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21 May 2024 – Basel, Switzerland
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Bringing lysosomal patch clamp recording to HTS
Science Spyglass High throughput organellar electrophysiology of TMEM175 and TPC2 from freshly isolated lysosomes recorded on the SyncroPatch 384 Application note Axxam S.p.A., MilanNanion Technologies GmbH, Munich Summary Intracellular ion channels are known to play an essential role in various signaling pathways for health and disease, considering that over 80% of transport processes occur inside the cells (1). Among the variety of organellar channels and transporters the proton leak channel transmembrane protein 175 (TMEM175) and the lysosomal two-pore channel (TPC) have received increasing attention in the field given their potential roles in connecting lysosomal homeostasis with pathophysiological conditions such as Parkinson’s disease and cancer (2-4). Consequently, the interest to explore intracellular ion channels as therapeutic targets has grown tremendously indicating a need for high-throughput electrophysiology including patch clamp. There has been some progress in alternative approaches such as solid supported membrane electrophysiology (SSME using the SURFE2R 96SE) recently (5), however, until now, HTS patch clamp has lacked the possibility to collect data from native lysosomes. Axxam and Nanion Technologies have now developed assays to investigate the function and pharmacology of lysosomal channels under native conditions, providing groundbreaking tools for the drug discovery industry. This is possible due to the development of special consumables (single- and multi-hole) dedicated to pursuing organellar recordings in combination with the high flexibility of the SyncroPatch 384 utilizing an ultra-low cell density approach that can use as low as 50k cells/ml, and small volumes of 1 ml for the whole of the 384-well plate, without a drastic reduction in success rate. This can be of extreme importance for expensive – as well as for samples of low quantity (cardiomyocytes, iPS cells or organelles) – to reduce costs and save time. Our approaches resulted in the construction of cumulative concentration response curves and even intraluminal solution exchange during the recording from freshly isolated lysosomes highlighting the broad range of applications possible with the SyncroPatch 384. ResultsTMEM175 Enlarged lysosomes incubated with 1 µM Vacuolin-1 were stained with 0.1 µM LysoTracker™ Red DND-99 (Invitrogen), a red fluorescent dye that stains acidic cellular compartments, such as lysosomes. The dye was added to the cells before isolation of the lysosomes and images were acquired at different magnifications and dilutions using the Operetta system (Perkin Elmer), resulting in an average diameter of 2.1 µm (Figure 1). Figure 1 A – Isolated lysosomes stained with LysoTracker™ Red DND-99 (Invitrogen); images at different magnifications were acquired using the Operetta (Perkin Elmer). B – Average diameter calculated at different dilutions: 3.0 ± 2.1 µm (1:10); 2.0 ± 1.3 µm (1:20); 2.4 ± 1.7 µm (1:50); data are presented as mean ± SD. The remaining lysosomes were used on the SyncroPatch 384 for recording TMEM175 channels expressed endogenously in HEK-293 cells. Critical for success was usage of Nanion’s “Organellar Chips”, a specialized consumable that supported and maintained the integrity of lysosomes throughout the recording and supported cumulative concentration response curves of DCPIB, a novel TMEM175 activator, able to mediate H+ and K+ currents (6) as highlighted in Figure 2. Since TMEM175 channels release luminal H+ into the cytosol, we developed assays using luminal solutions with different pH values, to enhance proton conductance, in addition to potassium flux. The seal resistance in “whole-lysosome” configuration was calculated before compound application and shows average values of 1.4 ± 0.2 GΩ and 2.1 ± 0.6 GΩ at pHluminal 4.0 and 7.0, respectively. TMEM175 activation was accompanied by a drop in Rseal, indicative for stimulation of a leak channel (Figure 2 A). We then executed cumulative concentration additions of DCPIB to activate endogenous TMEM175 channels using only part of the NPC-384 chip (32 wells per condition). Our analysis reveals an EC50 of 65.3 ± 17.5 µM (n=5) at pHluminal 4.0 and 21.5 ± 4.1 µM (n=3) at pHluminal 7.0 for outward currents (ion and proton flux from lumen to cytosol), as shown in Figure 2 D. Representative traces (Figure 2 B-C) clearly show a larger TMEM175 current evoked in the presence of the highest DCPIB concentration in an acidic luminal environment, suggesting enhanced proton flux at acidic luminal pH. Given the known pH dependence of TMEM175 activity (7) we also employed intraluminal solution exchange for the first time where we observed a current modulation after changes in luminal pH. During the experiment with pHluminal 7.0, TMEM175 current was first evoked by DCPIB application, then partially blocked by 4-AP (Figure 3 A). In the presence of 4-AP, acidification of the luminal solution, due to the internal exchange from pHluminal 