Biophysical characterization of compounds disrupting the SARS-CoV-2 Spike – ACE2 interaction using SPR
Association and dissociation constants give insight into an inhibitor's potential efficacy and safety The determination of the equilibrium dissociation constant, KD, for binding of a potential drug to a target is the first step for the identification of hits. For therapeutic options to prevent SARS-CoV-2 entry into host cells, the drug might either block the spike protein or the ACE2 receptor. The KD is an essential experimental triage parameter used to narrow down potential drug candidates initially identified by docking studies and molecular dynamics simulations. To get a deeper picture of how a drug candidate might actually perform clinically – in other words, some insight into its potential efficacy and safety – the KD needs to be accurately dissected into its more useful components, the association, and dissociation kinetic constants kon and koff. These constants relate to the equilibrium dissociation constant according to KD= koff/kon. The reason for this is that equilibrium kinetics describe a closed system where the drug and target are held at constant concentrations over time. Researchers need to know these on-off rates in order to feed more sophisticated models of what would happen when people take the drug. Getting good data upfront can only help downstream pharmacokinetic and pharmacodynamic investigations. The former, specified as concentration vs. time, captures what the body does to the drug (including rates at which it is metabolized and eliminated), while the latter, specified as effect vs. time, seeks to define what the drug does to the body. In an actual tissue microenvironment, where association rate often dominates over diffusion rate, and targets or receptors can be locally abundant, then significant rebinding effects are expected to occur. A fast kon can therefore significantly increase the duration of target occupancy even when the koff is faster than the elimination rate. In any realistic micro-pharmacokinetic accounting of the
Kinase inhibitors for treatment of COVID-19
Repurposing of kinase inhibitors to target SARS-CoV-2 In the face of proliferating viral variants which up the ante for vetting precise drug interactions, already approved small molecule kinase inhibitors showing efficacy against SARS-CoV-2 symptoms and sequelae are now comparatively easy to come by. To further flesh out this point, consider what is perhaps the smallest, and incidentally cheapest, kinase inhibitor one could ask for -- good old lithium chloride. University of Penn researcher Peter Klein recently published a quick note to the medRxivs reporting that patients taking lithium have a significantly reduced risk of COVID19 infection (odds ratio = 0.51 [0.34 - 0.76], p = 0.001). Conveniently, many people had previously been subjected to lithium therapy for a host of other reasons and therefore Klein and his fellow researchers could simply take a retrospective look to see how many got the virus. The tale of how the researchers then arrived at the probable mechanism for the lithium effect is a textbook case of well-executed, short-order, inferential drug discovery. Klein’s team had previously shown that the nucleocapsid (N) protein of SARS-CoV-2 contained GSK-3 consensus sites (known as RS domain for arginine-serine) that were identical to those found in the SARS-CoV-1 N protein. It was already known that lithium could block GSK-3 phosphorylation of the SARS-CoV-1 N protein, therefore suggesting a similar possibility for SARS-CoV-2. Previous phosphoproteomic analysis had also indicated that the RS domain of the SARS-CoV-2 N protein is highly phosphorylated, so Klein’s group performed in-vitro kinase assays with the Sars-CoV-2 N protein expressed in human kidney cells (HEK293T), and mouse lung epithelial (MLE12) cells to test different inhibitors. The main problem with lithium is its narrow therapeutic window in humans (peak level ~2 mM, trough levels ~1 mM). While there is some evidence for GSK-3 inhibition at 1 mM, behavioral effects can be seen already
Blog
Drug Discovery Blog Every drug discovery program has its own challenges. Scientists at Reaction Biology have 20 years of experience in problem-solving and decision making. Via blogs, we like to share some of our experiences and insight into our assays with the drug discovery community to support your journey to a preclinical drug candidate.
Spotlight: Cell-based kinase assay formats.
