VAV1, a pivotal guanine nucleotide exchange factor, plays a crucial role in immune cell signaling and its dysregulation is implicated in autoimmune diseases and cancers. Recent innovations in molecular glue degraders (MGD), exemplified by MRT-6160, are transforming the landscape of drug discovery by targeting previously undruggable proteins. These novel compounds leverage a unique mechanism to induce the formation of ternary complexes, leading to selective degradation of proteins like VAV1.

MRT-6160, a first-in-class VAV1-directed molecular glue degrader, has demonstrated remarkable potential in preclinical and early clinical studies. It effectively induces VAV1 degradation, attenuating immune-mediated inflammation and disease progression in conditions such as rheumatoid arthritis and colitis. Current advancements highlight its ability to reduce proinflammatory cytokine production, inhibit pathogenic T cell polarization, and mitigate autoimmune responses.

Our comprehensive platform supports the exploration of VAV1-targeting molecular glues by integrating cutting-edge in vitro assays. These include detection of binary and ternary complexes, intracellular protein interactions, and target degradation analysis using HiBiT assays. Complemented by functional assessments of T and B cell activities, cytokine profiling, and proteomics, this platform provides robust insights into the selectivity and off-target effects of these novel drugs.

We rapidly expressed and purified high-purity VAV1 protein, which was successfully utilized in subsequent biochemical and biophysical assays.

Figure 1: Analysis of Purified VAV1 Protein.

To comprehensively evaluate binary and ternary complex formation in molecular glue studies, we have developed and applied multiple complementary assays, including TR-FRET, Spectral Shift, and NanoBRET PPI.

1. CRBN Binary Assay Using TR-FRET

This assay measures the binding between CRBN and molecular glue degraders (MGDs). The mechanism involves MGDs competitively binding to CRBN, displacing fluorescently labeled substrates and resulting in a decrease in signal intensity.

Figure 2: A) Mechanism of CRBN binary assay; B) Example data of CRBN&MGD binding.

2. CRBN Ternary Complex Formation Assay

This assay utilizes HTRF to detect the formation of a ternary complex between VAV1 and CRBN. In the absence of MGDs, no FRET signal is observed as VAV1 and CRBN do not interact. However, MGDs bring VAV1 and CRBN into close proximity, enabling the detection of a measurable FRET signal.

Figure 3: A) Mechanism of CRBN ternary complex formation assay; B) Example data of CRBN/VAV1 ternary complex formation.

3. Spectral Shift Assay

The Dianthus Spectral Shift Assay is a label-free method using spectral scattering to detect small shifts under 590 nm excitation, measuring Kd values at 650 nm and 670 nm. It is ideal for identifying low-affinity ligands and weak interactions. This developed assay measures the binary binding affinity between CRBN and MGDs, as well as the ternary complex formation involving CRBN, MGDs, and VAV1.

Figure 4. A) Binary Binding: CRBN vs. VAV1 Degrader. B) Ternary Complex Formation: CRBN, VAV1, and Degrader.

4. NanoBRET PPI Assay

The NanoBRET PPI Assay offers high sensitivity and detection of protein-protein interactions in living cells. In this assay, VAV1 and CRBN are tagged with NanoLuc luciferase (donor) and HaloTag (acceptor), respectively. MGDs promote the formation of a stable ternary complex, bringing HaloTag ligands into proximity with NanoLuc. This induces BRET, generating a measurable fluorescent signal.

Figure 5: A) Mechanism of NanoBRET PPI assay. B) Example Data: NanoBRET PPI assay results for VAV1-CRBN interaction, demonstrating signal changes upon MGD treatment.

1. VAV1 Degradation by HiBiT Assay

The HiBiT assay uses a small 11-amino acid HiBiT tag, inserted at the start or stop codon of the target gene, to enable precise and functional tagging without altering the protein’s activity. This tag allows sensitive detection of endogenous protein levels, making it ideal for target protein degradation studies and high-throughput screening. We have developed the Jurkat-VAV1-HiBiT-KI cell line to facilitate high-throughput screening of VAV1 degraders.

