Biological Assay for Next Generation Analgesics (BANGA)

In the field of pain drug discovery, failure to develop new analgesic drugs is thought to stem from the use of cellular and animal models that don’t correspond well to the human disease (Pankevich et al., 2014). Drugs against validated genetic targets, such as SCN9A (NaV 1.7) and Tachykinin NK1, showed efficacy in pre-clinical models yet failed to produce pain relieving effects in human clinical trials (Yekkirala et al., 2017). Historically, drug discovery uses recombinant lines or animal cells that poorly correlate to human physiology which is likely a factor in the clinical failures (Hyman, 2012).

Therefore, we designed Biological Assay for Next Generation Analgesics (BANGA), a phenotypic approach using human stem cells (hiPSC, and/or directly converted neurons) to generate human cells involved in pain signaling such as sensory neurons; and reward and addiction such as dopaminergic (DA) neurons. Unique to our approach, cells are derived using methods that rely on reprogramming small molecules, specific extracellular matrix (ECM) components, and other factors that collectively recreate the native microenvironment niche in culture. In practice, this stabilizes the developmental potential of reprogrammed cells generated from patient blood or tissue sample. By contrast, genetic vector-based iPSC (and other reprogramming) methods have significant variability (i.e., not reproducible) and tend not to derive functionally mature neuronal cells (Farkhondeh et al., 2019).

BANGA includes HTS, HCS and follow up assays to support early drug discovery and later optimization phases. An unparalleled feature of BANGA is that compounds are also screened against DA neurons and other neurons of the Ventral Tegmental Area (VTA), thus identifying risk for addiction at an early stage. This novel approach, specifically aimed at discovery of nonaddictive analgesics can also be used to develop  clinical biomarker(s) as all neurons used in BANGA are personalized and patient specific. This type of clinical biomarker(s) could revolutionize clinical trials of future analgesics. A central tenant of the assay design is to use physiologically relevant assay parameters in 2D and 3D formats such that authentic, in-vivo like conditions and assay results are generated. This approach is a dramatic shift from the commonly used target-based assays and cell-based assays using recombinant lines or animal cells. These have historically failed to deliver alternative or improved analgesics (Pankevich et al., 2014; Taneja et al., 2017; Yekkirala et al., 2017).

We have developed a prototype 2D format of BANGA in 96-well microplates using iPSC-derived sensory neurons. This prototype assay takes advantage of neuronal hyperexcitability, veratridine-evoked depolarization of the nociceptors, and various fluorescent dye imaging (compatible with most high content screening analysis systems) and HPLC quantitation of dopamine released by DA neurons. ^  We then used this pilot assay to screen a panel of analgesic compounds. For addiction risk proof-of-principle, “VTA like assembloids” generated from dopaminergic, gabaergic, glutamatergic, and glial cells differentiated from iPSC and directly converted neurons were explored. Typical  results of this prototype of BANGA are shown: