NaLTSA

Target engagement using NanoLuc luciferase thermal shift assay (NaLTSA)

Target engagement through ligand induced thermal stabilization can be characterized by several approaches using biochemical and cellular approaches. However, these impose encumbering operational requirements, such as the availability of large amounts of purified proteins or selective antibodies. Appending the target protein of interest (POI) with a small luciferase (NanoLuc®) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive methodology for assessing target engagement. This method is referred as the NanoLuc luciferase TSA or NaLTSA. NanoLuc® is a registered trademark of Promega Corporation.

*(Image source – ACS Medicinal Chemistry Letters 2018 9 (6), 546-551)*

Figure 1. NaLTSA assay principle and workflow. Compounds that bind NanoLuc-POI fusion will lead to stabilization compared to controls resulting in differences in Tm as detected by luminescence.

At o2h, we have validated the NaLTSA approach using the NanoLuc (Nluc) tagged BRD4^BD2 protein and its small molecule inhibitors. Bromodomain-containing protein 4 (BRD4) protein represents an attractive target for cancer therapy due to its role in regulation of histone lysine acetylation. BRD4 interacts with acetylated lysine residues on histone using two N-terminal bromodomains (BD1 and BD2). First, we determined the melting temperature (Tm) of the Nluc:BRD4^BD2 protein. This was followed by evaluating the compound induced protein stabilization by measuring the apparent shift in Tm values (ΔTm). Higher ΔTm value indicates stronger ligand-mediated protein thermal stabilization, higher target affinity, and can be used for compound rank-ordering. This was in line with data obtained from alternate cell-based target engagement assay (NanoBRET assay)

Figure 2. Melting temperature (Tm) determination of Nluc:BRD4^BD2 using NaLTSA. Nluc:BRD4^BD2 protein showed temperature dependent unfolding in the presence of vehicle (1% DMSO) in NaLTSA assay. The Tm was found to be 53.6⁰C (mean of two independent experiments). The NaLTSA assay was found to be robust, producing excellent curve fitting (R² ≥ 0.98) and reproducibility across independent experiments (CVs < 10%)

Figure 3. Compound induced Nluc:BRD4^BD2 target engagement levels using NaLTSA. BRD4^BD2 ligands of varying potencies showed different apparent Tm shifts (ΔTm) compared to the vehicle control. Higher ΔTm value indicates stronger ligand-mediated protein thermal stabilization and higher affinity. ABBV-075 shows highest Nluc:BRD4^BD2 engagement with ΔTm = 4.3⁰C. The low binder compound shows negligible engagement with ΔTm = 0.1⁰C. Compound rank ordering can be performed using ΔTm values generated in the NaLTSA assay

Figure 4. Compound potency using NanoBRET target engagement assay. ABBV-075 shows highest inhibition of Nluc:BRD4^BD2 activity in NanoBRET target engagement assay with IC50 = 1.3 nM. The low binder compound shows no inhibition of Nluc:BRD4^BD2 activity. This matches with the NaLTSA target engagement data profile

To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.

Sunil Shah

CEO - o2h Ventures and Co-Founder - o2h discovery

Sunil's Biography

Sunil Shah

CEO - o2h Ventures and Co-Founder - o2h discovery

A serial entrepreneur having begun a career in the Life Sciences team at PA Consulting group followed by co-founding two companies in the information technology and life sciences sector. The second of these companies, Oxygen Healthcare Ltd was acquired by Piramal Enterprises Ltd (BSE: PEL). Sunil co-founded o2h ventures which involves discovery services / collaborations, seeding drug discovery, academic in-licensing and biotechnology incubation. Sunil has a degree in Biochemistry and an MBA from Cambridge University

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Prashant Shah

CEO - o2h discovery and Co-Founder - o2h group

Prashant's Biography

Prashant Shah

CEO - o2h discovery and Co-Founder - o2h group

Prashant is a serial entrepreneur in life sciences and tech in which one of those companies was acquired by a public company. He is currently active in seed investing (a portfolio of ~50 companies), product/IP development, services, and building lab/office infrastructure. The early career was with the Strategy group at Accenture. He has a BEng, an MSc, in which he worked on the Human Genome Program at the Sanger Centre, and an MPhil in Management from the Judge Institute. Prashant is also a General Partner in the o2h SEIS/EIS Human Health Funds.

