PROteolysis TArgeting Chimeras (PROTACs) are a specific type of molecule designed for Targeted Protein Degradation (TPD). TPD involves employing heterobifunctional small molecules known as “Degraders,” to effectively reduce the levels of specific target proteins within cells. PROTACs consist of a target binding handle bridged via a chemical linker to an E3 ligase handle, and hijacks the endogenous ubiquitin machinery to target proteins for ubiquitination and subsequent degradation.

PROTACs provide significant therapeutic potential with possible prolonged pharmacodynamics, improved potency, and ability to target proteins previously thought of as “undruggable”.

o2h discovery offers integrated PROTAC services as well as a bespoke and customisable “off-the-shelf” PROTAC toolbox to jump-start your targeted protein degradation program, along with our strong expertise in medicinal and synthetic chemistry. Alongside this, our experienced biology team is ready to help you get more out of your PROTAC and TPD molecule discovery with assays that can asses key features like ternary complex formation, screen for endogenous protein degradation and estimate DC50 (half maximum degradation concentration) values, providing a fully integrated offering.

protein degradation – biology

Our biology evaluation suite to estimate ternary complex formation and targeted protein degradation

The advantages of SPR for studying biophysical parameters of PROTAC binding are-

1. Capable of measurement of both binary and ternary affinity and kinetics
2. Reproducible results
3. Label-free assay readout
4. Medium but faster throughput compared to other biophysical techniques such as ITC
5. Results correlate with in-solution assays such as ITC, as well as cell-based approaches

Binary interaction between VCB complex and MZ1   

To study the binary interaction, we immobilized the His-tagged VCB complex (Ligand) on a biosensor surface (Ni²⁺ activated NTA chip) followed by flowing MZ1 molecule (Analyte) at increasing concentrations. The binary interaction between VCB and MZ1 was demonstrated in a dose-dependent manner evident from proportional increase in Response values (Figure 1). From the kinetic analysis of the data, we obtained a K_D value of the interaction to be 75.2 nM.

Figure 1: Studying the binary interaction between VCB complex and MZ1. The His-tagged VCB complex (15 µg/mL) was immobilized on the NTA sensor chip. MZ1 was passed onto the VCB-immobilized surface at increasing concentrations (1.6 nM to 1000 nM). The sensorgrams on the left exhibit MZ1 dose-dependent response. The binding response on the right is represented as a function of MZ1 concentration.

Binary interaction between BRD4^BD2 and MZ1    

To confirm the binary interaction in a reverse experiment, we immobilized His-tagged BRD4^BD2 protein on the chip surface (Ni²⁺ activated NTA sensor chip) and MZ1 molecule was passed over the chip surface in a range of concentrations. The binary interaction between BRD4^BD2 and MZ1 was evident from increase in Response in a dose-dependent manner (Figure 2). From the kinetic analysis of the data, we obtained a KD value of the interaction to be 13.8 nM.

Figure 2: Studying the binary interaction between BRD4^BD2 and MZ1. The His-tagged BRD4^BD2 (5 µg/mL) was immobilized on the NTA sensor chip. MZ1 was passed onto the BRD4^BD2-immobilized surface at increasing concentrations (1.6 nM to 1000 nM). The sensorgrams on the left exhibit MZ1 dose-dependent response. The binding response on the right is represented as a function of MZ1 concentration.

Ternary interaction

To study the ternary interaction, we immobilized the ligand His-tagged VCB complex (2.5 µg/mL) on the NTA sensor chip surface by His capture coupling. We then prepared a complex of MZ1+BRD4^BD2 (MZ1 concentrations: 1 nM to 100 nM; BRD4^BD2 concentrations: 6 nM to 500 nM) and was passed over the immobilized ligand. The formation of the ternary complex was evident from a concentration-dependent increase in response (Figure 3). Our data analysis revealed a stronger association (high affinity interaction) of the ternary complex evident from a KD value of 5.4 nM, which also indicate a high degree of cooperativity.

Figure 3: Analyzing the ternary interaction. The His-tagged VCB complex was immobilized on the biosensor surface followed by passing the preformed complex of MZ1+BRD4^BD2 over a range of concentrations. The ternary interaction increased in a dose-dependent manner with a saturation observed at 100 nM MZ1 concentration.

