PROTAC

PROTAC (PROteolysis TArgeting Chimera) is an exciting arena in intracellular Targeted Protein Degradation, which enables therapeutic targeting of disease-causing protein, through its proteasome-mediated selective degradation. A PROTAC is a bispecific chemical linker which binds to the target protein at one end and an E3 Ubiquitin ligase at the other end, thereby forming a ternary complex. This proximity enables rapid and selective ubiquitination of the intracellular target protein and subsequently its proteasome-mediated degradation.

PROTAC offers several therapeutic advantages during the drug discovery:

• The PROTAC molecule does not need to target the active site of a target protein. Instead, it can target any site.
• Analogous to the conventional knockdown, PROTAC depletes the target protein.
• PROTAC molecules are not degraded with target protein; instead, they are recycled to turn over more target molecules, enabling sub-stoichiometric dosing.
• Cooperative PROTAC’s can engage target proteins into a high affinity ternary complex, further enhancing the PROTAC potency.

Formation of high affinity and highly stable ternary complex is critical for the success of PROTAC’s as it ensures greater levels of target protein ubiquitination and its rapid rate of proteolysis. Moreover, PROTAC’s which form cooperative complexes often bind target proteins in a ternary complex with a higher affinity than their constituent target binder alone.

Studying protein-protein interactions (PPI’s)

To study protein-protein interaction (PPI), a number of biophysical and biochemical methods are available, which includes fluorescence polarization (FP), time-resolved fluorescence energy transfer (TR-FRET), and AlphaScreen/AlphaLISA technologies. All these methods make use of interaction between a target protein or ligase and a binding partner coupled to a probe. When in complex with the target protein, the probe produces a signal that is distinct from the signal produced when the probe is free in solution. However, these techniques are limited by the necessity of labeling the proteins as well as their inability to measure certain important biophysical parameters, such as the kinetic rate constants for ternary complex formation and the ternary complex t_1/2.

Alternatively, Isothermal Titration Calorimetry (ITC) can be used to measure thermodynamic properties and confirm binding stoichiometry. However, it is limited by very low throughput and high protein requirements, and it provides no information on binding kinetics.

Surface Plasmon Resonance (SPR)

Overcoming all the above limitations, Surface Plasmon Resonance (SPR) is advantageous in biophysical characterization of the PROTAC and it is currently the only available technique that enables measurement of PROTAC ternary kinetics with a medium throughput.

Some of the advantages of SPR for studying biophysical parameters of PROTAC are as below:

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

At o2h, we offer end-to-end custom PROTAC services from design, chemical synthesis, and biological evaluation. We also have a VHL and CRBN-based PROTAC tool box in place to expedite the PROTAC discovery services for client-based projects.

We screen a library of potential PROTAC molecules by assessing their affinity of interaction with the target protein by employing SPR. We perform binary (between PROTAC molecule and target protein) as well as ternary (E3 ligase : PROTAC : Target protein) interaction studies by SPR to identify a PROTAC molecule with the highest affinity.

Case study: Studying Binary and Ternary interactions of PROTAC MZ1 with VCB (VHL/Elongin B/Elongin C) complex and the target protein BRD4^BD2 by SPR

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.

To know more about our biology capabilities and our broader services, please reach out to us at discovery@o2h.com.