Montana Molecular

Two new posters from Merrilee Thomas, PhD and colleagues at Montana Molecular from the Society for Neuroscience 2025 meeting! (Link to PDF poster files in the Comments.) In the first, our new live cell painting tools are applied to neurons and glia (see picture). We developed bright fluorescent cell painting tools, delivered in next-generation BacMam vectors, that can be visualized in real-time in living cells producing a wealth of kinetic data to inform drug discovery and disease research. Using BrainXell’s iPSC-derived cortical glutamatergic neurons, astrocytes, and microglia we can identify numerous organelles and proteins, including: • Mitochondria • Nucleus and Nucleoli • F-Actin • Microtubules • Omegasomes • Endosomes • Lysosomes • Tau • Golgi In the second poster, a new TRPML1 activity sensor is described. This Ca2+ permeant channel plays a critical role in maintaining Ca2+ homeostasis in the lysosome. TRPML1 dysfunction leads to neurodegeneration resulting from impaired lysosomal activity and Ca2+ dyshomeostasis. Our new sensor enables direct measurement of Ca2+ release with high specificity. The assay provides a powerful approach for interrogating TRPML1 function in human cells and for identifying modulators of lysosomal Ca2+ signaling. Please get in touch with us to talk more about these new products!

You may be thinking, why should we be interested in signaling kinetics? Here's an answer! A video seminar from Sam Hoare at Montana Molecular, "Translational impact of GPCR signaling kinetics on the clinical pharmacology of therapeutics." The video provides an in-depth modern and historical perspective on GPCR signaling kinetics from bench to bedside. Understanding, measuring and applying signaling kinetics to drug discovery will aid the discovery of new and improved medicines. The goal here is to provide scientists the background necessary to apply this fundamental GPCR science to drug development. The link to the video is in the comments. Much of the beautiful signaling kinetic data were generated by Montana Molecular's CRO team, Luciana Leo, Anastasia Schultz and Katie Babin. We can provide you with impressive quality GPCR signaling data in support of your drug discovery projects. Please enjoy the video and feel free to leave comments here!

Dualling SfN posters by Merrilee Thomas, PhD tomorrow: Tues. Nov 18th 8am-10am - LP027.13 TLC for the Ca2+ Dyshomeostasis Neurodegeneration Model - A fluorescent biosensor, enables visualization of changes in TRPML1 activity in response to drug treatment. Capturing differences between agonists in living cells over time is the key to evaluating and prioritizing candidate drugs. #sfn2025 Tues. Nov 18th 1pm-5pm - PSTR367.03 Using Next Generation Viruses to Interrogate iPSC-derived Neurons & Microglia - Optimal gene delivery with unlimited cargo size to iPSC-derived microglia and neurons, and undifferentiated iPSCs has arrived. Jean-Francois Rolland Rishi Rakhit Martin Kampmann Derek Hernandez Nicholas Hertz, Ph.D R Scott Struthers Ardem Patapoutian James Summers Kate Herrera, Ph.D. Eric Karran Christina Thapa, PhD Greg Lemke Amanda McQuade Yamina Berchiche

Here's a dramatic example of the difference between endpoint and kinetic assays. At top is a bar graph of cAMP data at 45 minutes for three GLP-1 agonists at equally effective concentrations. At bottom is the time course data from which the bar graph was created, measured using Montana Molecular's cADDis cAMP biosensor. The time course reveals profound mechanistic differences between GLP-1 agonists. GLP-1 (7-36) stimulates a rapid burst of signaling, followed by a rapid decline and then a slower decay (likely resulting from receptor desensitization mechanisms). Orforglipron, the nonpeptide, more slowly stimulates cAMP but the response is completely sustained. for 12 hours, reflecting the absence of receptor desensitization demonstrated for this molecule. Danuglipron, a different class of nonpeptide, produces a rapid burst of cAMP followed by a sustained lower level of cAMP generation. These mechanistic insights could potentially translate to therapeutic efficacy. They are revealed in the kinetic assay but are completely concealed in the endpoint assay. At Montana Molecular we specialize in mechanism of action pharmacology. Our CRO team can run these kinetic assays to test your molecules of interest, or we can provide you the biosensors. We would be delighted to chat through your project requirements - get in touch!