Figure 6: A) Mechanism of HiBiT assay. B) VAV1 degradation data in Jurkat-VAV1-HiBiT-KI cells.

2. VAV1 Degradation Analysis Using the Jess System

The Jess system automates protein analysis, streamlining Western blot workflows with capillary electrophoresis for protein separation and quantitative immunodetection. It enables precise and reproducible quantification of protein expression levels. ICE has implemented the JESS system to measure VAV1 degradation across different samples.

Figure 7: A) VAV1 degradation assay in Jurkat, T and B cells. B) Time-dependent VAV1 degradation: data from PBMC cells.

VAV1 is a positive regulator of T cell receptor (TCR) and B cell receptor (BCR) signaling. Upon activation by stimuli such as TCR, BCR, and cytokine receptors, VAV1 undergoes rapid tyrosine phosphorylation, facilitating critical processes like gene transcription, immune cell development, and activation. Reducing VAV1 levels effectively decreases immune cell activation, proliferation, and cytokine production.

1. Degradation of VAV1 inhibits T- and B-cell activation and effector functions

Figure 8: Purified primary human pan-T cells were pre-treated with compound for 24 hrs followed by CD3/CD28 TCR stimulation & subsequent analyses after 24 hrs (A. CD69 expression), 48 hrs (B. IL-2 secretion), or 96 hrs (C. cell proliferation).

Figure 9: Purified primary human B-cells were pre-treated with compound for 24 hrs followed by BCR stimulation and subsequent analyses after 24 hrs (anti-IgM + IL-4: A. CD69 expression.  B.  IL-6 secretion).

2. Degradation of VAV1 inhibits CD4+ TH17 polarization in a concentration-dependent manner

Figure 10: Primary human CD4+ T cells were pre-treated with compound for 24 hrs, followed by CD3/CD28 TCR stimulation and polarization towards the TH17 subtype as indicated (IL-6, TGF β1, IL-1β, IL-21, IL-23, IL-4 and IFN-γ). After 5 days, IL-17A levels (pg/mL) in the supernatant were assessed by ELISA.

Data-Independent Acquisition (DIA) proteomics was used to assess the selectivity of the test compound in a dose-dependent manner. Volcano plot analysis of differential proteins at 50 nM and 500 nM showed VAV1 as the only consistent target. Manual confirmation of partially overlapping proteins found no highly credible off-target effects. Additionally, no binding to common CRBN neo-substrates was detected, confirming the compound's strong target degradation activity and selectivity.

Figure 11: Human PBMCs were treated with 50 nM or 500 nM VAV1 MGD or DMSO control for 24 hours. DIA proteomics confirmed the molecule's strong target degradation activity and high selectivity.

The common challenges of VAV1-like targets, such as complex ternary formation, stringent selectivity, and functional validation, are precisely the strengths of our platform. By addressing these commonalities through robust assay development and scalable workflows, we provide a universal solution for discovering and optimizing MGDs with broad therapeutic potential. Our platform doesn’t just target VAV1—it enables researchers to uncover and exploit the shared principles of molecular glue activity, driving innovation across a wide range of targets.

Reference

[1]. Neurath, M.F. and Berg, L.J. (2024) ‘Vav1 as a putative therapeutic target in autoimmune and chronic inflammatory diseases’, Trends in Immunology, 45(8), pp. 580–596. doi:10.1016/j.it.2024.06.004.

[2]. A vav1-directed molecular glue degrader, MRT-6160, reduces joint inflammation in a collagen-induced arthritis autoimmune disease model (no date) ACR Meeting Abstracts. Available at: https://acrabstracts.org/abstract/a-vav1-directed-molecular-glue-degrader-mrt-6160-reduces-joint-inflammation-in-a-collagen-induced-arthritis-autoimmune-disease-model/ (Accessed: 22 January 2025).