7.0 to 4.0, increases TMEM175 current (Figure 3 B). A similar experiment was repeated by inverting the luminal pH, starting from 4.0 and changing to 7.0, using the internal perfusion feature of the SyncroPatch 384. In the presence of 4-AP, the reduction of H+ in the luminal solution induces a reduction in TMEM175 current due to a lower proton contribution (Figure 3 C-D). Figure 2 A – Bar graph of seal resistance calculated before and after DCPIB application. B – Representative traces recorded in control and in the presence of increasing concentrations of DCPIB, using luminal solution with pH 4.0, and C pH 7.0. D – Concentration response curve of DCPIB application using different luminal solution, with pH 4.0 (red) and 7.0 (black); in both experiments, cytosolic solutionwas pH 7.0. Figure 3 A – Representative TMEM175 traces recorded in control and in the presence of 100 µM DCPIB (light green) and 2 mM 4-AP (dark green); pHluminal 7.0 – pHcytosolic 7.0. B – Effect of luminal solution exchange (from pH 7.0 to pH 4.0) on TMEM175 current in the presence of 4-AP. C – Representative TMEM175 traces recorded in control and in the presence of 100 µM DCPIB (light green) and 2 mM 4-AP (dark green); pHluminal 4.0 – pHcytosolic 7.0. D – Effect of luminal solution exchange (from pH 4.0 to pH 7.0) on TMEM175 current in the presence of 4-AP. ResultsTPC2 Enlarged lysosomes (Vacuolin, 1 µM) were freshly isolated as described in Schieder et al (8-9) from HEK cells either stably
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The dualism of the aging and disease
Science Spyglass Navigating the dualism of the aging and disease landscape with the pertinent tools An interview with Fernanda Ricci, High Content Screening Unit Manager at Axxam. Don’t miss her upcoming webinar: Forever young?Targeting the hallmarks of aging Request link for webinar Read Fernanda’s opinions on the subject of early drug discovery for diseases related to aging: Q. Why do you consider a webinar on aging important? In an era where life expectancy is increasing, the quest to understand aging and promote healthier, longer lives has become more critical than ever. Aging is a complex and multifaceted process that leaves its mark at the molecular, cellular, and systemic levels for all of us, increasing our risk of diseases and disability, and placing demands on health and social care services. However, recent scientific breakthroughs are shedding light on new approaches to unravel the mysteries of aging, such us metabolic regulators, mitochondrial functionality, inflammation pathways. What is truly exciting today is that these discoveries might pave the way for a future where aging is not just a process but a target for intervention; for a future where we may replace the word “aging” with “longevity.” Certainly, we are all committed to reaching a healthier state as much and as fast as possible. The road is still long, with many issues to solve, but starting with the right methods we can speed up the discovery process. For this reason, we are working to establish biological assays relevant to understanding the fundamental processes of aging. Fernanda Ricci, Axxam High Content Screening Unit Manager Q. What tools are you developing to study aging in the laboratory? We are operating on multiple fronts. Aging processes involve several significant molecular pathways, and our commitment extends to miniaturizing all the assays, enabling high-throughput drug screening campaigns to increase the likelihood of finding the right hits for the specific phenotype. Few examples of relevant cell-based assays include DNA damage, mitochondrial dysfunction, autophagy rate readouts, inflammation-based readouts. Q. What are the major relevant pathways or targets involved in aging progression? A real game-changer is certainly inflammation. Chronic inflammation is the troublemaker here, creating the basis for systemic dysfunction that can lead to issues ranging from cancer to heart problems. Some effects at the molecular and cellular levels, for example, involve the mislocalization of transcription factors, consequently altering the genetic script, or a decrease in the stem cell pool despite an increase in senescent cells, and in turn these cells release inflammatory cytokines, adding fuel to the systemic inflammation storm. Remarkably, our vital organelles such as lysosomes and mitochondria get damaged, and they are no longer able to perform their functions, such as clearing cellular toxic products, producing energy, and removing oxidative species. The “Free Radical Theory” of aging comes into play, increasing the probability of protein crosslinking, DNA damage, and a shuffle in gene expression, all contributing to the onset of metabolic and neurodegenerative disorders, as well as cancer in the long term. However, research is progressing rapidly, much like the aging process itself. Several targets, pathways, and phenotypes involved in aging have already been discovered that can be of therapeutic interest and can be addressed selecting the right tools and assays. Today, life science is approaching aging as a Pandora’s box; by treating aging, the incidence of other diseases can also be reduced, achieving a good and longer healthy old age. Aging is inevitable, but how we age could be our decision in the near future. Contact us Back
