*4.7. Molecular Networking*

The UHPLC-QTOF-MS/MS data of the prioritized extracts were visualized and subsequently analyzed using molecular networking. Established parameters [8,55] were used for the experiment. MSConvert (ProteoWizard package32) was used to convert the raw data (\*.d files) into plain text (\*.mgf) files, wherein all detected fragment ions are expressed as a list of mass/intensity value pairs sorted according to their parent ions (peak picking: vendor MS level = 1-2; threshold type = absolute intensity, value = 1000, orientation = most-intense). The networking algorithm itself, thus the calculation of cosine similarity values between parent ion vectors, was computed offline, using an in house server [38].

### **5. Conclusions**

In summary, a combination of (i) phenotypic activity screening assays and (ii) metabolic fingerprinting allowed a fast prioritization and dereplication of samples with the desired bioactivity for further processing. Applying our SeaPEPR pipeline, we were able to dereplicate the active component(s) of crude extracts responsible for the antimicrobial activities observed in primary screens. Thereby, a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative were dereplicated. Furthermore, inhibitory activity against the common plant pest *S. tritici* was discovered for natural products of marine origin. The pipeline represents a valuable tool for further bioprospecting projects, since only low sample volumes are needed that in turn renders extensive collection of limited bioresources for screening purposes (e.g., slow-growing macroorganisms like sponges) obsolete.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/12/649/s1, Pairwise similarities of clustered samples and resulting metabolic grouping; Morphological description and underwater documentation; Microfractionation bioassays and dereplication results.

**Author Contributions:** T.F.S.: conceptualization, study design and acquisition; F.G.I. and W.B.: study design and acquisition. S.I.M.W., F.J.R., W.B. collected sponge specimens and performed identification. R., M.M., M.A.P., C.H., performed experiments, analyzed and/or interpreted data. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the LOEWE program of the state of Hesse. Riyanti obtained a fellowship from the Indonesia Endowment Fund for Education (LPDP), grant number 20160222305487. W.B. was supported by the Indonesian Ministry of Research Technology and Higher Education for the Basic Research Grant No.01/PL30/P3M/P-DSR/2019. The Federal Ministry of Education and Research (BMBF) supported the work in the lab of T.F.S. with the grant 16GW0117K.

**Acknowledgments:** The authors thank Junhui Cho (Justus-Liebig-University Giessen) for his help in extract generation and Herjumes Atjin (Ucil) (Politeknik Negeri Nusa Utara) for sample collecting.

**Conflicts of Interest:** The authors declare no conflict of interest.
