2.2.2. Antibiofilm Activity

In a 96-well plate, biofilm grown cultures of *M. hydrocarbonoclasticus* and *C. marina* were incubated with napyradiomycins (**1**–**12**) at a concentration of 31.25 μg/mL. After discarding the media, biofilm inhibition was determined from OD600 measurements of crystal violet stained cells resuspended from the plate bottom with acetic acid. All napyradiomycins extracted from the actinomycete strain PTM-029 (**3** and **7**), (**9** and **12**), (**10**), and (**12**) showed significant biofilm inhibition towards *M. hydrocarbonoclasticus* that ranged from 25.1 ± 10.2% for (**10**) to 48.2 ± 1.9% for (**9** and **12**). Additionally, napyradiomycins (**3** and **7**) also showed significant biofilm inhibition towards *C. marina* (23.4 ± 4.9%) (Figure 4 and Table 2).

**Figure 4.** Biofilm inhibition assay performed using napyradiomycins (**1**–**12**), at a concentration of 31.25 μg/mL. After incubation for 18 h, planktonic cells were washed, and biofilm was stained using crystal violet and measured at OD600nm. The biofilm formation of *P. inhibens* was not inhibited by any of the napyradiomycins tested at this concentration. Percentage of biofilm inhibition refers to the percentage of biofilm that was inhibited in the presence of the napyradiomycins, when compared to biofilm formation with only DMSO. Shown are the average results of three replicates and error bars represent the standard error of the mean (SEM). N.I.—not inhibited; N.T.—not tested, results were statistically significant (\*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05; Dunnett's test).

**Table 2.** Results from the biofilm inhibition assay performed using napyradiomycins (**1**–**12**) at a concentration of 31.25 μg/mL. Shown are the average values of the percentage of biofilm inhibition of three replicates with the standard error of the mean (SEM). N.I.—not inhibited, N.T.—not tested.


All PTM-420 napyradiomycins, except (**1**) showed significant antibiofilm activity against *M. hydrocarbonoclasticus* (**4**, 56.9 ± 5.7%; **8** and **11**, 59.3 ± 3.8%; **5** and **6,** 43.4 ± 8.2%; **2**, 60.2 ± 5.7%).

As for the bacterial growth inhibition assays, compounds isolated from PTM-420 were tested against three other marine bacterial species, *M. hydrocarbonoclasticus, C. marina, M. luteus, P. batsensis,* and *P. inhibens* (Figure 4 and Table 2).

Napyradiomycin (**4**) showed significant antibiofilm activity against *M. luteus* and *P. batsensis* (97.0 ± 1.2%, and 13.4 ± 0.8%, respectively). Likewise, (**8** and **11**) also showed significant antibiofilm activity against *M. luteus* and *P. batsensis* (100.0 ± 0.3% and 26.2 ± 0.0%, respectively). Napyradiomycin (**1)** had high antibiofilm activity against *M. luteus* and *P. batsensis* (88.6 ± 2.9% and 87.2 ± 0.1%, respectively), while (**5** and **6**) inhibited the biofilm formation of *M. luteus* (87.3 ± 0.3%) (Figure 4 and Table 2).

PTM-420 napyradiomycins, were assayed at lower concentrations (serial 2-fold dilutions, 15.60 μg/mL to 0.98 μg/mL), against the bacterial species for which they showed antibiofilm activity at a concentration of 31.25 μg/mL (Table 3 and Figure 5).

**Table 3.** Percentage of inhibition of biofilm formation for di fferent marine bacteria in the presence of di fferent concentrations of napyradiomycins (**1)**, (**2)**, (**4**), (**5** and **6**)**,** and (**8** and **11**). Shown are the average values of the percentage of biofilm inhibition of three replicates with the standard error of the mean (SEM). N.I.—not inhibited.


Napyradiomycins (**8** and **11**) completely abolished (100%) biofilm formation of *M. luteus* at all tested concentrations. Biofilm formation by this species was nearly completely inhibited (>90%) by (**1**) at concentrations higher than 1.95 μg/mL, and at the lowest concentration of 0.98 μg/mL inhibition was of 59.0 ± 7.4%. At this concentration, biofilm formation was also nearly eliminated by (**4**) (>90%). Napyradiomycins (**5** and **6**) significantly inhibited the biofilm formation (~80%) of *M. luteus*, at a concentration of 3.91 μg/mL and higher. Biofilm formation of *M. hydrocarbonoclasticus* was inhibited by (**2**),(**4**),(**5**and**6**),and(**8**and**11**)byatleast50%atalltestedconcentrationsand80%inthecaseof(**4**).

 *P. batsensis* biofilm formation was inhibited by (**8** and **11**) at a concentration of 1.95 μg/mL and higher. Napyradiomycin (**1**) also e fficiently inhibited biofilm formation of *P. batsensis* (86.9 ± 1.0%) at a concentration of 15.60 μg/mL, and was moderately active at lower concentrations (Table 3).

