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.

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

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