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Intramembrane proteases hydrolyze peptide bonds within the cell membrane as the decision-making step of various signaling pathways. Sporulation factor IV B protease (SpoIVB) and C-terminal processing proteases B (CtpB) play central roles in cellular differentiation via regulated intramembrane proteolysis (RIP) process which activates pro-σ K processing at the σ K checkpoint during spore formation. SpoIVB joins CtpB in belonging to the widespread family of PDZ-proteases, but much remains unclear about the molecular mechanisms and structure of SpoIVB. In this study, we expressed inactive SpoIVB (SpoIVB S378A ) fused with maltose binding protein (MBP)-tag and obtained the solution structure of SpoIVB S378A from its small angle X-ray scattering (SAXS) data. The fusion protein is more soluble, stable, and yields higher expression compared to SpoIVB without the tag. MBP-tag not only facilitates modeling of the structure in the SAXS envelope but also evaluates reliability of the model. The solution structure of SpoIVB S378A fits closely with the experimental scattering data (χ 2 = 1.76). Comparing the conformations of PDZ-proteases indicates that SpoIVB adopts a PDZ-protease pattern similar to the high temperature requirement A proteases (HtrAs) rather than CtpB. We not only propose that SpoIVB uses a more direct and simple way to cleave the substrates than that of CtpB, but also that they work together as signal amplifiers to activate downstream proteins in the RIP pathway.

Diltiazem and glibenclamide are commonly used hypotensive and antidiabetic drugs. This study reports the discovery of the potential antitumor and antimetastatic effects of these two drugs using a structural dynamics-driven virtual screening targeting urokinase receptor (uPAR). Owing to uPAR’s high flexibility, currently resolved crystal structures of uPAR, all in ligand-bound states, provide limited representations of its physiological conformation. To improve the accuracy of screening, we performed a long-timescale molecular dynamics simulation and obtained the representative conformations of apo-uPAR as the targets for our screening. Experimentally, we demonstrated that diltiazem and glibenclamide bound uPAR with K D values in the micromolar range. In addition, both compounds effectively suppressed tumor growth and metastasis in a uPAR-dependent manner in vitro and in vivo. This work not only provides two potent uPAR inhibitors but also reports a proof-of-concept study on the potential off-label antitumor and antimetastatic uses of diltiazem and glibenclamide.