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Solute carrier transporters as therapeutic targets
Science Spyglass Solute carrier transporters and drug discovery: unlocking the “gatekeepers” as therapeutic targets Solute carrier (SLC) transporters comprise a family of more than 450 membrane-bound proteins that facilitate the transport of a wide array of substrates across biological membranes. They play a fundamental role in controlling the transport of molecules, such as ions and metabolites, across the cell membranes, and their dysfunction has been associated with a variety of diseases, such as diabetes, cancer, and central nervous system (CNS) disorders. Despite emerging as important targets for therapeutic intervention, SLCs are still under-investigated as therapeutic targets, one of the reasons being the lack of assays and tools suitable for running High Throughput Screening (HTS) of large compound collections, aiming to identify novel therapeutics. As member of the Innovative Medicines Initiative Consortiums RESOLUTE and REsolution, Axxam focused on the development of functional cell-based assays, suitable for running HTS campaigns, for SLCs (wild types and variants) belonging to different classes, by using different types of detection systems, including fluorescent dyes, genetically encoded biosensors, fluorescent substrates, imaging analysis. Watch the videos below to know more about Axxam contribution into the RESOLUTE and REsolution: Assays based on membrane potential dyes to study SLC function Characterization of SLC genetic variants involved in human disease If you are intersted in knowing more about our work, download the following posters on case studies: Fluorescent dyes and sensors for SLC assays Robust assays for SLCs HTS grade SLC assays SLC12A2 assay development SLC26A9 and SLC9B2 biosensor assays SLC30A8 imaging-based assay SLC30A8 live cell imaging assay Contact us RESOLUTE has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777372. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This article reflects only the authors’ views and neither IMI nor the European Union and EFPIA are responsible for any use that may be made of the information contained therein. Back
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From gene to validated and qualified hits
Science Spyglass From Gene to Validated and Qualified Hits,High-Throughput Screening at Axxam Despite a constantly expanding repertoire of therapeutic modalities, small molecules continue to account for the majority of new drug approvals and continue to attract attention and investigation, especially with the expansion of the knowledge on different mechanisms of action, including pharmacological chaperones, targeted protein degraders, covalent protein modification, etc. The small molecule drug discovery research is a complex endeavor that involves multiple stages, each with its own set of hurdles and uncertainties. The hit identification phase is a foundational step in the whole process; by providing the initial set of compounds that have the potential to be developed, it lays the groundwork for a successful drug development. The efficiency, quality, and diversity of hits identified during this phase significantly influence the success and progress of the entire drug development process. Adaptation to Automation: adapt experimental protocols for automated testing Hit Confirmation: re-test of cherry-picked hits from primary screening in triplicates at screening concentrations Activity Determination: full concentration-response in triplicates for confirmed hits At Axxam we are passionate about small molecules and eager to translate innovative target and disease biology into new therapies to patients in need. We offer clients access to a broad cutting-edge infrastructure for automated high-throughput screening, a comprehensive high-quality compound library with modern compound management logistics, a leading expertise in assay development and integrated discovery for hit selection. We believe that leveraging on Axxam’s knowledge and experience into clients’ drug discovery programs will facilitate the translation from Gene to Qualified Hit along the discovery workflow. Assay development High-Throughput Screening (HTS) assays come in many different flavours monitoring target binding or function in either biochemical or cell-based assay systems. Similarly, assays employ a large variety of (mostly optical) readouts.Independent of the many diverse technologies available, HTS assays share 3 main features: Specificity towards a molecular target and mode-of-action (MoA) Robustness combining reproducible low-noise signals with experimental protocols amenable to automation Sensitivity towards the desired molecular MoA Our assay development aims to optimally combine all features in the HTS assay. In-depth assay optimization to probe and fine-tune the assay parameters and protocols ensures assay