Reaction’s Assay Formats The NanoBRET Intracellular Target Engagement Assay technology enables the investigation of compound-target engagement (or binding) in the physiological environment of intact cells. NanoBRET is an energy transfer technique designed to measure the apparent affinity of test compounds by competitive displacement of a NanoBRET tracer, reversibly bound to a NanoLuc luciferase-kinase fusion construct expressed in cells. Upon binding of the drug to the target kinase, the fluorescent tracer is pushed away from the binding site, increasing the tracer's proximity to the luciferase-kinase construct, resulting in a decreased BRET signal. The Cellular Phosphorylation Assay is an assay for quantification of the activity of a kinase. The assay is performed with cells either expressing the endogenous kinase or a kinase construct. Substrate phosphorylation is quantified via detection with phospho-site specific antibodies. Upon kinase inhibition by a compound, the kinase stops phosphorylating the substrate, which is quantified as a readout for kinase activity. The BaF3 Cell Proliferation Assay, which is often called BaF3 Cell Transformation Assay, is an activity assay format designed to examine the transformation capacity of a kinase. The survival of BaF3 cells is dependent on signaling from extracellular Interleukin-3 (IL3). This dependency can be overcome by the expression of a transforming kinase — the expression of an oncogenic kinase that drives cell proliferation even in the absence of IL3. Inhibition of the kinase by a compound deprives the cells of the oncogenic driver resulting in cell death. Comparing the Cellular Phosphorylation Assay and NanoBRET Intracellular Target Engagement Assay It is recommended to conduct enzyme activity assays that quantitate substrate phosphorylation as well as target engagement assays that measure binding affinity and target residence time. Neither approach is flawless, but when used together, potential
Reaction Biology and Vyant Bio Announce Closing of Definitive Agreement to Acquire U.S. Operations of Vyant Bio, Inc. Subsidiary
~ Reaction Biology Acquires the Hershey, Pennsylvania-Based U.S. Operations of Vyant Bio Subsidiary vivoPharm PTY Ltd ~ ~ Transaction Expands Reaction Biology’s Capabilities to Provide Customers with Solutions in Both the Discovery and Development of New Drug Candidates ~ ~ Transaction Provides Vyant Bio with Additional Capital to Deploy into Further Development of its Therapeutic Pipeline in Genetic CNS Disorders ~ MALVERN, PA and CHERRY HILL, NJ, November 3, 2022 – Reaction Biology Corporation (“Reaction”), an industry-leading provider of drug discovery services, and Vyant Bio, Inc. (“Vyant Bio”) (Nasdaq: VYNT), an innovative biotechnology company reinventing drug discovery for complex neurodevelopmental and neurodegenerative disorders, today announced that the companies have closed on a definitive agreement for Reaction to acquire Vyant Bio’s subsidiary vivoPharm LLC, located in Hershey, Pennsylvania. Reaction will retain U.S. personnel from the acquired operations to establish its Hershey, Pennsylvania presence, which includes in vitro and in vivo capabilities. Through the acquisition, Reaction gains laboratory facilities, equipment, employees, cell lines and capabilities (in pharmacology, toxicology, pathology and bio analytics) that further expand the industry-leading suite of drug discovery services that Reaction provides for its biopharmaceutical customers. The purchase price for this transaction is $5.5 million in an upfront cash payment, subject to customary adjustments for working capital, closing cash, indebtedness and transaction expenses. After these closing adjustments were reflected, $5.5 million was paid at closing. Vyant Bio expects to net approximately $4.4 million in cash after tax and transaction related expenses, as well as incur $0.6 million in exit costs associated with this transaction. “vivoPharm’s U.S. operations are highly complementary to Reaction’s existing suite of drug discovery CRO services, enabling us to
Reaction Biology Acquires Bioassay GmbH Increasing Large Molecule Capabilities Within Its Robust Portfolio of Drug Discovery and Development Services
~ Bioassay GmbH, an Independent Contract Laboratory, Specializes in the Development, Validation and Routine Performance of Bioanalytical Assays ~ ~ Transaction Includes Acquisition of Bioassay GmbH Subsidiary, Peptide Specialty Laboratories GmbH, a Provider Specializing in the Synthesis and Production of Customized Peptides and Specific Antibodies ~ ~ Transaction Adds Large Molecule Talent and GMP and GLP Accredited Equipment and Facilities ~ Malvern, PA, December 2, 2022 – Reaction Biology Corporation (“Reaction”), an industry-leading provider of drug discovery and development services, today announced that the Company has closed on a definitive agreement for Reaction to acquire Bioassay Labor für biologische Analytik GmbH (“Bioassay”), including its wholly-owned subsidiary, Peptide Specialty Laboratories GmbH (“PSL”), located in Heidelberg, Germany. Bioassay offers Good Manufacturing Practices (GMP) accredited potency assays and functional bioassays, Good Laboratory Practices (GLP) compliant services, and through PSL, peptide synthesis services, including peptide-specific antibody production and neoantigen synthesis. Personnel, equipment, and facilities from the acquired companies will expand Reaction’s presence in Germany, while also enabling its global customers to leverage Bioassay’s expansive portfolio of regulated clinical and commercial services. “Market demand is increasing globally for large molecule services as biopharmaceutical companies search for new treatment options for patients suffering with cancer and other serious diseases,” said John H. Johnson, Chief Executive Officer of Reaction Biology. “This transaction enables Reaction Biology to add Bioassay GmbH’s impressive clinical phase and post-approval infrastructure, which includes large molecule capabilities, as well as its GMP and GLP accreditations, to our expanding suite of drug discovery and development services. We can now provide our customers with even broader
Reaction Biology Selected for Participation in National Chemical Biology Consortium to Support Drug Discovery Utilizing Surface Plasmon Resonance (“SPR”) Technology
