Bioguided Fractionation of Local Plants against Matrix Metalloproteinase9 and Its Cytotoxicity against Breast Cancer Cell Models: In Silico and In Vitro Study

Abstract

Matrix metalloproteinase9 (MMP9) is known to be highly expressed during metastatic cancer where most known potential inhibitors failed in the clinical trials. This study aims to select local plants in our state, as anti-breast cancer agent with hemopexin-like domain of MMP9 (PEX9) as the selective protein target. In silico screening for PEX9 inhibitors was performed from our in house-natural compound database to identify the plants. The selected plants were extracted using methanol and then a step-by-step in vitro screening against MMP9 was performed from its crude extract, partitions until fractions using FRET-based assay. The partitions were obtained by performing liquid-liquid extraction on the methanol extract using n-hexane, ethylacetate, n-butanol, and water representing nonpolar to polar solvents. The fractions were made from the selected partition, which demonstrated the best inhibition percentage toward MMP9, using column chromatography. Of the 200 compounds screened, 20 compounds that scored the binding affinity -11.2 to -8.1 kcal/mol toward PEX9 were selected as top hits. The binding of these hits were thoroughly investigated and linked to the plants which they were reported to be isolated from. Six of the eight crude extracts demonstrated inhibition toward MMP9 with the IC₅₀ 24 to 823 µg/mL. The partitions (1 mg/mL) of Ageratum conyzoides aerial parts and Ixora coccinea leaves showed inhibition 94% and 96%, whereas their fractions showed IC₅₀ 43 and 116 µg/mL, respectively toward MMP9. Using MTT assay, the crude extract of Ageratum exhibited IC₅₀ 22 and 229 µg/mL against 4T1 and T47D cell proliferations, respectively with a high safety index concluding its potential anti-breast cancer from herbal.

Keywords: MMP9; PEX9; ageratum; bioguided; cancer; fractionation; in silico; in vitro; screening.

Figure 1

The flow chart of the studies in bioguided fractionation of local plants to identify MMP9 inhibitors and breast cancer cytotoxic agents.

Figure 2

Superimposition of (a) 17 published PEX9 inhibitor and (b) top twenty ligand scores docked into PEX9 with deep pocket in the left side and shallow pocket in the right side. Inset is the control docking of sulfate ion with green stick is initiate pose and red stick is re-docked pose. The external ligands are colored in stick model with C = yellow; H = white; O = red; and N = blue. The protein is presented in a surfaced form with magenta area = hydrogen bond donor; white = neutral and green = hydrogen bond acceptor.

Figure 3

The histogram of inhibition percentage from 15 partitions from methanol extract of each plant measured by FRET-based assay against MMP9 activity in vitro. N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH) was used as the positive control by showing 100% inhibition at 1 µM. The bars described the standard error of triplicate assay.

Figure 4

The drug dose-dependent curves of two active fractions, i.e., (a) Ageratum aerial parts, and (b) Ixora leaves and (c) NNGH as the results of FRET-based assay toward MMP9. NNGH was used as the positive control by showing IC₅₀ 47.8 nM. The bars showed the standard error of triplicate assay as previously described.

Figure 5

The 4T1 cell morphologies after and before treating with four top active extracts as followed; (a) untreated cell, (b) Ixora leaves, (c) Turnera leaves, (d) Ageratum aerial part, and (e) Plumeria leaves. The arrow indicates the living cell for the white nodes whereas the death cell is black node surrounded by white line.

Figure 6

The drug dose-dependent curves of four top active extracts against (a) 4T1, (b) T47D, and (c) Vero cells.

Figure 7

The TLC chromatogram of the fraction 1–4 of Ageratum which are detected under (a) UV₃₆₅ and (b) UV₂₅₄. The fraction 1–3 of Ixora are indicated under (c) UV₃₆₅ and (d) UV₂₅₄.

Figure 8

The GC chromatogram of Ageratum active fraction showing four peaks in a different retention time. The peak at 2.250 min is of chloroform which is used as solvent.

Figure 9

The mass spectra of four peaks identified from GC chromatogram of Ageratum fraction 2. According to Figure 8, four peaks with R_t 8.825 min, 11.255 min, 12.175 min, and 14.460 min are detected as compounds having mass/ ion: 522 (base peak 175), 538 (base peak 205), 543 (base peak 201), and 539 (base peak 55), respectively. The base peak informs the most stable fragment during electron impact in MS characterization.