Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery

Abstract

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 cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons and stem cell-derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors.

Keywords::

• botulinum neurotoxin
• cell-based assays
• drug discovery
• ELISA
• embryonic stem cells
• high-content imaging
• high-throughput screening
• motor neurons
• MSD
• phenotypic screening

Acknowledgements

The authors thank LM Wanner, GY Gomba and HT Du for their assistance with figures.

Disclaimer

The views, findings, interpretations and recommendations are those of the authors and are not necessarily endorsed by the US Department of Health and Human Services or the US Army.

Financial & competing interests disclosure

This work was supported by grants from the Defense Threat Reduction Agency and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05). KP Kota is an employee of PerkinElmer, the manufacturer of the Opera Confocal Microscope. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Pluripotent human embryonic stem (ES) and induced pluripotent stem cells (iPS) cells possess self-renewal capacity and the ability to differentiate into motor neurons, the natural target of botulinum neurotoxins (BoNTs) and thereby offer a unique renewable cell source for BoNT studies.

• Novel technologies combining physiologically relevant cell-based assays and high-throughput screening (HTS) offer improved opportunities for identifying drug candidates to treat botulism (for which there is currently no therapeutic available for treating post-neuronal intoxication).

• Human motor neuron-based assays can be utilized to determine the mechanism of action of identified lead compounds.

• Physiologically relevant human motor neuron systems can be also utilized to increase our understanding of host cellular pathways involved in either BoNT intoxication and/or recovery, which is critical for developing novel methods to treat botulism.

• It is well established that BoNT serotypes A, B and E are responsible for the majority of human botulism cases, therefore drug screening efforts should be designed to identify compounds that can inhibit multiple serotypes.

• Immunoassays that quantify synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage are sensitive and specific means of evaluating BoNT activity in cellular environments and can be used to drive both molecular (direct) and empirical (indirect) BoNT/A screening approaches.

• The implementation of high-throughput homogenous assays may accelerate the discovery and development of effective BoNT/A inhibitors.

Notes