performance and sensitivity also throughout the adaptation to automation, which is finally assessed by testing a limited compound subset in a pilot run under HTS conditions. Hit validation and qualification Definitions of hit validation and hit qualification differ significantly in various organizations. Very often, no distinction is made at all. At Axxam hit validation includes actions to discriminate desired hit compounds from the unwanted ones that are inevitable selected during the HTS, while we refer to hit qualification as an additional post-HTS activity aiming to increase the value delivered with a hit list report. Hit validation The single HTS assay is typically not sufficient to ensure discrimination of the desired pharmacological modulators from the inevitable “by-catch” in bioassay testing, i.e. compounds acting through off-target or unspecific interference mechanisms. Therefore, well-designed screening cascades with additional tests tailored towards the individual project needs are required for a diligent hit validation ensuring specific target interaction via the desired molecular MoA. These include: Orthogonal assays: same target, but different assay format – for positive selection of hits Counter assays: same assay format, but different target – for hit de-seletion Selectivity assays: related target, frequently same assay format As a final step, purity of hit compounds will typically be probed by mass spectroscopy and re-tested from solid material. To further facilitate a data-driven hit prioritization process in the HTS follow-up, Axxam provides ad hoc services designed to deliver information on: Physicochemical properties, i.e. solubility lipophilicity, chemical stability Metabolic stability Membrane permeability Plasma protein binding Plasma stability Hit qualification In most cases, hit compounds derived from screening a diversity-oriented compound library provide only limited information on the underlying structure-activity relationships (SAR) to instruct further optimization. To explore hits in this respect, our medicinal chemistry experts have devised an efficient process generating “chemical context” around screening hits. Following the in-depth analysis of available data based on similarity, relevant substructures or functional groups potentially contributing to molecular recognition at the target receptor, the most attractive candidates will be further explored by the team. Starting from a resynthesis of the original hit to confirm its chemical structure bone fide, “strategic analogues” will be designed, synthesized and tested. The results provide initial clues on SAR determinants and help to identify the most suited candidates for further prosecution. Including in these experiments not only the primary (activity) assay, but also reference assays and tests probing either physicochemical and basic ADME properties further strengthen the specificity of the drug-target interaction and ensures hit selection to be based also on secondary compound properties relevant to evolve hit compounds towards a final target product profile (including for example the intended rout of administration). Finally the expert team will also explore the chemical IP space hit by our compound candidates by searching the patent literature databases. Our HTSPLUS package includes this thorough hit qualification phase on hit compounds derived from screening the Axxam compound collection AXXDiversity. The package generated will greatly facilitate the process of selecting the best candidates for further Hit-to-Lead optimization and enhance the chances for successful progression. Discover more about the hit qualification phase Infrastructural backbone: HTS automation and compound & data management While a comprehensive quality compound library and the smart design of the HTS assay and screening cascade greatly impact the result of individual screening projects, a sophisticated automation and IT infrastructure is another, equally important enabler of success. Laboratory robots and automation not only perform recurrent experimental tasks beyond regular office hours but also with superior precision and reproducibility. Similarly, in compound management oversized high-tech refrigerators combine optimized conditions of long-term sample storage of compound plates with flexible input and retrieval function. They interface with pipettors and acoustic dispensers to flexibly generate plate copies, cherry-pick hit compounds, and generate serial dilutions in variable formats and volumes (from nl to μl). Tracking each plate’s unique barcode throughout all steps of the process serves
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Thermal shift assays for early-stage drug discovery
