**4. Conclusions**

The bioprospection of marine-derived actinomycetes revealed napyradiomycins as potential antifouling agents. Given their potent antibacterial, antibiofilm, and antisettlement activities, they revealed the capacity to prevent growth or the primary adhesion of marine bacterial species to submersed abiotic surfaces as well as other subsequent fouling steps. Napyradiomycin (**1**) exhibited the higher antibacterial activity, (**4**) the higher microfouling inhibitory activity, and (**10**) the most potent antimacrofouling activity. However, napyradiomycins (**8** and **11**) would be our first choice for the development of antifouling paints, as they were active against all the assayed marine organisms. This broad spectrum activity is a clear advantage towards the commercialized products, including ivermictin and CuSO4, which are more limited, as they are directed for macrofouling inhibition only. Overall, in silico toxicity predictions of napyradiomycins sugges<sup>t</sup> toxicity similar to marketed drugs and antifouling biocides, low bioaccumulation factor, and no mutagenicity. Taken together with the absence of toxic e ffects against the assayed species, napyradiomycins could be considered for further investigation as active ingredients for the marine antifouling paints and coatings development pipeline. In particular, the 3-chloro-napyradiomycin sca ffold is indicated to be a key functional moiety for micro and macrofouling activities, especially having a bromine substitute at position C-16, such as (**8** and **11**). The correlation between napyradiomycin´s biosynthetic features and antifouling activities (micro and macro) opens prospects for their engineered biosynthetic enhanced yield production and future commercialization.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/1/63/s1, napyradiomycins (**1**–**12**) structural characterization description; Table S1. Toxicity end point predictions for seven Prestwick approved drugs; Table S2. Toxicity end point predictions for two antifouling approved drugs; Table S3. Aquatic toxicity, environmental fate data and classification of copper and arsenic, experimental data.

**Author Contributions:** Bioresources: actinomycetes isolation and characterization, S.P.G.; fouling bacteria, R.G.S.; mussel larvae I.C. and J.R.A.; crude extract preparation, fractionation, compound isolation, and structure elucidation S.P.G., F.P., and M.P.; SAR analysis, S.P.G. and F.P.; in silico model, F.P.; in silico data analysis, S.P.G. and F.P.; antibacterial and antibiofilm screening R.G.S., I.R.G., and H.M.; antifouling screening V.V., I.C., and J.R.A.; writing—original draft preparation, S.P.G., F.P., R.G.S., I.R.G., I.C., and J.R.A.; writing—review and editing, S.P.G., F.P., R.G.S., I.R.G., I.C., and J.R.A.; supervision, S.P.G., R.G.S., and V.V.; project administration, S.P.G., R.G.S., V.V., I.C. and J.R.A.; funding acquisition, S.P.G., R.G.S., V.V., I.C. and J.R.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). It was also supported by CIIMAR which is financed by national funds from FCT/MCTES (UID/Multi/04423/2019). Funding from the 7th Framework Programme (FP7/2007–2013) under gran<sup>t</sup> agreemen<sup>t</sup> PCOFUND-GA-2009-246542, DFRH/WIIA/102/2011 and SFRH/BI/52130/ 2013. Financial support provided by FCT/MCTES through grants IF/00700/2014 and SFRH/BPD/110020/2015. Funding also provided by the projects PTDC/QUI-QUI/119116/2010, PTDC/BIA-MIC/31645/2017, PTDC/BTA-GES/ 32359/2017, and PTDC/BTA-BTA/31422/2017 (POCI-01-0145-FEDER-031422), financed by FCT/MCTES, COMPETE2020 and PORTUGAL2020. The NMR spectrometers are part of The National NMR Facility, supported by FCT (RECI/BBB-BQB/0230/2012).

**Acknowledgments:** SPG is indebted to W. Fenical, P. R. Jensen, and C. A. Kau ffman from Scripps Institution of Oceanography, San Diego, USA, for the sustenance given to perform the sediment sample collection. P. Castilho from Madeira University and M. Freitas from Funchal Marine Biology Station are acknowledged for their hospitality and logistic support during the field expedition. To M. Wilson for English revision.

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