Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis.

P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.

Investigating the applicability of the CYP102A1-decoy-molecule system to other members of the CYP102A subfamily.

Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1.

Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP-FC , comprising the ligand-binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0 ) expressing TRAP-FC ubiquitously under the control of the chicken-beta-actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc-1. Ablation of each ERBB family member, especially the loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration, and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.