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Posters

Different neurotransmitters and drugs can produce very different effects on the same neuron, even though they all signal through an increase in cAMP. There are now biophysical measurements and models that help explain how this occurs.

• The timing (dynamics) of GPCR signaling impacts the physiological function and therapeutic activity of ligands activating the receptors.
• Bright fluorescent biosensors enable continuous recording of GPCR signaling in live cells, with high read frequency (seconds) for long durations (hours)
• Kinetic parameters can be extracted from the waveform data by curve fitting using a plug-in for the popular program GraphPad Prism
• This platform is applied to quantify the kinetics of partial agonism and bias at the μ-opioid receptor, and the dynamics of arrestin recruitment to multiple GPCRs.

• Signaling dynamics is a new frontier in drug discovery, enabling drugs to signal at the right time and in the right place.
• Signaling dynamics can be measured routinely in high throughput using biosensors, which enable continuous detection of signaling molecules over time, and the FDSS/µCell, which enables high frequency simultaneous reads of entire microtiter plates.

Montana Molecular has developed biosensors of GPCR signaling with unprecedented performance, enabling sub-second read frequency, total read times of over five hours, and miniaturization to 384-well format, for Gs, Gi, Gq and arrestin pathways. Signaling dynamics are quantified using simple curve fitting methods using a free plug-in to the commonly-used program GraphPad Prism. Here these technologies are applied to quantify the signaling and desensitization dynamics of endocrine and metabolic GPCRs, to provide high quality, actionable results to advance research and drug discovery programs.

Signaling dynamics is a new frontier in GPCR drug discovery, involving the design of drug molecules that signal for the right time in the right place. Montana Molecular has developed biosensors of GPCR signaling with unprecedented performance, enabling sub-second read frequency, total read times of over five hours, and miniaturization to 384-well format, for Gs, Gi, Gq and arrestin pathways.

• The timing (kinetics) of GPCR signaling impacts the physiological function and therapeutic activity of ligands activating the receptors.
• Bright fluorescent biosensors enable continuous recording of GPCR signaling in live cells, with high read frequency (seconds) for long durations (hours).
• Kinetic parameters can be extracted from the waveform data by curve fitting using a plug-in for the popular program GraphPad Prism.
• This platform is applied to quantify the kinetics of partial agonism and bias at the μ-opioid receptor, and the dynamics of arrestin recruitment to multiple GPCRs

• GENIe is a bright sensor compatible with standard fluorescent plate readers that produces a robust assay.
• GENIe can be paired with R-cADDis to compare cGMP and cAMP concentrations in living cells on a fluorescence plate reader.
• GENIe can detect the action of PDE inhibitors
• A comparison of the GENIe and cADDis sensors reveals the specificity of PDE inhibitors.

Montana Molecular has developed a new suite of fluorescent probes that deliver bright cell painting tools that can be visualized in real-time in living cells producing a wealth of kinetic data to inform drug discovery and disease research.

The Montana Paint Box can:

• Use live cell imaging, no fixation or staining required
• Quantify drug effects on subcellular compartments
• Monitor metabolic activities
• Identify disease-related phenotypes
• Produce robust signal that can be measured using standard fluorescent microscopes and automated plate readers

Biosensors for G-protein signaling & arrestin recruitment

• Genetically-encoded fluorescent biosensors enable continuous recording of signaling kinetics in live cells
• Gs, Gi, Gq and arrestin
• Easy workflow – add ligand and read in plate reader

The SARS-CoV-2 main protease (Mpro) remains a critical target for antiviral therapeutics despite vaccine availability. While Nirmatrelvir (PaxlovidTM) showed early promise, emerging mutations in Mpro pose significant challenges. Clinical data from Pfizer’s EPIC-HR trials and surveillance of the GISAID database have identified multiple treatment-emergent substitutions, particularly around the S1-S4′ subsites, that confer resistance while maintaining enzymatic function. Current methods for screening Mpro inhibitors face key limitations. Biochemical assays, while efficient, fail to capture cellular complexity and transport dynamics. BSL3 viral assays provide definitive results but are resource-intensive and low-throughput. A critical gap exists for assays that can demonstrate target engagement in living cells while maintaining safety and scalability.

BrainXell iPSC-derived neurons:

• iPSC-derived neurons are increasingly valuable in drug discovery because they can model human-specific genetics and disease phenotypes.
• BacMam vectors are commonly used for rapid gene expression in transformed cell lines, but their optimal use in iPSC-derived neurons is not fully understood.
• We investigated BacMam delivery timing, vector efficacy, and the necessity of HDAC inhibitors in BrainXell iPSC-derived glutamatergic cortical neurons (catalog #BX-0300-30).

We found that:

• Transduction was effective post-seeding at any time over four weeks, and HDAC inhibitors were not necessary.
• Montana Molecular’s BacMam Big Sky vector showed optimal long-term expression and cell health over three weeks in living neurons.
• Cell painting tools showed no mislocalization.
• Montana Molecular’s fluorescent sensors and cell painting tools can be introduced at different iPSC differentiation stages, enabling continuous monitoring of cellular dynamics.
• The biosensors provide for real-time tracking of cAMP, DAG, calcium dynamics, membrane potential, and protein-protein interactions in living neurons.

• Signaling dynamics is a new frontier in drug discovery, enabling drugs to signal at the right time and in the right place.
• Signaling dynamics can be measured routinely in high throughout using biosensors, which enable continuous detection of signaling molecules over time, and the FDSS/µCell, which enables high frequency simultaneous reads of entire microtiter plates.
• However, for time course data to be useful for drug discovery, drug parameters need to be extracted from the data by curve fitting to drive optimization of new molecules.
• Here we present a simple curve fitting platform for analyzing time course data that operates as a plug in to the program GraphPad Prism and apply it to measure signal generation and desensitization rates for a variety of receptor systems.