Malvern, PA, December 19, 2022 – Reaction Biology Corporation (“Reaction”), an industry-leading provider of drug discovery services, today announced that it has been selected to participate as a Specialized Center in the Chemical Biology Consortium (CBC), the discovery engine of the National Cancer Institute (NCI) Experimental Therapeutics (NExT) Program, administered through the Frederick National Laboratory for Cancer Research (FNLCR), a federal national laboratory sponsored by the NCI, part of the National Institutes of Health, and currently operated by Leidos Biomedical Research, Inc. The CBC brings together a multi-disciplinary team from government, industry, and academia to develop and advance new anticancer therapies to the clinic. This latest contract is the third that Reaction has been chosen for participation in the CBC. Under this latest collaboration agreement, Reaction will utilize its expertise in Surface Plasmon Resonance (“SPR”). The scope of the project includes developing a robust SPR assay to measure the binding affinity of small molecules to human target proteins and conducting measurements to determine KD values for novel small molecules provided by the project team. “Reaction Biology is thrilled to build upon its longstanding participation in the CBC, which began in 2016, to help identify and advance early stage drug discovery compounds through to the clinical candidate stage for cancers where great unmet needs remain,” said Rebecca Eells, Ph.D., Director of Biophysics at Reaction Biology. “As a leading service provider, our multidisciplinary research team will bring unparalleled expertise in fundamental and applied cancer research, backed by Reaction’s fully-integrated oncology and immuno-oncology drug discovery services and highly qualified technicians using our state-of-the art laboratories.” Members of the CBC contribute their expertise in high throughput screening, structural biology, medicinal chemistry, compound
Reaction Biology to Present Data at AACR 2023 Showcasing Expansive Capabilities in Immuno-Oncology Drug Discovery and Development
~ New Data will be Presented on the Company’s Innovative Assay Technologies, Preclinical Profiling Models and Proprietary Screening Platforms ~ ~ Reaction Biology will be at Booth #1527 in the AACR 2023 Exhibit Hall ~ Malvern, PA – April 12, 2023 – Reaction Biology (“Reaction” or the “Company”), an industry-leading provider of drug discovery and development services, today announced that eleven abstracts highlighting data from the Company’s expanding suite of research offerings will be presented at the American Association for Cancer Research (AACR) Annual Meeting 2023, held April 14-19, 2023, in Orlando, Florida. “The immuno-oncology landscape has undergone profound and encouraging progress in recent years and there is a growing demand for large molecule services in the search for new treatment options for patients with difficult-to-treat cancers,” said Haiching Ma, Ph.D., Chief Scientific Officer of Reaction Biology. “At AACR 2023, we look forward to showcasing data from our expansive suite of in vitro assays and in vivo tumor models that can provide our customers with solutions to help accelerate the identification and validation of important new drug candidates.” New data will be presented on the Company’s innovative assay technologies, preclinical profiling models and proprietary screening platforms. The full range of data presented at AACR include: Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents (Sunday, April 16, 2023, 1:30 - 5:00 PM; Poster Section 2, Poster Board Number: 1, Permanent Abstract Number: 33) Exploiting macrophage differentiation and plasticity as an immunomodulating strategy (Sunday, April 16, 2023, 1:30 - 5:00 PM; Poster Section 23, Poster Board Number: 11, Permanent Abstract Number: 671) Characterization of the selective inhibitory effect of KRas inhibitors in different cellular assay formats (Sunday, April 16, 2023, 1:30 - 5:00 PM; Poster Section 19, Poster Board
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Our Privacy Policy 1. How we protect your personal data This data protection policy (“Policy”) informs you how we (Reaction Biology Corporation) process your Personal Data when you visit our website, order our products, or receive marketing materials from us. “Personal Data” is any information relating to an identified or identifiable natural person (a data subject), such as your name, physical address, IP or email address that we receive either directly from you, or from our affiliated companies. While this Policy refers to the EU General Data Protection Regulation (“GDPR”), we provide the same standard of protection for Personal Data from individuals outside of the European Economic Area (“EEA”). Who is responsible for the data collection on this website? The party responsible for the processing of the Personal Data (data controller) is: Reaction Biology Corporation One Great Valley Parkway, Suite 2 Malvern, PA 19355 Attn: Customer Service Phone: +1 877.347.2368 Email: dataprotection@reactionbiology.de How do we collect your data? Some Personal Data are collected when you provide them to us to perform our services to you or sell our products online to you. This could be the case, for example, for Personal Data that you provide via an online contact, email contact, or order form. Other data sets are collected automatically by our IT systems through cookies etc. when you visit the website. These data are primarily technical data such as the browser and operating system you are using or when you accessed the page. These data are collected automatically as soon as you enter our website. We use Hotjar in order to better understand our users’ needs and to optimize this service and experience. Hotjar is a technology service that helps us better understand our users’ experience (e.g. how much time they spend on which pages, which links they choose to click, what users do and don’t like, etc.) and this enables us to build and maintain our service
FAQ
FAQs See our FAQ list below for a quick answer to the most common questions. Our global team will be happy to talk to you directly about any questions and requests.