Science Spyglass Unlocking the Power of Thermal Shift Assays for Early-Stage Drug Discovery Thermal-shift assay (TSA) represents a powerful tool for protein-stability analysis and for discovering and characterizing new protein binders. Understanding protein stability is crucial for drug development, protein engineering, and other biotechnology applications. Among the various methods available for assessing protein stability, TSA has emerged as an effective technique. TSA provides valuable insights into protein stability, ligand interactions, and quality control, making it indispensable for drug development and protein engineering. Here, we outline the principles, methodology, and applications of TSA, as well as our recommendations for use in early-stage drug discovery and our TSA capabilities to identify and characterize protein binders for your target protein. What are thermal shift assays? TSA is a highly parallelizable, cost-effective technique for studying protein stability and protein binders. It measures the thermal denaturation of a target protein by monitoring the fluorescence intensity of a dye that binds to exposed hydrophobic regions of the protein during heating. As the protein unfolds, the exposed hydrophobic regions increase, leading to an increase in fluorescence signal. Compounds, such as inhibitors or in general modulators, stabilize the three-dimensional conformation of the target protein and as a consequence the temperature needed to denature the protein is higher (see purple curve in left figure). The figure on the right below shows fluorescence intensity curves (top panel). The blue curve is the target protein with the dye; the other traces correspond to the addition of increasing concentration of a binder compound that causes a shift of the melting temperature of the protein complex. The melting temperature can be calculated as the inflection point of the fluorescence intensities or as the first derivative of the curves (peaks in the bottom right panel). TSA and differential scanning fluorimetry (DSF) can generally be used interchangeably, a primary distinguishing feature between the two lies in their denaturation methods. TSA specifically refers to the denaturation of a target protein triggered solely by temperature intensification. On the other hand, DSF encompasses techniques that employ various physicochemical agents, including but not limited to temperature intensification, in order to induce denaturation in the target protein. In this article, our focus will be directed towards exploring the specific aspects of TSA. Key steps in TSA experiments The TSA methodology involves a series of simple steps that make it suitable for routine laboratory use. The following is a brief outline of the key steps involved in performing a TSA experiment: Protein sample preparation: purify the protein of interest ≥ 90% using established protocols, ensuring its quality and concentration. Fluorescent dye binding: add a fluorescent dye, such as SYPROTM Orange (a gold standard dye), to the protein solution. The dye selectively binds to hydrophobic regions exposed during protein unfolding. Thermal gradient generation: set up a thermal gradient by using a thermocycler. Typically, the temperature is increased in small increments, allowing for gradual protein denaturation. Fluorescence detection: monitor the fluorescence intensity of the dye at each temperature increment. The increase in fluorescence corresponds to protein denaturation. Data analysis: analyze the thermal-shift data to determine the protein melting temperature (Tm), which represents the temperature at which 50% of the protein is unfolded. Tm provides insights into protein stability and ligand interactions. TSA applications Overall, TSA offers numerous applications in various fields, including: Drug discovery: TSA enables screening of compound libraries for potential drug candidates. By measuring changes in the protein’s thermal stability in the presence of small molecules, compounds that stabilize or destabilize the target protein can be identified. Protein engineering: by assessing the thermal stability of protein mutants, TSA aids in the optimization of protein engineering strategies. It provides insights into how specific mutations or modifications affect the protein’s stability, aiding in the design of more robust variants. Ligand interaction studies: TSA can evaluate the binding affinity and thermodynamics of protein-ligand interactions. By monitoring the shift in protein melting temperature upon ligand binding, the binding strength can be quantified and the impact of different ligands on protein stability can be assessed. Affinity and inhibition parameters: affinity parameters (Kd) can be directly extrapolated varying ligand concentrations. We demonstrated that these affinity parameters directly correlate with, for example, inhibitor parameters (IC50, Ki). Quality control: in biopharmaceutical production, TSA plays a vital role in assessing the stability and quality of protein-based drugs. It ensures the consistency and integrity of protein formulations, preventing potential degradation during storage and transportation. Additionally, TSA enables the ranking of compounds based on their affinity to the target protein, providing valuable insights into their binding properties. By using TSA, researchers can confidently evaluate compounds and identify those with a higher or lower affinity to the target protein, aiding in drug discovery and optimization processes. Recommendations for TSA in