against cytotoxicity.

The invention belongs to the field of medicines. Danshinolic acid B has functions of inhibition of islet amyloid polypeptide aggregation and treatment of diabetes. The in-vivo islet amyloid polypeptide aggregation can destroy a beta-cell membrane structure and a normal form function of mitochondria to cause beta-cell apoptosis and damage a beta-cell function, thereby being taken as an important nosogenesis of type-2 diabetes. By danshinolic acid B, the aggregation of the islet amyloid polypeptide can be inhibited, and the process of damaging the beta-cell membrane structure and the normal form function of mitochondria during aggregation is changed over to protect the beta-cells, so that danshinolic acid B can be used for preventing and treating the diabetes.

Xiaochao Li

Papers

Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide

Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptide and protects pancreatic beta-cells against cytotoxicity.

Application of danshinolic acid B in inhibition of islet amyloid polypeptide aggregation and treatment of diabetes