A Conserved Allosteric Site on Drug-Metabolizing CYPs: A Systematic Computational Assessment

Abstract

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44 μs of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques.

In vitro and In silico studies of interactions of cathinone with human recombinant cytochrome P450 CYP(1A2), CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5

“…The in vitro results showed uncompetitive as well as noncompetitive inhibition and therefore, the docking results are only applicable to represent interactions of competitive inhibition that occurred within the active sites, as the allosteric site of these CYPs were still actively being investigated. A recent computational modelling study investigated the conserved allosteric site on drug metabolising CYPs in prokaryotes and human CYPs, denoted as hotspot 1 (H1), located among helices C, E and H [28] . CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6 were capable to bind small molecules or organic solvents at their H1 sites except CYP2C9, CYP2E1 and CYP3A4 [28] .…”

“…A recent computational modelling study investigated the conserved allosteric site on drug metabolising CYPs in prokaryotes and human CYPs, denoted as hotspot 1 (H1), located among helices C, E and H [28] . CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6 were capable to bind small molecules or organic solvents at their H1 sites except CYP2C9, CYP2E1 and CYP3A4 [28] . Further studies are recommended to investigate whether cathinone inhibits drug metabolising CYPs and subsequently be metabolised by CYPs or otherwise.…”

Protein-Ligand Identification and In Vitro Inhibitory Effects of Cathine on 11 Major Human Drug Metabolizing Cytochrome P450s

“…What is more, the allosteric sites of CYP enzymes are an area of active research, although locations among helices C, E and H have recently been proposed. 64 It is worth mentioning that in vitro and docking predictions are lacking in biotransformation capabilities, 65 and the lack of in vivo biological mechanisms has probably stronger impact on the accuracy of toxicity evaluations as cathine inhibitory effects on CYPs were performed in vitro. 66 This study answers some previous questions regarding the inhibitory effects of cathine on major human drug metabolizing CYP enzymes but also raises new questions to explore.…”

“…What is more, the allosteric sites of CYP enzymes are an area of active research, although locations among helices C, E and H have recently been proposed. 64…”

Discovery of a Novel 3site State as the Multi-Substrate Bound State of P450cam

“…The newly posited allosteric site was found to be conserved across several members of CYP superfamily. 15 However, substrate binding at allosteric site further complicates the channel-1 dynamics. Current standing on this field concludes that two camphor bind to P450cam in the active and allosteric sites.…”