Target Validation
Target Validation Target validation is a crucial early step for developing a drug against a new cancer target. There is no universally applicable process for target validation; therefore, our team will create a research plan with the client tailored to the target of interest. When designing the project we account for potential pitfalls such as results confounded by off-target effects. Target validation at Reaction Biology is mainly based on our suite of cellular and in vivo oncology models. Here are a few examples of target validation approaches: Target knockout is one approach to determine if the target is related to the growth or transformation of cancer cells Cells with target knockout can be subjected to phenotypic assays such as the cell transformation assay, migration assays, invasion assays, or proliferation assays Cells with target knockout can also be investigated in mice to find out if the lack of target expression has any impact on the tumor growth in vivo Cellular target engagement assays and cellular enzyme activity assays will be performed to determine the drug concentration necessary to reach relevant target occupation or inhibition. This information is crucial for proper interpretation of results with phenotypic assays or tumor models preventing toxicities that originate in too high a dosage. Knockdown of the target can be achieved in three ways: Transient knockdown is performed with target-specific RNA interference. The well-established Tet-On/Off technology can be employed to modulate target expression. Complete knockout is achieved with CRISPR technology. Cell lines with CRISPR-mediated knockdown will be generated by a partner CRO.
Protein Production
Protein Production The creation of a drug against a novel target is often challenged by the unavailability of existing recombinant proteins and assays. Our Reaction Oncology Platform comprises a multidisciplinary team of chemists and biologists to plan the screening needs tailored to your target and nature of the compound. Our protein production unit specializes in creating recombinant proteins for various assay formats, including enzymatic activity assays, binding assays, biophysical assays, fluorescence/luminescence assays, or protein-protein interaction assays. We may make multiple constructs of your target for reaching an optimal expression level and solubility or for testing various tags or tag cleavage options. Visit our Protein Production and Assay Development webpage
Medicinal Chemistry Integration
Medicinal Chemistry Integration Modern drug discovery is a multi-disciplinary team effort for creating small molecules as well as more complex modalities such as dual-target small molecules, protein degraders, or cyclopeptides. A skilled chemistry team is the main driver for developing new chemical entities to create new IP and value for our customers. The Reaction Oncology Platform enables a team approach by bringing together highly skilled chemists and our experienced biologists.
Cardiac Safety
Cardiac Safety Every drug candidate filed with the FDA or EMA for clinical approval has to be evaluated for their potential to delay cardiac repolarization and cause heart arrhythmia. We perform cardiac testing in two laboratories – our US-based lab for high- and medium-throughput testing of compounds in biochemical and cellular assay formats. Advanced studies are performed by our partner PharmaCore Labs covering compound testing on a broad cardiac ion channel panel under CiPA regulations as well as tissue- and organ-based studies. Together we cover all necessary cardiac tests during the pre-clinical evaluation of new drug candidates. Cardiac liability testing is performed under non-GLP conditions for exploratory evaluation of new potential therapeutics only. Cardiac liability assessment at Reaction Biology and Pharmacore Labs is performed at different steps during the drug discovery process. During hit-to-lead phase, first tests are performed in medium to high-throughput mode via biochemical binding studies of new compounds to hERG – the most frequently affected cardiac ion channel. Patch clamp studies on hERG expressing cell lines are part of the late hit-to-lead phase to discard unsuitable drug candidates early in the discovery process. Promising drug candidates are further screened on our core cardiac panel, including hERG, Nav1.5, and Cav1.2 via patch clamp during the lead optimization phase. Testing on the full cardiac ion channel panel is performed on cells via patch clamp following CiPA guidelines, including 6 or 7 ion channels. Further testing on cell culture can be conducted on human ventricular iPS (induced pluripotent stem cell)-derived monocytes via patch clamp to test the intracellular action potential. Promising lead candidates are tested in freshly isolated cardiac tissue such as Purkinje fibers, sinoatrial nodes, or papillary muscles for modulation of the intracellular action potential. ECG readings on freshly isolated beating rabbit hearts
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Our clients are seeking answers in their quest to discover effective treatments for patients. Finding these answers is our mission. Our method is to accelerate the drug discovery process with the widest possible range of assays and services but also to maintain an integrated view of the journey to a lead drug candidate.
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Let's Discover Together. Our growth story began over 20 years ago and since then we have evolved into a dynamic organization where hundreds of talented innovators and achievers are working together as ONE Reaction team across the globe. With strong core values and a passion to put people first, we are committed to delivering the highest quality data, through customer service excellence to secure our position as the discovery partner of choice in our areas of scientific expertise.
