Decoding the selective chemical modulation of CYP3A4

Abstract

Drug-drug interactions associate with concurrent uses of multiple medications. Cytochrome P450 (CYP) 3A4 metabolizes a large portion of marketed drugs. To maintain the efficacy of drugs metabolized by CYP3A4, pan-CYP3A inhibitors such as ritonavir are often co-administered. Although selective CYP3A4 inhibitors have greater therapeutic benefits as they avoid inhibiting unintended CYPs and undesirable clinical consequences, the high homology between CYP3A4 and CYP3A5 has hampered the development of such selective inhibitors. Here, we report a series of selective CYP3A4 inhibitors with scaffolds identified by high-throughput screening. Structural, functional, and computational analyses reveal that the differential C-terminal loop conformations and two distinct ligand binding surfaces disfavor the binding of selective CYP3A4 inhibitors to CYP3A5. Structure-guided design of compounds validates the model and yields analogs that are selective for CYP3A4 versus other major CYPs. These findings demonstrate the feasibility to selectively inhibit CYP3A4 and provide guidance for designing better CYP3A4 selective inhibitors.

Fig. 1. Primary screen against CYP3A4.

a Scatter plot of percent inhibition determined using the biochemical P450-Glo assay. Data were normalized to 15 µM ritonavir as positive controls (100% inhibition, green) and DMSO as negative controls (0% inhibition, orange). Screened compounds are in blue. b Clustering by structural similarities of the 956 compounds selected for dose–response studies against CYP3A4 and CYP3A5, with the three lead selective CYP3A4 inhibitors indicated as blue, green and pink. The positional spread of the three compounds in the circular clustering tree indicates variety in chemical scaffold. c Chemical structures of the selective CYP3A4 inhibitors SCM-01, SCM-02, and SCM-03, respectively. The colors represent their labeled locations in (b). Source data for (a) are provided as a Source Data file.

Fig. 2. The C-terminal loop plays an important role in selective CYP3A4 inhibition.

a–c Concentration-dependent inhibition of CYP3A4 and CYP3A5 for (a) SCM-01, (b) SCM-02, and (c) SCM-03, respectively. Percentage inhibition data were normalized against their DMSO controls (as 0% inhibition) and against 30 µM ketoconazole controls (as 100% inhibition). The reported numerical values are the means and standard deviations of the IC₅₀ values derived from triplicate experiments (n = 3). d–f Crystal structures of CYP3A4 in complex with (d) SCM-01, (e) SCM-02, and (f) SCM-03 highlighting the interactions between ligands and adjacent residues. g Superimposition of the three selective inhibitor–bound CYP3A4 crystal structures (green, blue, and pink) and all five published CYP3A5 structures (yellow), showing that the selective inhibitors would clash with the CYP3A5 C-terminal loop (circled region, purple) if bound to CYP3A5 in the same conformation as CYP3A4. h Representative CYP3A5-apo crystal structure showing H-bonds associated with the C-terminal loop residues. i Representative CYP3A4–SCM-01 crystal structure showing that the D214-G481 is the only H-bond associated with the C-terminal loop in CYP3A4. The inset shows the sequence alignment of C-terminal loop residues in CYP3A4 and CYP3A5. The heme groups in all panels are colored in wheat. Source data for (a–c) are provided as a Source Data file.

Fig. 3. Structure-activity relationship analysis of SCM-01 analogs indicates that the region A group is important for selective CYP3A4 inhibition.

a The chemical scaffold of SCM-01 analogs. The R group represents the region A group to be modified. b Dose–response curves showing concentration-dependent inhibition of CYP3A4 and CYP3A5 by SCM-01 and its analogs. The structure of the region A group of each compound is shown in the inset. Percentage inhibition data were normalized against their DMSO controls (as 0% inhibition) and 30 µM ketoconazole controls (as 100% inhibition). The reported numerical values are the means and standard deviations of the IC₅₀ values derived from triplicate experiments (n = 3). c Superimposition of CYP3A4–SCM-01 (gray) and CYP3A4–SCM-08 (green) structures showing that SCM-08 binds to CYP3A4 the same as SCM-01, with the region A cyclobutane group residing on surface 1. Source data for (b) are provided as a Source Data file.

