Glycoside hydrolase family 18 and 20 enzymes are novel targets of the traditional medicine berberine

Abstract

Berberine is a traditional medicine that has multiple medicinal and agricultural applications. However, little is known about whether berberine can be a bioactive molecule toward carbohydrate-active enzymes, which play numerous vital roles in the life process. In this study, berberine and its analogs were discovered to be competitive inhibitors of glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidase (GH20 Hex) and GH18 chitinase from both humans and the insect pest Ostrinia furnacalis Berberine and its analog SYSU-1 inhibit insect GH20 Hex from O. furnacalis (OfHex1), with K_i values of 12 and 8.5 μm, respectively. Co-crystallization of berberine and its analog SYSU-1 in complex with OfHex1 revealed that the positively charged conjugate plane of berberine forms π-π stacking interactions with Trp⁴⁹⁰, which are vital to its inhibitory activity. Moreover, the 1,3-dioxole group of berberine binds an unexplored pocket formed by Trp³²², Trp⁴⁸³, and Val⁴⁸⁴, which also contributes to its inhibitory activity. Berberine was also found to be an inhibitor of human GH20 Hex (HsHexB), human GH18 chitinase (HsCht and acidic mammalian chitinase), and insect GH18 chitinase (OfChtI). Besides GH18 and GH20 enzymes, berberine was shown to weakly inhibit human GH84 O-GlcNAcase (HsOGA) and Saccharomyces cerevisiae GH63 α-glucosidase I (ScGluI). By analyzing the published crystal structures, berberine was revealed to bind with its targets in an identical mechanism, namely via π-π stacking and electrostatic interactions with the aromatic and acidic residues in the binding pockets. This paper reports new molecular targets of berberine and may provide a berberine-based scaffold for developing multitarget drugs.

Keywords: berberine; chitinase; glycoside hydrolase; human; inhibitor; insect; protein crystallization; β-N-acetyl-D-hexosaminidase.

Figure 1.

Structure of berberine and its analogs.

Figure 2.

Inhibition kinetics of berberine and its analogs toward GH20 Hexs. A, inhibition kinetics of SYSU-1 toward OfHex1; B and E, inhibition kinetics of berberine toward OfHex1 and HsHexB; C and F, inhibition kinetics of thalifendine toward OfHex1 and HsHexB; and D and G, inhibition kinetics of palmatine toward OfHex1 and HsHexB.

Figure 3.

Inhibition kinetics of berberine and its analogs toward GH18 chitinases. A, D, and G, inhibition kinetics of berberine toward OfChtI, HsCht, and AMCase; B, E, and H, inhibition kinetics of thalifendine toward OfChtI, HsCht, and AMCase; and C, F, and I, inhibition kinetics of palmatine toward OfChtI, HsCht, and AMCase.

Figure 4.

Inhibition kinetics of berberine and its analogs toward GH84 and GH63 enzymes. A and D, inhibition kinetics of berberine toward HsOGA and ScGluI; B and E, inhibition kinetics of thalifendine toward HsOGA and ScGluI; and C and F, inhibition kinetics of palmatine toward HsOGA and ScGluI.

Figure 5.

Crystal structures of OfHex1 in complex with berberine. A, surface representations of OfHex1 complexed with berberine. B, binding mode of berberine in the active pocket of OfHex1. Electrostatic potential between −6 kT/e and 6 kT/e was shown as a colored gradient from red (acidic) to blue (basic). The 2F_o − F_c electron-density map around the ligand is contoured at the 1.0 σ level. C, amino acid residues involved in the binding of berberine in the active pocket of OfHex1. The hydrophobic recess accommodating the 1,3-dioxole group of berberine is shown in pink. The hydrogen bonds are shown as dashed black lines.

Figure 6.

Crystal structures of OfHex1 in complex with SYSU-1. A, surface representations of OfHex1 complexed with SYSU-1. B, binding mode of SYSU-1 in the active pocket of OfHex1. Electrostatic potential between −6 kT/e and 6 kT/e was shown as a colored gradient from red (acidic) to blue (basic). The 2F_o − F_c electron-density map around the ligand is contoured at the 1.0 σ level. C, superimposition of the berberine-complexed and SYSU-1–complexed OfHex1. Residues of the berberine-complexed and SYSU-1–complexed OfHex1 are shown in wheat and white, respectively.

Figure 7.

Modeled structures of berberine in complex with GH20 and GH18 enzymes. Electrostatic potential between −6 kT/e and 6 kT/e was shown as a colored gradient from red (acidic) to blue (basic). A, binding mode of berberine in the active pocket of HsHexB. B, locations of the key residues for berberine binding in HsHexB. C, binding modes of berberine in the active pocket of HsCht. D, binding modes of berberine in the active pocket of OfChtI. E, binding modes of berberine in the active pocket of AMCase. F, superimposition of the binding modes of berberine with three chitinases. Residues of the HsCht and its berberine are shown in green. Residues of the OfChtI and its berberine are shown in yellow-orange. Residues of the AMCase and its berberine are shown in cyan.

Figure 8.

Modeled structures of berberine in complex with GH84, GH63, and GH13 enzymes. Electrostatic potential between −6 kT/e and 6 kT/e was shown as a colored gradient from red (acidic) to blue (basic). A, binding mode of berberine in the active pocket of HsOGA. B, binding modes of berberine in the active pocket of ScGluI. C, binding modes of berberine in the active pocket of PPA.

Figure 9.

In vivo activity of berberine and SYSU-1. A, larvae before exposed to the compounds. B, larvae of DMSO-fed group 6 days later. C, larvae of berberine-fed group 6 days later. D, larvae of SYSU-1-fed group 6 days later.

Figure 10.

Aromatic residues and negatively charged residues involved in the binding of berberine to OfHex1 (PDB code 5Y0V) (A), QacR (PDB code 3BTI) (B), BmrR (PDB code 3D6Y) (C), and RamR (PDB code 3VW2) (D). Berberine is shown in green. Aromatic and negatively charged residues are shown in yellow and white, respectively.

Figure 11.

Analysis of the structural characteristics of berberine and its analogs. The energy-minimized structures of berberine and its analogs were generated with MM2 on ChemBio3D (PerkinElmer Life Sciences). The electrostatic potential surfaces for berberine and its analogs were generated with DelPhi on Accelrys Discovery Studio 2016 (Dassault Systèmes). Red and cyan represent the electronegative and electropositive potentials, respectively, and green represents a potential halfway point between the two extremes. The pK_a of tetrahydroberberine was predicted by Marvin Beans (ChemAxon).