Bukunmi Adediran

Collagen prolyl 4-hydroxylase (C-P4H) is an essential enzyme in collagen synthesis and known to be a potential target for drugs that prevent excess collagen formation. Currently known C-P4H inhibitors target the catalytic site of C-P4H, being analogues of 2-oxoglutarate (2OG). However, in mammalian cells there are many other 2OG-dependent dioxygenases with a highly similar catalytic domain, which limits the selective specificity of drugs targeting C-P4H activity. The peptide-substrate-binding (PSB) domain is unique for the C-P4H family and known to be important for the catalytic efficiency of C-P4H. Therefore, interfering with peptide binding to the PSB domain might allow more specific inhibition of the hydroxylation activity of C-P4Hs. We developed a robust FRET-based high-throughput screening assay (Z’ > 0.73) based on PSB-peptide interactions. This assay was used to screen a peptidomimetic library of 15614 compounds with the PSB domains of C-P4H isoforms I and II. A hit compound (OUL-PSBi-001) was identified with IC ₅₀ s of 40 µM and 62 µM for PSB-I and PSB-II, respectively. We also showed that this compound indeed inhibits the catalytic activity of the full-length CP4H-I and -II. This FRET assay provides a new strategy for finding selective inhibitors for the treatment of fibrotic diseases and cancer.

Collagen prolyl 4‐hydroxylase (C‐P4H) catalyzes the 4‐hydroxylation of Y‐prolines of the XYG‐repeat of procollagen. C‐P4Hs are tetrameric α₂β₂ enzymes. The α‐subunit provides the N‐terminal dimerization domain, the middle peptide‐substrate–binding (PSB) domain, and the C‐terminal catalytic (CAT) domain. There are three isoforms of the α‐subunit, complexed with a β‐subunit that is protein disulfide isomerase, forming C‐P4H I‐III. The PSB domain of the α‐subunit binds proline‐rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline‐rich peptides (PPII, polyproline type‐II, conformation) to the PSB‐I domain has previously been reported for the PPG‐PPG‐PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG‐PRG‐PPG, PPG‐PAG‐PPG) (where x, at Y‐position 5, is not a proline) bind to the PSB‐I domain differently, more deeply, in the peptide‐binding groove. The latter mode of binding has previously been reported for structures of the PSB‐II domain complexed with these PxGP‐peptides. In addition, it is shown here by crystallographic binding studies that the POG‐PAG‐POG peptide (with 4‐hydroxyprolines at Y‐positions 2 and 8) also adopts the PxGP mode of binding to PSB‐I as well as to PSB‐II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB‐I than for PSB‐II, with, respectively, K_D values of about 70 μM for PSB‐I and 20 μM for PSB‐II. The importance of these results for understanding the reaction mechanism of C‐P4H, in particular concerning the function of the PSB domain, is discussed.