Kinetic continuous read assays are great because we can change the conditions over time in the same assay. This is super-helpful with compound profiling (is the compound an inhibitor, activator, allosteric modulator?) and with kinetic mechanism of action studies (washout, adding inhibitors and so on). In one experiment we can get information that would take several experiments using single-time-point endpoint assays. Ideal for this are the Montana Molecular, LLC biosensors like cADDis (for cAMP) and Borealis (for arrestin), where the signals are stable for many hours. This enables reliable multi-step signaling assays that can quickly provide information on compound activity and reveal new insight into the compound’s mechanism of action. We provide the sensors for you to do these experiments, and also provide a CRO service to test your compounds. The figure shows how this can be applied to identify compound mechanisms in a single assay, from inverse agonism (panel A) to full agonism (D). The test compound is applied first and activity measured for 30 minutes. In this phase we can identify any agonist or inverse activity. After this the reference agonist is added (EC80 in A-D and EC20 in E). In this second phase we can see how test compound affects the reference agonist, so we can see antagonism (A,B) or allosteric enhancement (E). We have also done complex multistep assays over 12 and even 24 hours to evaluate mechanisms of activity, including several additions and subsequent testing of medium from the cells. This is just one example of how signaling kinetics can be evaluated using our continuous read assays. We have made videos on the numerous benefits of kinetic assays (see Comments). Please contact us for more information or a consultation!

Long lasting and wash resistant signaling by orforglipron (1), the nonpeptide GLP-1 agonist. In this study from Montana Molecular, LLC, we discovered that cAMP signaling by the molecule was completely resistant to extensive washout. Other compounds were tested in this assay and the results are available (2, links in Comments). In the experiment, HEK cells expressing the GLP-1 receptor were exposed to either orforglipron or GLP-1 (7-36) at EC90 concentrations. After 45 minutes of recording cAMP, using the cADDis biosensor, the cells were extensively washed. After this the cAMP level was recorded for 16 hours. The response to GLP-1 (7-36) declined quickly and was largely gone by four hours. However, the cAMP signal in response to orforglipron did not decline. If anything it continued to rise after the washout. The cAMP signaling was completely sustained for 16 hours after washout. The mechanism of this effect requires further study. A nice review of persistent signaling mechanisms was provided by Daniel Hothersall and colleagues (3). Two great reviews on signaling dynamics of the GLP-1 receptor are also recommended, from Drs Ben Jones, Alejandra Tomas Catala and colleagues at Imperial College London (4,5). An example of a highly successful wash-resistant, persistently-signaling drug is the beta agonist salmeterol (Advair) used for asthma and COPD (6,7). Here the persistent signaling enables a convenient dosing schedule of the drug. Another example is the new antipsychotic xanomeline (Cobenfy) (8). Biosensors provide ideal tools for continuously recording GPCR signaling in live cells. The Montana Molecular cAMP biosensor is likely unique in the duration of signaling that can be recorded. In our CRO service we can often provide 16-24 hours of read time for cAMP signaling. An example report for the GLP-1 receptor is available (9) employing our in house data. References are in the comments.

We are presenting a poster on GLP-1 signaling kinetics at ELRIG Drug Discovery 2025 today and tomorrow (no. 135, from 13:00 - 13:45). This shows how signaling kinetics dramatically distinguishes between agonists acting at the GLP-1 receptor. Sam Hoare will be available to talk with you about Montana Molecular's fluorescent biosensors, reporters, and CRO services in support of drug discovery and research. Looking forward to see you there!

A warm welcome to Grace Baker and Eric Merkel who joined our research team in Bozeman this past month. Both are graduates of Montana State University-Bozeman, in Biomedical Engineering and Chemical Engineering, respectively. Eric is actually returning after a year's break, climbing mountains in South America. His next challenge is the design of new biosensors for GPCRs in cancer (G12/13). Grace is implementing new software systems for our Electronic Laboratory and inventory management. For me, working with Grace and Eric, and the exceptional team at Montana Molecular, LLC is the privilege of a lifetime!

Controlling the level of GPCR expression is critically important. With Montana Molecular, LLC's BacMam technology this is so easy - we simply change the amount of GPCR BacMam we add to the cells. We use this for mechanism of action pharmacology studies and also for compound profiling. In multi-receptor profiling (e.g. comparing species variants) it enables us to very rapidly develop assays. This is transformative, enabling us to get data in weeks that would take many months by traditional stable cell line approaches. Check out the video here (https://lnkd.in/eUp66cDP) to see how this works.

We'll be at ELRIG 2025! Be on target with your biosensor and reporter technology! Catch up with Sam Hoare and see our GLP-1 signaling kinetics poster #135. Contact Sam at shoare@montanamolecular.com or through the conference app. We can chat about how our team can support yours.