Fig. 4. The identification of two distinct ligand binding surfaces of CYP3A4/5 by docking studies reveals the mechanism of selective CYP3A4 inhibition.

a Illustration of the favorable CYP3A4–SCM-01 docking pose 1 and its corresponding surface 1. b Illustration of the clash between SCM-1 and CYP3A5 (circled region) if SCM-01 were to bind to CYP3A5 the same way as CYP3A4. c Illustration of the less favorable CYP3A4–SCM-01 docking pose 2 and its corresponding surface 2. d Illustration of the CYP3A5–SCM-01 docking pose 2 and its corresponding surface 2. For stick representations of SCM-01 in all panels, carbon atoms are colored in yellow, fluoride atoms colored in cyan, nitrogen atoms colored in blue, and oxygen atoms colored in red. The heme group is colored in wheat.

Fig. 5. SCM-18 is a highly selective and non-suicide CYP3A4 inhibitor.

a The chemical structure of SCM-18. The region A group is colored red. b Dose–response curves showing concentration-dependent inhibition of CYP3A4/5 by SCM-18. c Dose–response curves showing time-dependent inhibition of CYP3A4/5 indicate that SCM-18 is not a suicide (irreversible or mechanism-based) inhibitor. For panels b and c, percentage inhibition data were normalized against DMSO controls (as 0% inhibition) and 30 µM ketoconazole controls (as 100% inhibition). The reported numerical values are the means and standard deviations of the IC₅₀ values calculated from triplicate experiments (n = 3). d, e Normalized UV-vis spectra showing spectral changes upon SCM-18 binding to CYP3A4/5. Insets are difference spectra calculated by subtracting the spectrum of ligand-free CYP3A4/5 from each of the ligand-bound state. Spectra of the ligand-free state are shown as the black solid lines, and spectra at the highest ligand concentration are shown as red solid lines. f ΔA_peak – ΔA_trough vs compound concentration plots showing the effect of selective inhibitor binding on CYP3A4/5 spectral signals. The reported numerical values are the means and standard deviations of K_s values derived from triplicate experiments (n = 3). g Overlay of CYP3A4–SCM-01 (gray), CYP3A4–SCM-18 (pink), and CYP3A5–apo (yellow) crystal structures showing that SCM-18, like SCM-01, would clash with the CYP3A5 surface 1 formed by C-terminal loop residues. h CYP3A4–SCM-18 docking results showing that SCM-18 binds CYP3A4 in 2 poses facing two distinct surfaces (green and blue). i Overlay of the CYP3A4–SCM-18 pose 2 and CYP3A5-apo crystal structure showing that ligand binding to CYP3A5 in pose 2 is possible but not favorable due to polar residues. For stick presentations in (h, i), fluoride atoms are colored in cyan, chloride atoms are colored in green, nitrogen atoms are colored in blue, and oxygen atoms are colored in red. The heme group is colored in wheat. Source data for (b–f) are provided as a Source Data file.

Fig. 6. Trade-off between potency and selectivity of SCM-18 analogs.

a–c Chemical structures of (a) SCM-24, (b) SCM-25 and (c) SCM-26 and dose–response curves showing their concentration-dependent inhibition of CYP3A4/5. Percentage inhibition data were normalized against DMSO controls (as 0% inhibition) and 30 µM ketoconazole controls (as 100% inhibition). The reported numerical values are the means and standard deviations of the IC₅₀ values calculated from triplicate experiments (n = 3). d–f Overlay of CYP3A4–SCM-18 (pink) with (d) CYP3A4–SCM-24 (yellow), (e) CYP3A4–SCM-25 (yellow), (f) CYP3A4–SCM-26 (yellow) crystal structures highlighting unique interactions associated with these analogs. For stick representations in (d–f), fluoride atoms are colored in cyan, chloride atoms are colored in green, nitrogen atoms are colored in blue, and oxygen atoms are colored in red. Source data for (a–c) are provided as a Source Data file.

Fig. 7. SCM-08 is a highly selective CYP3A4 inhibitor that does not target other CYPs.

Dose–response curves showing concentration-dependent inhibition of a panel of CYPs as determined using the P450-Glo and the Vivid P450 assays. Percentage inhibition data were normalized against DMSO controls (as 0% inhibition) and 30 µM control inhibitors (as 100% inhibition). The reported numerical values are the means and standard deviations of the IC₅₀ values calculated from triplicate experiments (n = 3). Source data are provided as a Source Data file.

Fig. 8. Molecular basis for the selective CYP3A4 inhibition by SCM-1 analogs.

a SCM-1 analogs bind preferentially to the mostly hydrophobic surface 1 of CYP3A4 when region A groups (indicated as R in red) are hydrophobic of limited size. b Due to a wall created by the C-terminal loop in CYP3A5, SCM-1 analogs are forced to interact with the alternative surface 2, but the highly polar environment discourages interactions with analogs containing hydrophobic region A groups.