Discovery of a Novel Allosteric–Covalent Strategy to Target PDI Provides a New Therapeutic Avenue for Glioblastoma

Protein disulfide isomerase (PDI) is a key endoplasmic reticulum oxidoreductase and molecular chaperone that maintains protein folding homeostasis. Increasing evidence indicates that PDI is highly expressed in many cancers, particularly in glioblastoma (GBM), where it supports tumor cell survival, invasion, and resistance to therapy. Consequently, PDI has long been considered an attractive therapeutic target. However, most reported PDI inhibitors act by directly modifying the catalytic cysteine residues, a strategy that often suffers from limited selectivity and a high risk of off-target reactivity, thus restricting further drug development and clinical translation. How to achieve both potent inhibition and high family selectivity has remained a central challenge in the field.

A recent study delivered by Dr. AI Youwei’s lab at the Institute of Genetics and developmental Biology of the Chinese Academy of Sciences provides a solution to this long-standing problem by uncovering a fundamentally new mode of PDI inhibition. Instead of directly targeting the catalytic cysteines, the newly identified inhibitors operate through a previously unrecognized “allosteric–covalent” mechanism. This work establishes a new paradigm for selectively targeting PDI and opens a promising therapeutic direction for glioblastoma treatment.

In this study, a high-throughput screening of a drug-like compound library identified TC8026 as a PDI-active hit. Guided by systematic medicinal chemistry optimization, a new series of inhibitors based on a pyrrolo[2,3-d]pyrimidin-4-one scaffold was developed, achieving up to a 20-fold improvement in potency compared with the initial hit. Representative compounds, including 30w, 30z, 30aa, and 30ab, showed strong inhibition of PDI enzymatic activity in vitro and robustly induced endoplasmic reticulum stress and apoptosis in multiple glioblastoma cell lines. Among them, compound 30z displayed particularly promising in vivo efficacy, significantly suppressing tumor growth in a U251 xenograft mouse model.

More importantly, mechanistic investigations revealed that these compounds act through a mode of inhibition entirely different from conventional PDI inhibitors. Rather than reacting directly with the catalytic cysteines, the small molecules first bind to an allosteric pocket located in the b′ domain of PDI. This interaction involves key residues such as H256 and F304 and perturbs the substrate-binding interface. Binding at this site induces conformational rearrangements in PDI that expose a normally buried, non-catalytic cysteine residue, C312. Only after this structural reorganization does the inhibitor form a covalent bond with C312, resulting in stable and persistent inhibition of PDI.

Because C312 is unique to PDI among members of the PDI family, this two-step “allosteric–covalent” mechanism confers markedly enhanced target selectivity. In contrast to traditional covalent inhibitors that broadly react with catalytic cysteines shared by many related proteins, this strategy achieves precision by using an allosteric site to guide covalent modification to a PDI-specific residue. This discovery therefore resolves a major limitation that has hindered the development of PDI-targeted therapeutics.

Conceptually, this work establishes a new framework for covalent drug design: covalent engagement does not have to start from a reactive catalytic site, but can instead be enabled by prior allosteric binding that reshapes protein conformation and reveals a unique covalent handle. Such an approach provides a powerful route to combine the high potency of covalent inhibition with the high selectivity of allosteric targeting.

Functionally, the study demonstrates that targeting PDI through this novel mechanism effectively induces endoplasmic reticulum stress and triggers apoptotic death of glioblastoma cells, leading to strong antitumor activity both in vitro and in vivo. These results validate PDI as a viable and druggable target in GBM and highlight the translational potential of this new inhibitor class.

Together, this discovery defines a new chemotype of PDI inhibitors and introduces an innovative allosteric–covalent strategy that may be broadly applicable to other challenging drug targets. It provides not only promising lead compounds for glioblastoma therapy, but also an important conceptual advance for the design of selective covalent drugs in cancer and beyond.

The paper entitled“Discovery of 2-Chloro-pyrrolo[2,3-d]pyrimidin-4-one Derivatives as Protein Disulfide Isomerase Inhibitors with a Novel Allosteric-Covalent Binding Mode and Anti-Glioblastoma Activity” was published online in the Journal of Medicinal Chemistry on January 23, 2026 (doi: 10.1021/acs.jmedchem.5c03058).

This work was supported by the National Natural Science Foundation of China (22307139, 32321004, and 32422023), the National Key R&D Program of China (2022YFA1304500), and the Beijing Natural Science Foundation – Changping Frontier Project (L244054).

Contact:

Dr. AI Youwei

Institute of Genetics and developmental Biology, Chinese Academy of Sciences

Email: aiyouwei@genetics.ac.cn