Title: Targeting ALK for cancer therapy: high throughput screening assays
Abstract: Event Abstract Back to Event Targeting ALK for cancer therapy: high throughput screening assays Mohamad Fairus Abdul Kadir1, Pratheev Alagapan1, Yang Jin Lim1, Wai Mun Kong1, Dharvind Balan1, Mathura Subangari Dorasamy1, Wesley Roy Balasubramanian2, Sivapriya Marappan2, Umarani Subramaniam1*, Kavitha Nellore2 and Thomas Anthony2 1 Aurigene Discovery Technologies Sdn Bhd, Malaysia 2 Aurigene Discovery Technologies (India), India Background Drug discovery is a multi-step, interdisciplinary process which involves target identification, lead optimization, pre-clinical and clinical studies prior to first human exposure. Thousands of chemical compounds designed to interact with/block a specified target are synthesized and isolated at the early stage of a drug development. Subsequently, combinatorial chemistry and assay development are involved to screen selected compounds in a high throughput screening platform. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase which plays a critical role in proliferation, survival, and differentiation of normal cells. However, continuous activation of ALK signaling results from overexpression and mutation has emerged as a novel tumorigenic player in several human cancers. Crizotinib is the first ALK inhibitor approved by Food and Drug Administration (FDA) followed by ceritinib and alectinib. Nevertheless, the development of resistance towards crizotinib treatment due to the mutations in ALK (L1196M, C1156Y, and F1174L) triggered the development of alternative pharmacologic agents that inhibit the mutant forms as well. Therefore, in vitro ALK assays using Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and In-Cell Western (ICW) technology were developed to identify potential compounds that could inhibit the activity of both the ALK wild-type and its mutant form. Methods In TR-FRET assay, enzyme was added to a reaction mix containing buffer, substrate and ATP in a 384-well plate. For ICW assay, Karpas-299 cells were seeded in the microtiter plates followed by fixation and permeabilization. The inhibitory potency of the standard (NVP-TAE684, crizotinib and staurosporine) and test compounds was detected by the phospho-ALK with state-specific antibodies and conjugated secondary antibodies. The intensity of light emission is proportional to the level of substrate/ALK phosphorylation and the percentage inhibition of the compounds was then determined. Results In general, the standard and test compounds were more potent for WT ALK as compared to the L1996M mutant. The biochemical potency for WT and mutant ALKs correlated well with the cell potency in Karpas-299 cells. Conclusion The TR-FRET and ICW assays established with standard inhibitors can be used to screen compounds for their potential to inhibit ALK activity. The assays are simple, cost effective and ideal for high throughput screening to develop potential ALK inhibitors for cancer therapy. Acknowledgements This work was funded by Aurigene Discovery Technologies Limited, Bangalore, Karnataka, India. Keywords: TR-FRET, In-cell Western, kinase, Drug Discovery, Cancer Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Poster Presentation Topic: Cancer Citation: Abdul Kadir M, Alagapan P, Lim Y, Kong W, Balan D, Dorasamy M, Balasubramanian W, Marappan S, Subramaniam U, Nellore K and Anthony T (2019). Targeting ALK for cancer therapy: high throughput screening assays. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Umarani Subramaniam, Aurigene Discovery Technologies Sdn Bhd, Kuala Lumpur, Malaysia, [email protected] Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mohamad Fairus Abdul Kadir Pratheev Alagapan Yang Jin Lim Wai Mun Kong Dharvind Balan Mathura Subangari Dorasamy Wesley Roy Balasubramanian Sivapriya Marappan Umarani Subramaniam Kavitha Nellore Thomas Anthony Google Mohamad Fairus Abdul Kadir Pratheev Alagapan Yang Jin Lim Wai Mun Kong Dharvind Balan Mathura Subangari Dorasamy Wesley Roy Balasubramanian Sivapriya Marappan Umarani Subramaniam Kavitha Nellore Thomas Anthony Google Scholar Mohamad Fairus Abdul Kadir Pratheev Alagapan Yang Jin Lim Wai Mun Kong Dharvind Balan Mathura Subangari Dorasamy Wesley Roy Balasubramanian Sivapriya Marappan Umarani Subramaniam Kavitha Nellore Thomas Anthony PubMed Mohamad Fairus Abdul Kadir Pratheev Alagapan Yang Jin Lim Wai Mun Kong Dharvind Balan Mathura Subangari Dorasamy Wesley Roy Balasubramanian Sivapriya Marappan Umarani Subramaniam Kavitha Nellore Thomas Anthony Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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