Amgen Discovers MTA-Cooperative PRMT5 Inhibitors via Cofactor-Directed DNA-Encoded Library Screening

Jun 21, 2025 10:29 CST Updated 10:29

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DOI: 10.1073/pnas.2425052122

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Amgen's AMG 193 has recently entered Phase 1/2 clinical trials for the treatment of tumors associated with the overexpression of protein arginine methyltransferase 5 (PRMT5); this is another example of a discovery originating from DEL and advancing to the clinic, involving the transition from DEL Hit to the identification of the truncated molecule AM-9959, as well as the optimization of lead compounds AM-9934 and AM-9747.

In mid-March, Amgen J. Med. Chem. The discovery surrounding AM-9959 and the process of optimizing the left side, then the right side, leading to the discovery of AM-9934 and AM-9747 were disclosed (see related sharing at the end of the article). However, the previous article did not provide a detailed explanation of the DEL screening setup.

Recently (May 16), Amgen PNASA detailed report was made on the detailed DEL screening and the study of AM-9934.J. Med. Chem.The report was supplemented.

Next time, let's talk about another article by Amgen at the end of March. J. Med. Chem.Detailed Discovery Process of AMG 193 in China.

Original text open source, click at the end of the article"Read the original text"Can reach the original journal directly.

1. MTA Synergizes with PRMT5 Inhibition

Biological Significance and Inhibition of PRMT5

Deletion of the methylthioadenosine phosphorylase (MTAP) gene is very common in human cancers, and this deletion significantly increases the level of methylthioadenosine (MTA) within cells.

MTA is an essential enzyme for cells.Protein Arginine Methyltransferase 5 (PRMT5)'s natural specific inhibitor, therefore, causes insufficient PRMT5 activity in MTAP-deficient cells.

PRMT5 is the major type II arginine methyltransferase, highly expressed in various cancers and promoting tumorigenesis. PRMT5 is a transcriptional target of MYC, maintaining MYC-driven lymphoma through the regulation of RNA splicing.

The Catalytic Mechanism of PRMT5As follows: Endogenous PRMT5 forms a heterooctameric complex with methylosome protein 50 (MEP50), wherein PRMT5 binds to the arginine residues of substrate proteins and from the cofactor substratesS-Adenosylmethionine (SAM)Transfer methyl to target arginine.

SAM is easily degraded into methylthioadenosine (MTA), and MTA is a SAM-competitive inhibitor of PRMT5.

Although several PRMT5 small molecule inhibitors have entered clinical trials, they are associated with adverse reactions such as gastrointestinal symptoms and pancytopenia, andPRMT5 is important in normal cell and organism biology, which may lead to a narrow therapeutic window.

Research Objectives and Methods

Therefore, given that PRMT5 overexpression promotes tumorigenesis while a certain level of PRMT5 must be maintained in the body, the research team designed and developedIn the presence of MTA, inhibitors that synergistically suppress PRMT5 can selectively target MTAP-deficient cancer cells, reducing adverse effects on normal tissues.。

One major advantage of DEL screening is the ability to simultaneously screen immobilized target proteins against vast chemical spaces comprising hundreds of millions of compounds. However, its potential limitation lies in the fact that the enriched compounds during screening are only highlighted due to their affinity binding with the target, without undergoing functional evaluation.

To this end, the research team adopted a cofactor-directed DNA-encoded library (DEL) screening, which can directly identify binders of the target mechanism.

2. Cofactor-Oriented DEL Screening and Validation

Cofactor-Oriented DEL Screening

DEL Library: Two libraries were selected, collectively containing approximately 226.4 million DEL molecules. The composition of the two DELs is shown in the figure below.

Among them, the composition of DEL91 in the reported data is as follows.

Dual-track DEL Screening Setup: The research team employed cofactor-directed DNA-encoded library (DEL) screening, specifically using a His tag-labeled PRMT5:MEP50 heterotetrameric complex for DEL screening in the presence of MTA or SIN (SAM analogs), with parallel screening controls set up in the absence of cofactors.