The most promising compounds for antimicrofouling are those that inhibit biofilm formation without killing the bacteria. Compounds (**4**), (**5** and **6**), (**8** and **11**), and (**3** and **7**) have potential in this respect, as all inhibit biofilm formation of at least two of the marine bacterial species assayed without inhibiting their growth at the same concentration.

Specifically, (**4**) inhibited the growth of only *M. luteus* but was able to inhibit biofilm formation of *M. luteus* and *M. hydrocarbonoclasticus* (Figure 4 and Table 2). In addition, at the lowest tested concentration (0.98 μg/mL), the antibacterial activity against *M. luteus* was 38.7 ± 7.7%, while biofilm inhibition was over 90%. Therefore, (**4**) showed antimicrofouling e ffectiveness at low concentrations.

**Figure 5.** Inhibition of biofilm formation of *M. hydrocarbonoclasticus, M. luteus,* and *P. batsensis* by napyradiomycins (**1**), (**2**), (**4**), (**5** and **6**), and (**8** and **11**). Napyradiomycins were added to the biofilm growth medium of the different bacteria and after 18 h incubation planktonic cells were washed, and biofilm was stained using crystal violet and measured at OD600nm. Percentage of biofilm inhibition refers to the percentage of biofilm that was inhibited in the presence of the napyradiomycins, when compared to biofilm formation with only DMSO. Shown are the average results of three replicates and error bars represent the standard error of the mean (SEM). N.I.—not inhibited; results were statistically significant (\*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05, n.s. *p* > 0.05, Dunnett's test).

Napyradiomycins (**5** and **6**) showed no antibacterial activity, but high biofilm inhibition of *M. luteus* and *M. hydrocarbonoclasticus* (e.g., for a concentration of 3.91 μg/mL, biofilm formation of *M. luteus* was inhibited by 82.5 ± 5.1%, while biofilm formation by *M. hydrocarbonoclasticus* was inhibited by 43.8 ± 9.4%).

Napyradiomycins (**8** and **11**) showed inhibitory activity of biofilm formation and a variable degree of antibacterial activity against three of the five tested marine bacteria. For *M. hydrocarbonoclasticus*, (**8** and **11**) did not inhibit growth but significantly inhibited biofilm formation (~60%) for all tested concentration. For *M. luteus*, growth inhibition and complete biofilm abolishment (100%) was observed for all tested concentrations, however for the lowest concentration, growth inhibition was lower (70.5 ± 1.9%), indicating selective antibiofilm activity. For *P. batsensis*, growth inhibition (25.3 ± 0.6%) was observed only for the highest tested concentration (31.25 μg/mL), but significant inhibition of biofilm formation was observed at lower concentrations (37.8 ± 0.7% at a concentration of 15.60 μg/mL), with no detected antibacterial activity).

Finally, napyradiomycins (**3** and **7**) significantly inhibited (>20%) biofilm formation of *M. luteus* and *C. marina*, at a concentration of 31.25 μg/mL, while not showing any antibacterial effect on these marine bacterial species.

The antibiofilm activity of these napyradiomycins is not dependent on their antibacterial activity, which means that these compounds could be used as AF agents that will not contribute to antibiotic/biocide resistance.

The biofilm formation of *P. inhibens* and *P. batsensis* was inhibited by CuSO4 (12.9 ± 6.4% and 41.4 ± 0.9%). *M. luteus, M. hydrocarbonoclasticus,* and *C. marina* biofilm formation was not inhibited.

### *2.3. Antifouling Evaluation against Mytilus Galloprovincialis Larval Settlement*

Napyradiomycins (**1**–**12**) were screened against *M. galloprovincialis* plantigrade larval settlement to assess antimacrofouling activity. Most reports use tropical species and only a few involve temperate or cold-water species [59,60], we recognized the need to use European fouling species able to grow in temperate climate in bioassays.

The compounds revealed EC50 values ranging from 0.10 to 6.34 μg/mL (Table 4).

**Table 4.** Response of *M. galloprovincialis* plantigrade larvae settlement following incubation with napyradiomycins (**1**–**12**), after a 15 h acute exposure assay. Pearson goodness-of-fit (Chi-Square-χ2) significance was considered at *p* < 0.05 and 95% lower and upper confidence limits (95% LCL; UCL) were presented. Therapeutic ratio (LC50/EC50) was used to evaluate the effectiveness of each compound vs. its toxicity. Negative control: dimethylsulphoxide (DMSO) = 100% settlement; Positive control: CuSO4 0.16 μg/mL (5 μM) = 0% settlement.


