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

Protein disulfide isomerase (PDI) is an endoplasmic reticulum
oxidoreductase/chaperone,
and its dysregulation contributes to cancer progression, particularly
glioblastoma. A high-throughput screen identified TC8026 as a PDI-active hit, and further optimization afforded a pyrrolo[2,3-d]pyrimidin-4-one series with up to 20-fold improved potency.
Representative analogues (30w, 30z, 30aa, and 30ab) potently inhibited PDI, induced
endoplasmic reticulum stress-mediated apoptosis in glioblastoma cells,
and 30z significantly suppressed tumor growth in a U251
xenograft model. Mechanistic studies revealed a previously unrecognized
allosteric-covalent binding mode. The inhibitors initially engage
an allosteric pocket within the b′ domain involving residues
H256 and F304, thereby perturbing the substrate-binding interface
and inducing conformational changes that expose the noncatalytic cysteine
C312 for covalent capture. This b′-directed allosteric covalency,
distinct from conventional catalytic cysteine modification, confers
enhanced selectivity within the PDI family. These findings define
a novel allosteric-covalent chemotype of PDI inhibitors with a unique
binding mechanism and promising antiglioblastoma potential.