Challenges and solutions in handling In Vitro Assays
Sponsored by: GVK Biosciences
- Vitro Assay Development
Date: 25 October
Days to go: 8
Time: 3PM London/10AM New York
Novel in vitro label-free medium - to high - throughput screening assays by RapidFire – Mass Spectrome
Small molecule screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays.
In this webinar, we describe novel in vitro methods that have been developed at GVK BIO using RapidFire-Mass Spectrometry (RF-MS) to quantify bioactive lipids and sterols; that can be challenging due to their relatively short lifetimes and structural diversity. The presentation will cover the novel assays developed for evaluating the efficacy of NCE’s on Prostaglandin D2 (PGD2) and Dihydrolanosterol.
Biochemical and cellular assays were developed to quantify PGD2 and in turn assess the efficacy of NCE’s on PGD2 using RF-MS. The principle of the biochemical assay is based on the measurement of PGH2 conversion to PGD2 by PGDS. In the assay format, the substrate (PGH2) is formed in situ by the action of Cox-2 on arachidonic acid. The PGH2 is then converted to PGD2 by the PGDS enzyme. The reaction is quenched with tin chloride, which converts any remaining PGH2 to the more stable PGF2 alpha. Relative levels of PGD2 and PGF2 alpha, which acts as a surrogate to the substrate, are measured. Inhibitors are characterized as NCE’s which lower the conversion of PGH2 to PGD2. The principle of the cellular assay is based on intracellular calcium mediated PGD2 production and secretion used for NCE evaluation. Immune system cells release small molecule inflammatory mediators in response to stimulants such as calcium ionophore. The stimulated cells release bioactive lipids through enzymatic transformation of membrane phospholipids. During this process, phospholipases are largely responsible for the production of arachidonic acid, which is subsequently transformed into leukotrienes and prostaglandins through hydrolase and synthase enzymatic reactions. In the assay, Rat Basophilic Leukemia (RBL) cells were used as an in vitro test system to evaluate the inhibitory potential of NCE’s on PGD2 release in the presence of a calcium ionophore.
In addition, we also developed a unique cell based assay to quantitate the levels of various sterols involved in post-squalene cholesterol biosynthesis pathway using RF-MS. In principle, there are two rate limiting and committed steps involved in cholesterol biosynthesis that include; the formation of HMG-CoA (Mevalonate pathway) and lanosterol metabolism (post squalene pathway). Lanosterol is metabolized to cholesterol by 19 enzymatic steps and the challenge in quantitating the intermediates in this pathway is the rapid conversion of downstream metabolites. Hence, developing a method to quantitate sterol intermediates was critical to ascertain the inhibition of NCEs to the target class of enzymes.
The ultimate maturation of these techniques/methodologies will enable drug discovery researchers to screen NCE’s against minimally manipulated biological systems.
Dr. Premkumar Arumugam,
Associate Vice President – In Vitro Biology
Dr. Arumugam Premkumar received his PhD degree in Molecular virology from Madurai Kamaraj University, India. After his graduation he joined as a Post Doctoral Fellow at Weizmann Institute of Science in Israel. At Weizmann, he conducted a research on neurodegenerative disorders and acquired skills in Molecular Neuroscience and Molecular Pharmacology.
His research focus was on the role of dopamine in the progression of Parkinson disease and his work resulted in the identification of key molecules associated with cell death and differentiation induced by Dopamine.
To further his research on Neuroscience and GPCR biology he then joined the Memorial Sloan-Kettering Institute (MSKCC) at New York and carried out research on the role of Opioid receptors (family of G protein coupled receptors) in pain management and drug addiction.
At MSKCC, he also investigated the protein-protein interactions among different GPCR’s using various molecular and biochemical techniques. Besides his own projects, he has also actively collaborated with various research groups within and outside USA.
Having established himself as an expertise Molecular Neuro-Pharmacologist and pain receptor biologist in the academia, Dr. Premkumar joined GVK Biosciences in the year 2007 and he was instrumental in establishing In vitro pharmacology and reagent generation services and capabilities. Currently he is heading in vitro biology and reagent generation team and his team activities include assay development, HTS screening and reagent generation for targets like GPCR, Enzymes, Ion channel and Transporter etc.
For the past 11 years he worked closely with more than 100 global pharmaceutical companies and biotech organizations. His team contributed 4 IND filings for various clients and some of his works were highly appreciated by clients. In addition to his leadership role in in vitro pharmacology, he chairs IBSC committee at GVK Biosciences and serving as a Radiation Safety Officer for the past 9 years at GVK Biosciences.