This present study reports some natural products and one hydroxamic acid synthetic compound which were previously reported as matrix metalloproteinase-9 (MMP-9) inhibitors to be evaluated for their inhibition toward severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro). This enzyme is one of the proteins responsible for this coronaviral replication. Two herbal methanolic extracts i.e., Averrhoa carambola leaves and Ageratum conyzoides aerial part demonstrate >50% inhibition at 1000 µg/mL. Interestingly, apigenin, one of flavonoids, demonstrates 92% inhibition at 250 µg/mL (925 µM) as well as hydroxamic acid compound, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH), which shows 69% inhibition at 100 µM. The in vitro results are supported by the docking studies revealing that the binding mode of both compounds is mainly by interacting with GLU166 residue in the hydrophobic pocket of the 3CLpro. Pharmacophore mapping further supported the results by confirming that the in vitro activities of both compounds are due to their pharmacophore features employing hydrogen bond acceptor (HBA), hydrogen bond donor (HBD) and hydrophobic. Gas Chromatography-Mass Spectrometry (GC-MS) analysis reported chromene compounds in Ageratum conyzoides aerial part methanolic extract are potential to be this enzyme inhibitor candidate. These all results reflect their potencies to be SARS-CoV-2 inhibitors through 3CLpro inhibition mechanism.
In our previous work, the partitions (1 mg/mL) of Ageratum conyzoides (AC) aerial parts and Ixora coccinea (IC) leaves showed inhibitions of 94% and 96%, respectively, whereas their fractions showed IC50 43 and 116 µg/mL, respectively, toward Matrix Metalloproteinase9 (MMP9), an enzyme that catalyzes a proteolysis of extracellular matrix. In this present study, we performed IC50 determinations for AC n-hexane, IC n-hexane, and IC ethylacetate partitions, followed by the cytotoxicity study of individual partitions against MDA-MB-231, 4T1, T47D, MCF7, and Vero cell lines. Successive fractionations from AC n-hexane and IC ethylacetate partitions led to the isolation of two compounds, oxytetracycline (OTC) and dioctyl phthalate (DOP). The result showed that AC n-hexane, IC n-hexane, and IC ethylacetate partitions inhibit MMP9 with their respective IC50 as follows: 246.1 µg/mL, 5.66 µg/mL, and 2.75 × 10-2 µg/mL. Toward MDA-MB-231, 4T1, T47D, and MCF7, AC n-hexane demonstrated IC50 2.05, 265, 109.70, and 2.11 µg/mL, respectively, whereas IC ethylacetate showed IC50 1.92, 57.5, 371.5, and 2.01 µg/mL, respectively. The inhibitions toward MMP9 by OTC were indicated by its IC50 18.69 µM, whereas DOP was inactive. A molecular docking study suggested that OTC prefers to bind to PEX9 rather than its catalytic domain. Against 4T1, OTC showed inhibition with IC50 414.20 µM. In conclusion, this study furtherly supports the previous finding that AC and IC are two herbals with potential to be developed as triple-negative anti-breast cancer agents.