# Molecular networking derived from untargeted LC-MS/MS analysis to discover inhibitors of RANKL-induced osteoclastogenesis from Egyptian marine sponge-associated fungi

## TL;DR

Researchers discovered two natural compounds from marine sponge fungi that inhibit bone breakdown, offering potential for osteoporosis treatments.

## Contribution

The study introduces two new natural compounds with anti-osteoclastogenic activity from Egyptian marine fungi.

## Key findings

- Compound 1 and altenuene (8) inhibited RANKL-induced osteoclast formation without cytotoxicity.
- Compound 8 showed better solubility and lower toxicity compared to compound 1.
- Molecular networking identified eight new and thirteen known compounds from fungal extracts.

## Abstract

In continuous search for RANKL induced osteoclastogenesis inhibitors, twenty-six fungal isolates were obtained from ten Red Sea marine sponges collected from Egypt and the ethyl acetate fractions of their cultures’ methanol extracts were assessed in RAW264 macrophages. Active fractions were profiled via LC-MS/MS, followed by untargeted molecular networking, leading to the tentative identification of eight unreported compounds (1, A2, C1, C2, C4-C7), and thirteen known compounds. The two active fungi were identified and deposited in GenBank with accession numbers PQ423742 and PQ423748 for Aspergillus flavus and Cladosporium colombiae, respectively. Bioassay-guided isolation afforded two bisphenol diglycidic ethers, 1 and 2, and two diketopiperazines, 3 and 4 from A. flavus, while C. colombiae yielded cinnamic acid (5), two diketopiperazines (6 and 7), and altenuene (8). Structures were elucidated by NMR and mass spectroscopic analyses, which revealed 1 to be isolated for the first time from natural sources. Isolated compounds were biologically evaluated. Only 1 and 8 inhibited RANKL-induced formation of mature multinucleated osteoclasts with IC50 57.14 and 38.35 µM, respectively, without cytotoxicity against RAW264 macrophages. The ADMET properties for 1 and 8 were predicated using SwissADME and pkCMS platforms. 8 showed superior solubility and lower toxicity than compound 1.

The online version contains supplementary material available at 10.1038/s41598-025-12456-y.