Case Study 2: NanoBRET® assay system to detect the formation of the ternary complex formation mediated by PROTACs

Using a NanoBRET® assay system, where the target protein is tagged with Nanoluciferase and the recruited E3 tagged with a Halotag, we are able to quantify the interaction that occurs between them when a PROTAC is introduced to the cell by measuring the level of fluorescence emitted by the Halotag which increases upon close proximity with the nano luciferase (Figure 1).

Figure 1: A schematic illustration of the NanoBRET® assay system used to detect the formation of the ternary complex mediated by a PROTAC compound. The Target protein is fused to nano luciferase which emits light. When it comes into close proximity with an E3 ligase tagged with the HaloTag protein, the light from the nanoLuc excites the HaloTag leading to fluorescent light being emitted. In our system, this close proximity is induced by the introduction of a PROTAC molecule.

An example of PROTAC induced ternary complex formation and kinetics of targeted protein degradation using gold standard commercial PROTAC molecules.

Figure 2: PROTAC-induced BRD4-VHL ternary complex formation and BRD4 protein degradation. HEK292 cells transfected with NanoLuc-BRD4 donor plasmid and Halo-Tag-VHL acceptor plasmid at 1:100 ratios. Subsequently cells treated with 1uM MZ1 or AT1 PROTAC and compared with untreated DMSO control.

(A, B) Indicates ternary complex formation at 6h (C, D) BRD4-BD2 protein levels at 6h (E, F) real-time ternary complex formation with BRD4-B2/VHL following PROTAC treatment (G, H) real-time kinetics of BRD4-BD2 protein degradation following PROTAC treatment. MG132, a potent, reversible and cell permeable proteasomal inhibitor rescues BRD4 degradation in all condition.

Using the NanoBRET assay, we screened a set of o2h proprietary “PROTAC toolkit” and shortlisted compounds based on their varying ability to form a ternary complex and compare their degradation patterns of the target protein, BRD4.

Figure 3: Ternary complex formation between VHL E3 ligase and BRD4 when HEK293T cells are treated with 1μM of the indicated PROTAC compounds or DMSO vehicle. MZ1 is a gold standard tool compound with established activity, while cmpd 167, 169 and 173 are compounds created from the o2h PROTAC toolkit. As can be seen in the graph, cmpd 169 shows excellent ternary complex formation, cmpd 173 shows moderate complex formation and cmpd 167 shows weak ternary complex formation. This graph shows that we are able to measure ternary complex formation and differentiate between compounds that have varying abilities to form a ternary complex between E3 ligase and target protein.

Figure 4: Changes in luminescence from BRD4-linked nanoluc when treated with the indicated toolkit compounds or gold standard tool compound MZ1 at a uniform concentration of 1μM, with DMSO as a vehicle. As can be seen, cmpd 169 showed excellent degradation of the target, similar to MZ1, cmpd 173 showed a reduced degradation of the target over the time course while cmpd 167 showed essentially no degradation, with similar activity to the DMSO vehicle condition.

To accurately estimate DC50 values, we study loss of expression of a tagged version of the endogenous protein. For this, we utilize stably expressing LgBit nanoluciferase fragment, and knock-in the corresponding HiBit fragment at the endogenous locus of BRD4. When BRD4 is expressed, this creates an active luciferase protein with intensity of produced light that directly propotional to the BRD4 protein levels within the cells. With this, we are able to screen compounds over a concentration range to find accurate DC50 values with rapid turnaround time and provide medium-to-high throughput screening of PROTACs. Apart from HiBit-LgBit system providing a very bright and sensitive detection signal, it allows one to flexibly study different mechanisms of targeted protein degradation and not simply limited to PROTACs.

Figure 5: Degradation of endogenous BRD4 by VHL PROTAC molecules measured by loss of luminescence of a linked nanoluciferase. Results are shown as a percentage reduction in luminescent signal normalised to a vehicle (DMSO) treated control. DC50 of MZ1 = 25nM, DC50 of cmpd 169 = 22nM, DC50 of other compounds was unable to be determined accurately.

Together, these complementary cell-based assays are able to provide a full picture of target protein and E3 ligase interaction and target protein degradation for PROTACs.

If you want to Jump-start your PROTAC based drug discovery program (targeted protein degradation), or would like to get a quote, write to us at discovery@o2h.com