Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site

Abstract

2-ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.

Figure 1. Determination of the type of reversible inhibition of CYP2B4-dependent 7-EC (A), BZP (B), 7-PR (C), and 7-BR (D) metabolism by 2EN

The data points represent, no inhibitor (■), 0.25 μM (△), 0.5 μM (▼), 0.75 μM (◊), 1.0 μM 2EN (●); as for BZP, the legend is: no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM (●). The y-axes are expressed as the reciprocal of the rates (pmol product/min/nmol P450) except for bzp which is expressed as nmol product/min/nmol P450. The assays were performed as described in Experimental Procedures.

Figure 1. Determination of the type of reversible inhibition of CYP2B4-dependent 7-EC (A), BZP (B), 7-PR (C), and 7-BR (D) metabolism by 2EN

The data points represent, no inhibitor (■), 0.25 μM (△), 0.5 μM (▼), 0.75 μM (◊), 1.0 μM 2EN (●); as for BZP, the legend is: no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM (●). The y-axes are expressed as the reciprocal of the rates (pmol product/min/nmol P450) except for bzp which is expressed as nmol product/min/nmol P450. The assays were performed as described in Experimental Procedures.

Figure 1. Determination of the type of reversible inhibition of CYP2B4-dependent 7-EC (A), BZP (B), 7-PR (C), and 7-BR (D) metabolism by 2EN

The data points represent, no inhibitor (■), 0.25 μM (△), 0.5 μM (▼), 0.75 μM (◊), 1.0 μM 2EN (●); as for BZP, the legend is: no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM (●). The y-axes are expressed as the reciprocal of the rates (pmol product/min/nmol P450) except for bzp which is expressed as nmol product/min/nmol P450. The assays were performed as described in Experimental Procedures.

Figure 1. Determination of the type of reversible inhibition of CYP2B4-dependent 7-EC (A), BZP (B), 7-PR (C), and 7-BR (D) metabolism by 2EN

The data points represent, no inhibitor (■), 0.25 μM (△), 0.5 μM (▼), 0.75 μM (◊), 1.0 μM 2EN (●); as for BZP, the legend is: no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM (●). The y-axes are expressed as the reciprocal of the rates (pmol product/min/nmol P450) except for bzp which is expressed as nmol product/min/nmol P450. The assays were performed as described in Experimental Procedures.

Figure 2. Reversible inhibition of CYP2B4-dependent (A) PNA and (B) 7-EFC metabolism by 2EN at higher concentrations

The data points represent, no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM 2EN (●) for PNA; no inhibitor (■), 0.5 μM (△), 1 μM (▼), 1.5 μM (◊), 2 μM 2EN (●) for 7-EFC. The inset of B shows competitive inhibition of CYP2B4-dependent 7-EFC deethylation by 2EN at or below 1 μM – no inhibitor (■), 0.5 μM (△), 1 μM (▼). The y-axis for PNA is expressed as the reciprocal of the rates (pmol product/min/nmol P450); 7EFC is expressed as nmol product/min/nmol P450. The data points at the highest 2EN concentrations (at the lowest substrate concentrations) were missing due to not being detectable.

Figure 2. Reversible inhibition of CYP2B4-dependent (A) PNA and (B) 7-EFC metabolism by 2EN at higher concentrations

The data points represent, no inhibitor (■), 1 μM (△), 2 μM (▼), 3 μM (◊), 4 μM 2EN (●) for PNA; no inhibitor (■), 0.5 μM (△), 1 μM (▼), 1.5 μM (◊), 2 μM 2EN (●) for 7-EFC. The inset of B shows competitive inhibition of CYP2B4-dependent 7-EFC deethylation by 2EN at or below 1 μM – no inhibitor (■), 0.5 μM (△), 1 μM (▼). The y-axis for PNA is expressed as the reciprocal of the rates (pmol product/min/nmol P450); 7EFC is expressed as nmol product/min/nmol P450. The data points at the highest 2EN concentrations (at the lowest substrate concentrations) were missing due to not being detectable.

Figure 3. Potential substrate binding sites identified from the crystal structure of CYP2B4

Three potential substrate binding sites were derived from the CYP2B4 crystal structure (34). The white enclosure represents the distal binding site above the heme (Volume=594 ų). A second potential binding site with a volume of 330 ų is separated from the primary binding site and is shown in grey. A site on the surface of the CYP2B4 molecule, yet still in the vicinity of the heme binding region is shown in gold and occupies a volume of about 275 ų. Each of these enclosures is of sufficient size to bind the inhibitor 2EN.

Figure 4. Docking of benzphetamine and 2EN molecules in the primary common cavity site of CYP2B4

(A) The docked configuration of a single BZP (shown in magenta) nearly fills the primary binding site. (B) In contrast, the smaller size and architecture of 2EN (shown in green) readily accommodates low-energy configurations of two-2EN ligands above the heme.

Figure 4. Docking of benzphetamine and 2EN molecules in the primary common cavity site of CYP2B4

(A) The docked configuration of a single BZP (shown in magenta) nearly fills the primary binding site. (B) In contrast, the smaller size and architecture of 2EN (shown in green) readily accommodates low-energy configurations of two-2EN ligands above the heme.

Figure 5. Estimates of the relative number of binding configurations for substrates and inhibitors within the primary binding site

(A) The percentage of substrate bound configurations in the binding site above the heme as opposed to other cavities correlated with substrate volume. (B) Percentage of co-docked low-energy configurations of two ligands in the primary binding site on the distal site of the heme. Above a certain substrate volume, co-occupation of 2EN and substrate in the heme binding site is no longer possible---indicating a size threshold for the transition to lack of inhibition to a competitive interaction.

Figure 5. Estimates of the relative number of binding configurations for substrates and inhibitors within the primary binding site

(A) The percentage of substrate bound configurations in the binding site above the heme as opposed to other cavities correlated with substrate volume. (B) Percentage of co-docked low-energy configurations of two ligands in the primary binding site on the distal site of the heme. Above a certain substrate volume, co-occupation of 2EN and substrate in the heme binding site is no longer possible---indicating a size threshold for the transition to lack of inhibition to a competitive interaction.

Figure 6. A hierarchical docking result showing a low energy configuration of PNA (magenta) and 2EN (green)

A single 2EN and the PNA are in a “common-cavity” arrangement above the heme (red) shown in the common cavity. A second 2EN is shown in a “separate cavity” adjoining the heme binding site. The residues within 2.8 Å of 2EN and PNA in the closed cavity arrangement are PNA [E301,V367,I363,V477,G478] and 2EN[I101,V104, I114,I209,F297,E301,V367,V477]. The residues within 2.8 Å of 2EN and PNA in the separated cavity configuration are: PNA[V104,F115,F297,E301,I363,V367] and 2EN[H172,F203,F206,S207,S210, T306,L362,G478,N479,V480,I486]. Residues have been removed for clear visualization of the occupancy; both of these sites are buried.