Structural-Guided Identification of Small Molecule Inhibitor of UHRF1 Methyltransferase Activity

Abstract

Ubiquitin-like containing plant homeodomain Ring Finger 1 (UHRF1) protein is recognized as a cell-cycle-regulated multidomain protein. UHRF1 importantly manifests the maintenance of DNA methylation mediated by the interaction between its SRA (SET and RING associated) domain and DNA methyltransferase-1 (DNMT1)-like epigenetic modulators. However, overexpression of UHRF1 epigenetically responds to the aberrant global methylation and promotes tumorigenesis. To date, no potential molecular inhibitor has been studied against the SRA domain. Therefore, this study focused on identifying the active natural drug-like candidates against the SRA domain. A comprehensive set of in silico approaches including molecular docking, molecular dynamics (MD) simulation, and toxicity analysis was performed to identify potential candidates. A dataset of 709 natural compounds was screened through molecular docking where chicoric acid and nystose have been found showing higher binding affinities to the SRA domain. The MD simulations also showed the protein ligand interaction stability of and in silico toxicity analysis has also showed chicoric acid as a safe and nontoxic drug. In addition, chicoric acid possessed a longer interaction time and higher LD50 of 5000 mg/kg. Moreover, the global methylation level (%5 mC) has been assessed after chicoric acid treatment was in the colorectal cancer cell line (HCT116) at different doses. The result showed that 7.5 µM chicoric acid treatment reduced methylation levels significantly. Thus, the study found chicoric acid can become a possible epidrug-like inhibitor against the SRA domain of UHRF1 protein.

Keywords: SRA domain; UHRF1; chicoric acid; global methylation (5 mC); molecular docking; molecular dynamics simulation.

FIGURE 1

The crystal structure of the SRA domain of E3 ubiquitin-protein ligase UHRF1. SiteMap surface “red, blue and yellow colour” of the SRA domain of E3 ubiquitin-protein ligase UHRF1(PDB: 3BI7) “Green colour".

FIGURE 2

Molecular Docking of chicoric acid with UHRF1. (A) Molecular surface display with an electrostatic potential color scheme for UHRF1-Chicoric acid complex and the close-up view presented. (B) Putative binding mode of Chicoric acid in the bindin site of UHRF1(PDB: 3BI7) (C) Chicoric acid was displayed as green ball-and-sticks. And the amino acid residues of the are represented as grey sticks, and H-bonds are described in yellow dotted lines. (D) 2D depiction of the ligand-protein interactions.

FIGURE 3

Molecular Docking of nytose with UHRF1. (A) Molecular surface display with an electrostatic potential color scheme for UHRF1- Nystose complex and the close-up view presented. (B) Putative binding mode of Nystose in the binding site of UHRF1(PDB: 3BI7). (C) Nystose was displayed as green ball-and-sticks. And the amino acid residues of the binding site are represented as grey sticks, and H-bonds are expressed in yellow dotted lines. (D) 2D depiction of the lig-and-protein interactions.

FIGURE 4

Molecular dynamics simulation analysis of chicoric acid. (A) The RMSD plot was obtained for compound Chicoric acid complexed with SRA domain of E3 ubiquitin-protein ligase UHRF1(PDB: 3BI7). The 100 ns simulation time reaffirmed the stability of the complex without any significant changes in the structure. (B) Stability of the secondary structure UHRF1 over the 100 ns of MD simulation when complexed with Chicoric acid. Protein secondary structure elements (SSE) like alpha-helices and beta-strands were monitored throughout the simulation. The top plot reported SSE distribution by residue index throughout the protein structure. The middle plot summarized the SSE composition for each trajectory frame throughout the simulation, and the plot at the bottom monitored each residue and its SSE assignment over time.

FIGURE 5

Molecular dynamics simulation analysis of nytose. (A) The RMSD plot was obtained for compound Nystose complexed with SRA domain of E3 ubiquitin-protein ligase UHRF1(PDB: 3BI7). The 100 ns simulation time reaffirmed the stability of the complex without any significant changes in the structure. (B) Stability of the secondary structure UHRF1 over the 100 ns of MD simulation when complexed with Nystose. Protein secondary structure elements (SSE) like alpha-helices and beta-strands were monitored throughout the simulation. The top plot reported SSE distribution by residue index throughout the protein structure. The middle plot summarized the SSE composition for each trajectory frame throughout the simulation, and the plot at the bottom monitored each residue and its SSE assignment over time.

FIGURE 6

The interaction analysis of chicoric acid with UHRF1. (A) UHRF1 interactions with Chicoric acid throughout the simulation. The interactions between the ligand and protein were classified into hydrophobic, ionic, hydrogen bonds, and water bridges. Each classification can be further sub-grouped and noticed in the “Simulation Interactions Diagram” panel. The stacked bar charts were normalized over the trajectory’s course: for example, a value of 0.7 suggested that the specific interaction was maintained 70% of the simulation time. Values over 1.0 were possible, as some protein residue may make multiple contacts of the same subtype with the ligand. (B) The schematic diagram showed the detailed atomic interaction of Chicoric acid with UHRF1. Interactions occurred more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns). It is possible to have interactions with >100% as some residues may have multiple interactions of a single type with the same ligand atom. (C) A timeline representation of UHRF1- Chicoric acid interactions is presented in (A). The top panel showed the number of specific contacts that the protein made with the ligand throughout the trajectory. The bottom panel showed which residues interacted with the ligand in each trajectory frame. According to the scale of the plot, some residues made more than one specific contact with the ligand, which was represented by a darker shade of orange.

FIGURE 7

The interaction analysis of nytose with UHRF1. (A) UHRF1 interactions with Nystose throughout the simulation. (B) The schematic diagram showed the detailed atomic interaction of Nystose with UHRF1. (C) A timeline representation of UHRF1- Nystose interactions is presented in (A).

FIGURE 8

The global genomic methylation level study in chicoric acid treated HCT116 cell line. The percentage of global methylation (5mC) of HCT116 cell line treating with chicoric acid. The concentration of 7.5 µm showed the lowest percentage of methylation (5mC) level. 5Azadc; 5Azacytidine.