As shown in the figure below, the process begins with target protein immobilization, followed by cofactor-assisted DEL library co-incubation (no cofactor is involved in parallel screening). Afterward, washing and elution are performed. This two-round screening process is followed by sequencing and barcode deconvolution to identify the enriched molecules.

Screening Logic: In the presence of MTA, compounds that preferentially inhibit PRMT5 may represent a promising therapeutic approach for MTAP-deficient cancers, with minimal adverse effects on normal tissues.

Therefore, in the presence of MTA or SAM analogs (SIN), DEL screening of PRMT identifies compounds that depend on specific cofactors binding to PRMT5. Compounds dependent on MTA inhibit PRMT5 activity in an MTA-cooperative manner and selectively kill MTAP-null cancer cells while sparing MTAP wild-type cells.

Post-screening Data Analysis

First, remove the enriched DEL molecules with independent sequence counts less than 3, then use a scatter plot to cluster and visualize the remaining DEL molecules after truncation.

The cube in the figure below represents the visualization of the three-cycle library DEL91 after screening and truncation, where yellow, brown, and blue represent the three main characteristic sequences after clustering. The left figure shows the result of MTA-directed selection, and the right figure shows the result of SIN-directed selection.

Yellow Series 1Representatives are mainly enriched in MTA-directed selection and also show detectable enrichment in SIN-directed selection. This suggests that the series has minimal selectivity. The series was found to be metabolically unstable during subsequent extensive medicinal chemistry optimization (not shown in the text).

The Brown Series 2 also did not show selection dependency on MTA, and no further description was provided.

Blue Series 3Enrichment was observed only in the presence of MTA in the graph, indicating that this series selectively binds to the MTA-bound form of PRMT5. This series previously J Med ChemThe report provided a detailed analysis (as shown in the figure below).

The series was found in subsequent validation to have quinoline as the key pharmacophore at POS2. Additionally, the truncated molecule with a retained amino group was located at POS3. This led to the initial DEL Hit AM-9959, which was optimized to obtain AM-9934.

As shown in the figure below, it represents the visualization of the enrichment and truncation of the two cyclic libraries DEL123. The left side shows MTA-directed selection, and the right side shows SIN-directed selection. The clustering results indicate that one major characteristic structural series was generated, and this series was enriched only in SIN-directed selection, as shown in brown in the figure below.

In addition, there is a small chemical series, as shown in yellow, which is non-selective; several representatives were synthesized, but they failed to demonstrate any inhibitory activity against PRMT5, and the article did not provide specific details.

DEL123 is primarily enriched in a series strongly associated with the PRMT5 inhibitor EPZ015666 (also known as GSK3235025) discovered by GSK in 2016. Therefore, researchers did not conduct an in-depth study of this series but instead used the known EPZ015666 as a tool molecule for research.

DEL Hit and Optimization Product Characterization

On-DNA Validation

First, to validate the selectivity of the DEL91 Series 3 for MTA-bound PRMT5, the research team synthesized an oligonucleotide conjugate of a representative Hit compound, AM-9959, to mimic the DEL molecule, using an oligonucleotide conjugate of EPZ015666 as the tool molecule control, as shown in the figure below.

Simulate DEL screening by performing a round of wash-elution on oligonucleotide conjugates using the apolipoprotein (APO) form or cofactor-bound form of the PRMT5:MEP50 complex, and quantify the enrichment level of compounds relative to unbound oligonucleotides using compound-specific primers through qPCR, as shown in the figure below.

The results showed that the EPZ015666 oligonucleotide conjugate had little enrichment for the MTA-bound PRMT5 complex, consistent with previously reported data; whereas it exhibited a strong enrichment preference for the SIN-bound complex.

In contrast, the AM-9959 oligonucleotide conjugate exhibited a clear enrichment preference for the MTA-binding complex, with lower enrichment for SAM-binding enzymes and even less enrichment in the SIN environment.

