Small molecule ubiquitin-proteasome system inhibitor library screen reveals botulinum neurotoxin type a inhibitors

Şen, Edanur
Botulinum neurotoxins (BoNTs), known as the most potent bacterial toxins, cause potentially lethal disease botulism. BoNTs are classified as category A bioterror agents by many countries because they are easy to produce and have extreme toxicities. On the other hand, BoNTs are considered a therapeutic marvel in medicine because they have been extensively utilized not only for cosmetic purposes to get rid of wrinkles but also to treat various conditions in clinics, including movement disorders. These toxins specifically target cholinergic nerve terminals and block acetylcholine release by cleaving Soluble N-ethylmaleimidesensitive Factor Attachment Protein Receptor (SNARE) complex proteins, which are crucially important for neuroexocytosis. There are eight serologically distinct serotypes of BoNTs (BoNT/A–G, BoNT/X); however, only serotypes A, B, E, and F lead to human botulism. Among these, BoNT/A is the most widely used serotype in clinics and also the most studied serotype as more than half of botulism cases are due to BoNT/A poisoning. BoNT/A has the longest-lasting effect as compared to other serotypes. For example, both BoNT/A and BoNT/E serotypes cleave the same protein, SNAP-25, and the half-life of BoNT/E is limited to days in cells while BoNT/A can be active for up to 6 months in the neuronal cytosol. The underlying mechanisms for the half-life differences between different serotypes have not been well understood. However, there has been heightened research interest in understanding how BoNT/A manages to escape destructive protein degradation machinery in neurons. Recent studies have identified the E3 ligases and deubiquitinases (DUBs) critical for the destruction of BoNT/A. More specifically, one specific E3 ligase, HECTD2, leads to ubiquitination of the enzymatic component of BoNT/A Light Chain (LC) in cells, but the dominant DUB activity of VCIP135 inhibits the proteolytic degradation of the LC. In addition to this, another DUB, USP9X, indirectly affects the stability of BoNT/A in cells. Therefore, the persistence of BoNT/A in the cell can be potentially modified by affecting these factors using small molecule modulators. Modulation of BoNT half-life in cells by small molecules can be important for research purposes to understand intoxication/recovery mechanisms, as well as for the generation of effective countermeasures against botulism. In this study, the main goal was to screen a focused ubiquitin-proteasome system (UPS) inhibitor library to reveal compounds modulating BoNT/A activity and half-life in cells. Our screen utilizing mouse embryonic stem cell-derived motor neurons identified 10 potential lead compounds affecting BoNT/A mediated SNAP-25 in neurons. Then, tested dosedependent effects of the selected compounds and further tested their potential toxic effects in the cell. Following, we explored the effects of the lead compounds on the stability of LC in cells. Small molecules WP1130, b-AP15, NSC632839, PR-619, Celastrol, MDBN, PYR-41, and SL-01 exhibited efficacy against BoNT/A LC action and half-life in cells. Among these, PR-619 appears to be highly crucial as it is an inhibitor of VCIP135 that has been identified as a crucial DUB affecting the half-life of BoNT/A LC. To the best of my knowledge, this is the first study in the literature showing that UPS targeting small molecules can modulate BoNT/A LC action and half-life in cells.


Microbial detoxification of groundnut meal naturally contaminated with aflatoxin using rhodococcus erythropolis
Doğan, Önay Burak; Çekmecelioğlu, Deniz; Bozoğlu, Faruk; Department of Food Engineering (2015)
Aflatoxins are highly mutagenic toxins with carcinogenic effects produced as secondary metabolites by fungal species Aspergillus flavus and Aspergillus parasiticus under certain conditions. Chronic or acute consumption of aflatoxins found in food and feed products possesses great health risks. It is particularly an important problem in animal feed from food waste and by-products. Therefore there is growing need to eliminate aflatoxins from contaminated products. In this study, first the optimum growth condi...
Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery.
Kiriş, Erkan; Burnett, JC; Soloveva, V; Kane, CD; Bavari, S (2014-03-01)
Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and costefficient manner. Consequently, cell-based systems including immortalized cells, primary neurons, and stem-cell derived neurons h...
In vivo interaction of carcinogenic acrylamide with cytochrome p450 isozymes and phase II enzymes in rabbit liver, kidney and lung
Nuyan, Mine; Arınç, Emel; Department of Biochemistry (2008)
Acrylamide is an industrially produced chemical with known neurotoxic, reproductive toxin and carcinogenic effects. The carcinogenicity associated with acrylamide is mostly attributed to its metabolism by liver CYP2E1. However, studies investigating the effects of acrylamide on CYP2E1 enzyme are limited. In this study, it was aimed to investigate in vivo interaction of carcinogenic acrylamide on microsomal cytochrome P450 enzyme activities, and protein levels, and on cytosolic NQO1 and GST enzyme activities...
Reconstruction of the temporal signaling network in salmonella infected human cells
Budak, Güngör; Aydın Son, Yeşim; Tunçbağ, Nurcan; Department of Bioinformatics (2016)
Salmonella enterica is a bacterial pathogen whose mechanism of infection is usually through food sources. The pathogen proteins are translocated into the host cells to change the host signaling mechanisms either by activating or inhibiting the host proteins. In order to obtain a more complete view of the biological processes and the signaling networks and to reconstruct the temporal signaling network of the human host, we have used two network modeling approaches, the Prize-collecting Steiner Forest (PCSF) ...
Development of a new immobilization procedure for detection of stphylococcal enterotoxin B (SEB) and Candida Albicans
Ertürkan, Deniz; Özgen, Canan; Külah, Haluk; Department of Chemical Engineering (2012)
Fast and accurate detection of pathogens such as bacteria, their toxins and viruses at low concentrations is very important. The conventional techniques are time consuming where expensive equipment is required with a consumption of excess amount of blood from patients. Recently, immunosensors are used for the detection of pathogens because they are miniature, sensitive, biocompatible and require low power. According to the Centers for Disease Control and Prevention (CDCP), 76 million people become ill due t...
Citation Formats
E. Şen, “Small molecule ubiquitin-proteasome system inhibitor library screen reveals botulinum neurotoxin type a inhibitors,” M.S. - Master of Science, Middle East Technical University, 2021.