Screening & Hit-to-Lead

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

From gene to validated and qualified hits Read More »

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

Thermal shift assays for early-stage drug discovery Read More »

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

shRNA for gene knock down in screening campaigns Read More »

Scroll to Top