How Genentech Used Direct Measurement of ATP Hydrolysis for the Discovery of a Clinical RIP1 Inhibitor

Drug discovery campaigns are often remembered for the final molecule that reaches the clinic. Less visible—but equally important—is the assay strategy that made the discovery possible. The identification of GDC-8264 by researchers at Genentech revealed a potent and selective RIP1 kinase inhibitor now in Phase 2 clinical development. This provides a clear example of how assay design can shape the trajectory of an entire program.

At the foundation of this effort was a straightforward decision: measure enzymatic activity directly.

Beginning with Catalysis

Receptor-interacting protein kinase 1 (RIP1) regulates inflammatory cell death pathways, particularly necroptosis. Because its kinase activity drives pathological signaling, the discovery team chose to screen for compounds that inhibited ATP hydrolysis rather than relying on binding assays or indirect reporters.

To do this, they conducted a high-throughput screen of approximately 2.7 million compounds, monitoring RIP1 kinase domain–catalyzed hydrolysis of ATP at Km(app) concentrations. Compounds demonstrating greater than 50% inhibition at screening concentrations were advanced for follow-up titration, and apparent inhibition constants (Ki(app)) were determined using the same biochemical ATP hydrolysis assay.

This approach ensured that every hit represented functional inhibition of catalytic turnover.

Rather than asking whether a compound bound to RIP1, the assay asked a more direct question: does it stop ATP from being converted to ADP?

That distinction proved important.

An Allosteric Opportunity Revealed

The initial screening hit, a substituted pyrazole, showed micromolar inhibition of RIP1 activity.

Structural studies later revealed that the compound did not bind in the canonical ATP hinge region. Instead, it occupied a deep allosteric pocket near the αC helix in a DLG-out inactive conformation.

Because the primary screen measured ATP hydrolysis directly, the discovery team was not limited to hinge-binding chemotypes. Any molecule capable of suppressing catalytic activity could be identified, regardless of binding location.

This opened the door to a structurally novel and highly selective inhibitor series.

Had the campaign relied solely on hinge-focused binding assays, this allosteric opportunity might have been overlooked. By focusing on catalytic output, the screening format allowed the chemistry to evolve without being constrained by predefined binding assumptions.

Driving Medicinal Chemistry with Quantitative Enzyme Data

Once the initial hit was identified, the same ATP hydrolysis assay became the central tool for medicinal chemistry optimization. Compounds were evaluated through consistent Ki(app) measurements under defined biochemical conditions.

Through iterative scaffold refinement, solubility optimization, and structure-guided design, the team improved potency by more than two orders of magnitude. The campaign progressed from an early hit with Ki(app) ≈ 0.084 μM to a final clinical candidate with human RIP1 Ki(app) = 0.00071 μM.

This quantitative framework allowed potency, ligand efficiency, and drug-like properties to be evaluated in parallel. Because the readout reflected direct catalytic inhibition, SAR decisions were grounded in mechanistic data rather than indirect surrogates.

The biochemical assay was not simply a gatekeeper at the beginning of the campaign—it was the backbone of optimization throughout.

From Enzyme Inhibition to Biological Impact

The optimized compound, GDC-8264, demonstrated sub-nanomolar biochemical inhibition and strong selectivity for RIP1 across broad kinase panels

Importantly, this enzymatic inhibition translated into cellular activity, including blockade of RIP1-dependent necroptotic cell death and inhibition of inflammatory cytokine production in human whole blood

Further studies confirmed favorable pharmacokinetics, in vivo efficacy in inflammatory models, and an acceptable safety profile, ultimately supporting advancement into clinical trials

A notable mechanistic finding was that GDC-8264 inhibited RIP1 kinase activity without interfering with RIP1’s scaffolding functions involved in NF-κB signaling. This separation of catalytic inhibition from scaffolding activity underscores the importance of measuring the correct biochemical function during screening.

When catalytic turnover is the primary endpoint from the outset, downstream biology is more likely to align with the intended therapeutic mechanism.

A Broader Lesson in Assay Strategy

The RIP1 program illustrates a broader principle in enzyme drug discovery: when the therapeutic objective is to inhibit catalysis, the primary screen should measure catalysis directly.

Direct nucleotide detection provides a universal readout for kinases and ATPases. It avoids bias toward specific binding motifs and enables identification of unconventional inhibitor classes, including allosteric modulators.

In this case, a decision made at the earliest stage of screening shaped the outcome of the entire program. The biochemical ATP hydrolysis assay enabled the identification of a novel scaffold, guided quantitative optimization, and ultimately contributed to the advancement of a selective RIP1 inhibitor into clinical development.

The molecule’s journey to the clinic is a reminder that robust assay design is not merely a technical detail—it is often the first determinant of discovery success.

Learn more about the Transcreener Kinase Assay used in the study.

Read the publication from Genentech.