early-stage drug discovery When considering the use of TSA in early-stage drug discovery, we offer the following recommendations: TSA is highly effective in primary screening scenarios that involve small, focused or unbiased compound libraries. By utilizing TSA during the initial screening process, promising compounds can be quickly identified for further investigation. TSA can serve as secondary assay for hit validation and target engagement. By employing TSA in this capacity, additional insights can be gained into the interactions between compounds and target proteins, further validating potential hits. TSA is particularly useful in characterizing lead compounds during the hit-to-lead phase of drug discovery. These assay provides crucial information on the stability and binding properties of lead compounds, aiding in the selection of the most viable candidates for further development. For the identification of proteolysis targeting chimera (PROTACTM) scaffold molecules, TSA can be a valuable tool. The interactions between PROTACs and target proteins can be assessed by utilizing TSA, facilitating the development of effective protein-degradation strategies. TSA can also be employed to characterize protein-mutant variants. By subjecting mutant proteins to TSA, the impact of specific mutations on protein stability and ligand interactions can be evaluated, providing crucial insights into protein engineering and optimization. TSA offers several advantages over traditional methods of protein
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shRNA for gene knock down in screening campaigns
Science Spyglass Why is shRNA better than siRNA for gene knock down in screening campaigns? One of the first challenges to face in the drug discovery process, is the generation of reliable in-vitro models that mimic the pathological conditions of the disease being studied. Many diseases are caused by the loss of function of a gene, with consequences on downstream pathways and detrimental effects on cell functions. Different approaches can be used in-vitro to obtain a loss of function model at the DNA, RNA or protein level. We are not new to the use of RNA interference for silencing the mRNA transcripts of such genes, having worked for many years with small interfering RNA (siRNA) transfection. Despite allowing for a great flexibility and ease of use, this approach poses some issues in terms of throughput and variability, when screening large compound libraries to find molecules able to revert the pathological conditions into healthy ones, mainly due to the need to transfect many batches of cells during the screening process. An alternative to siRNA transfection, well-suited for high-throughput screening (HTS) and hit-to-lead campaigns, is the development of assays based on inducible short hairpin RNA (shRNA) expression, to knock down the gene of interest. How does it work? In our experimental model, we treat the cells with lentiviral particles encoding for inducible shRNA directed against the gene of interest, in order to generate a stable cell line able to mimic the pathological condition, upon gene downregulation triggered by doxycycline. What are the advantages? Compared to siRNA transfection, that is a transient approach, the shRNA viral transduction generates a stable cell line, and the gene downregulation can be sustained over time by treatment with doxycycline, making this approach very suitable for HTS campaigns and hit-to-lead programs. Indeed, the entire process is particularly fit for automation as it requires fewer handling steps. One additional important advantage of this approach is that the shRNA induction with doxycycline is dose- dependent (the possibility to modulate the expression is especially important when studying essential and lethal genes) and time-dependent (reversible upon withdrawal of doxycycline). When to apply this approach? Independently of the therapeutic area, this approach is particularly meaningful when studying diseases characterized by a depletion or mutation of a “disease causing gene” (loss of function) with the final aim of identifying small molecules that revert such pathological conditions to the normal state. The experimental disease-related model can be used also to study “downstream genes” regulated or at least influenced by the disease causing gene that will be knock down by specific shRNA. A case study:focus on a disease caused by the mis-splicing of a target gene We developed a disease model transducing shRNA expressing lentivirus into a human neuroblastoma cell line, to silence a splicing factor (the disease-causing gene), whose downregulation causes a mis-splicing in a target gene (downstream gene). The downstream gene was measured by multiplex TaqManTM q-PCR in dose response, quantifying normal and aberrant slice variants at the transcriptional level. In addition, the downstream gene was monitored at the protein level with Nano-Glo® HiBiT detection system by Promega. Related content Drugging the RNA world at cellular and subcellular levels Webinar available on demand Contact us Back
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