All compounds revealed a good level of effectiveness, which is defined by an EC50 value < 25 μg/mL [61]. The most effective napyradiomycins, showing an EC50 below 1 μg/mL were (**1**) (EC50 0.66 μg/mL), (**8** and **11**) (EC50 = 0.73 μg/mL), (**9** and **12**) (EC50 = 0.45 μg/mL), (**10**) (EC50 = 0.10 μg/mL), and (**12**) (EC50 = 0.95 μg/mL) (Table 4). Interestingly, the EC50 of (**10**) was better than the commercial agen<sup>t</sup> ivermectin (EC50 = 0.4 μg/mL value against *M. edulis*) [62].

Regarding toxicity, none of the tested compounds caused mortality of *M. galloprovincialis* larvae at the highest tested concentration (12 μg/mL). Thus, LC50 values were considered higher than 12 μg/mL. The EC50 and LC50 values were used to calculate the therapeutic ratios (LC50/EC50). To meet the standard requirement for efficacy level of natural antifouling agents the US Navy program established a cut-off above 15 for the therapeutic ratio [21]. Therefore, the most promising antifouling agents towards *M. galloprovincialis* larvae are napyradiomycins (**1**) (LC50/EC50 = 18.3), (**8** and **11**) (LC50/EC50 = 16.5), (**9** and **12**) (LC50/EC50 = 26.6), and (**10**) (LC50/EC50 = 117.3) (Table 4).

### *2.4. Napyradiomycins in Silico Ecotoxicity Evaluation*

The ecotoxicity of diverse pharmaceuticals, biocides and chemical compounds have forced regulatory authorities to recommend the application of in silico risk assessment to predict the fate of these molecules and their potential ecological and indirect human health effects. Using the Toxicity Estimation Software Tool (T.E.S.T.) [63], napyradiomycins (**1**–**12**) were evaluated for potential ecotoxicity, the prediction results are in Table 5.


**Table 5.** Toxicity end point predictions for napyradiomycins **1**–**12**.

1 96 hour LC50 (mg/L). 2 48 hour LC50 (mg/L). 3 48 hour IGC50 (mg/L), the Nearest Neighbor model, the other models are unable to predict this end point. 4 LD50 (mg/kg). 5 DT: developmental toxicant. 6 MN: mutagenicity negative.

In accordance with the European Union Directive 2001/59/EC and the Regulation on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) 1272/2008, the in silico TEST results classified compounds **1**–**12**, with the danger symbol N ("dangerous for the environment"), risk phrase 50 ("very toxic to aquatic organisms" to "toxic to aquatic organisms"), acute toxicity estimate (ATE) category 4 ("practically non-toxic and not an irritant"), and as developmental toxicants with low bioaccumulation factor and mutagenicity negative [64–67].

In comparison, predictions of toxicity were performed for seven approved drugs: bimatoprost (**S1**), a topical medication used for controlling the progression of glaucoma or ocular hypertension; alfuzosin (**S2**), a nonselective alpha-1 adrenergic antagonist used in the therapy of benign prostatic hypertrophy; lovastatin (**S3**), a fungal metabolite isolated from cultures of *Aspergillus terreus* and a potent anticholesteremic agent; antimycin A (**S4**), an antibiotic produced by *Streptomyces* sp.; oxethazaine (**S5**), an anesthetic; calcipotriene (**S6**), a synthetic derivative of calcitriol or Vitamin D used for the treatment of moderate plaque psoriasis in adults; and latanoprost (**S7**), a prostaglandin F2alpha analogue and a prostanoid selective FP receptor agonist with an ocular hypertensive effect, (Supplementary Table S1), two antifouling agents (ivermectin B1b (**S8**) and ivermectin B1a (**S9**), (Supplementary Table S2), and arsenic and copper substances used in marine paints (Supplementary Table S3).

The predicted values related with environmental toxicity for (**1**–**12**) (Table 5) are in the same order of magnitude and have the same classification than those obtained for the Prestwick approved drugs (**S1**–**S7**) (Supplementary Table S1) and the two antifouling approved biocides **S8**, **S9** (Supplementary Table S2). Except for ATE, in which our napyradiomycins showed lower toxicity than **S1**–**S9** (category ≤ 4).

Comparing the toxicity predictions for (**1**–**12**) (Table 5) with the values for copper (Supplementary Table S3), we may predict that these MNP are less toxic than copper, which is widely used in antifouling paints and coatings [64,65]. Arsenic showed lower aquatic toxicity values than (**1**–**12**) but higher acute toxicity. Overall, the in silico results sugges<sup>t</sup> napyradiomycins as a suitable model to test for Naval Sea Systems Command (NAVSEA) standards and proceed in the antifouling coatings development roadmap (http://www.nstcenter.biz/navy-product-approval-process/navy-communitycoatings-roadmap/, accessed on 6th January 2020).