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Andy Morley

Chief Scientific Officer

Andy's Biography

Andy Morley

Chief Scientific Officer

Andy is a highly experienced and accomplished Medicinal Chemist with over 25 years of experience in major pharmaceutical companies such as Sanofi-Aventis and AstraZeneca. He has extensive experience across all phases of drug discovery and has played a key role in the development of five candidates that have reached clinical trials. Andy is a prolific author and inventor, with over 55 publications and patents to his name. Since 2013, he has been working full-time with o2h Limited, where he leads the scientific evaluation of investment opportunities and provides scientific support. He has also served as CSO for two early-stage collaborations within the o2h Ventures portfolio, demonstrating his ability to successfully guide drug discovery projects from concept to clinical development.

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Nilesh Dagia

Chief Special Projects Officer

Nilesh's Biography

Nilesh Dagia

Chief Special Projects Officer

Nilesh leads the global operations of o2h group covering a wide range of innovation led investment, life-science and technology businesses. He is also overseeing the development and execution of the new o2h discovery Shirish Research Centre in Ahmedabad, India. Prior to joining o2h group, Nilesh worked with Piramal Group in various capacities including as an Alliance Manager for a risk-share oncology-based collaboration with a US Big Pharma and has also worked as the Head of Biology in Piramal Discovery solutions. Nilesh obtained his Ph.D. from Ohio University and completed a post-doc in Immunology, Stem Cells and Regenerative Medicine at Harvard Medical School. He is the author and inventor of >30 life science patents and publications. He received the Young Scientist of India award from OPPI in 2010.

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Meet our team

what our clients say

Brian Dymock

Head of QEDDI

Our relationship with o2h over many years supporting our medicinal chemistry projects in QEDDI has been highly productive. With o2h’s help we have progressed our portfolio to an advanced stage ready for partnering. We are very pleased with the efficiency, productivity and communication with o2h chemists and look forward to further positive outcomes.

Prof Kirill Alexandrov

CSIRO, QUT Synthetic Biology Alliance

We have worked with o2h team on a range of synthetic projects and had consistently positive experience .The combination of competence, effective project management and detailed documentation made us go back again and again.

Sharon Shacham

Co-Founder & MD, E44 Ventures

My relationship with o2h Discovery is now more than a decade. I have had many opportunities to collaborate with the o2h team for my drug discovery efforts at Karyopharm, Protai and Sporos Bioventures. I have always been impressed by their efficiency to deliver novel chemical compounds that helps to achieve targeted project milestones. Real time and insightful communication is also their key differentiator.

Julian Blagg

Executive Vice President, Drug Discovery

NeoPhore has worked closely with o2h for over 4 years, consistently supporting NeoPhore with small and medium scale synthesis and characterisation of novel small molecule therapeutics; work that has enabled significant Series A and Series B investment. NeoPhore highly values the synthetic expertise, efficiency, project management and clear communication at o2h. Importantly, o2h benefits from highly integrated analytical and compound purification groups to enable consistent and smooth delivery of high quality target compounds to tight deadlines. Furthermore, flexibility of resourcing at o2h is ideally suited to support an evolving small biotech company like NeoPhore.

Bharat Majeti

Head of LNP Technology, RVAC Medicines

We understand that lipid chemistry is complex and requires significant expertise in synthesis, purification, and analysis. Therefore, we looked for a competent and reliable CRO partner to support our requirements and found o2h Discovery. We have done several projects with o2h.

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