Given that SAM is prone to degradation into MTA, it can be inferred that the moderate enrichment of the conjugate for SAM may be due to potential contamination of SAM by MTA.

Off-DNA Validation

Thereafter, the research team synthesized compounds free of oligonucleotide conjugation to test whether the DEL hit would retain binding properties, as shown in the figure below.

The binding of compounds to PRMT5 was studied using Thermal Stability Analysis (TSA). As shown in Figure A below, the results are consistent with the on-DNA findings.

To verify whether this combination inhibits the enzymatic activity of PRMT5, the researchers then tested the catalytic activity of the enzyme in the presence or absence of MTA, as well as with the addition of a certain concentration of the compound, as shown in Figure B below.

The results showed that AM-9959 inhibited the independent PRMT5 activity with an IC₅₀= 520 nM, while the addition of MTA further enhanced the inhibitory activity of AM-9959 to 160 nM.

EPZ015666 can also effectively inhibit the enzymatic activity of PRMT5, IC₅₀= 78 nM, however, differently, the addition of MTA did not further enhance its inhibitory activity, but slightly weakened it, IC₅₀= 120 nM, consistent with TSA data.

AM-9959 exhibits MTA selectivity characteristics but lacks the potency for cellular entry and in vivo efficacy. Medicinal chemistry optimization (see previous sharing at the end of the article) led to AM-9934, whose oligonucleotide conjugate also demonstrates a strong preference for the MTA-binding enzyme complex, with enhanced affinity (TSA assay) and enzymatic activity inhibition (standalone IC).₅₀= 40 nM, enhanced to 5 nM with MTA participation, as shown in Figure B below).

Orthogonal Method Evaluation of AM-9934 MTA Synergistic Affinity

First, construct the AM-9934 conjugated fluorescent molecule and use fluorescence polarization (FP) to analyze the affinity of the fluorescently labeled compound in APO, SIN, and MTA environments, as shown in Figure C above, where KD = 754 nM under APO, not applicable in SIN, and KD = 38 nM under MTA.

Secondly, microscale thermophoresis (MST) was used to detect AM-9934 directly binding to 25 nM PRMT5, as shown in Figure D above. In the presence of MTA, the compound binding KD was 55 nM.

Comprehensive FP and MST assays demonstrate that AM-9934 exhibits high selectivity for MTA-bound PRMT5.

As shown in Figure E, AM-9934 and EPZ015666 can potently inhibit PRMT5 without the addition of excess MTA, with IC50 values of 12 nM and 10 nM, respectively. Moreover, they show no significant inhibition of other methyltransferases, indicating high target selectivity.

Cell Tests and Animal Tests

In cellular experiments, AM-9934 demonstrated selective inhibitory effects on MTAP-deficient cancer cells, with minimal impact on MTAP-expressing cells.

In animal models, AM-9934 specifically inhibits the growth of MTAP-deficient tumors while exhibiting low toxicity to normal tissues.

3. Summary and Outlook

This study not only provides a new method for discovering small molecule inhibitors with specific mechanisms but also offers a novel strategy for the precision treatment of MTAP-deficient cancers.

By exploiting the differential levels of the intracellular metabolite MTA, selective killing of tumor cells can be achieved while minimizing damage to normal tissues.

In addition, this study also demonstrated the potential of DEL technology in discovering multi-specific drugs.Demonstrates a cofactor-oriented DEL screening strategy for the targeted discovery of inhibitors with specific mechanisms of action., providing a widely applicable method for drug development.

These drugs can achieve the desired pharmacological effects by interacting with multiple components.

AMG 193, a clinical candidate optimized based on AM-9934Has shown preliminary anti-tumor responses in MTAP-deficient tumors in Phase I clinical trials. Other studies on MTA-cooperative PRMT5 inhibitors also support this therapeutic approach, with multiple studies advancing to Phase II clinical trials.

Continue to follow the latest developments of clinical candidates from DEL.

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