2.2.1. Antibacterial Activity

As a first screening approach, liquid cultures of *M. hydrocarbonoclasticus,* and *C. marina* in a 96-well format were analyzed for bacterial growth inhibition by percent decrease in OD600 after incubation with napyradiomycins (**1**–**12**) at 31.25 μg/mL.

No PTM-029 napyradiomycins had activity against *M. hydrocarbonoclasticus* or *C. marina*, while PTM-420 napyradiomycins (**1**) and (**8** and **11**) revealed antibacterial activity against *C. marina.* Therefore, PTM-420 napyradiomycins were further tested against three other marine bacterial species, *M. luteus, P. batsensis,* and *P. inhibens*.

Napyradiomycin (**1**) inhibited the growth of *M. luteus, P. batsensis,* and *C. marina* (96.1 ± 0.5%, 53.9 ± 0.3%, and 19.8 ± 0.6%, respectively), while (**8** and **11**) inhibited the growth of the same three species in addition to *P. inhibens* (95.9 ± 0.1%, 25.3 ± 0.6%, 20.8 ± 1.3%, and 13.0 ± 3.7%, respectively). Napyradiomycin (**4**) only inhibited the growth of *M. luteus* (72.5 ± 7.7%), at this concentration (Figure 2). The remaining napyradiomycins (**2**) and (**5** and **6**) had no effect on bacterial growth of these marine bacterial species, at a concentration of 31.25 μg/mL.

**Figure 2.** Growth inhibition assay performed for napyradiomycins (**1**–**12**), at a concentration of 31.25 μg/mL. After incubation for 18 h, bacterial growth was assayed by measuring OD600nm. The growth of *M. hydrocarbonoclasticus* was not inhibited by any of the napyradiomycins tested at this concentration, while (**2**), (**3** and **7**), (**5** and **6**), (**9** and **12**), (**10**), and (**12**) did not inhibit growth of any of the bacteria tested. Percentage of growth inhibition refers to the percentage of growth that was inhibited in the presence of napyradiomycins, when compared to growth 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, Dunnett's test).

Napyradiomycins (**1**), (**4**) and (**8** and **11**) were further 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 antibacterial activity at a concentration of 31.25 μg/mL (Figure 3 and Table 1).

**Figure 3.** Bacterial growth inhibition of *M. luteus*, *C. marina,* and *P. batsensis* by napyradiomycins (**1**), (**4**), and (**8** and **11**). Napyradiomycins were added to the growth medium of the different bacteria and after 18 h incubation, bacterial growth was assayed by measuring OD600nm. Percentage of growth inhibition refers to the percentage of growth that was inhibited in the presence of the napyradiomycins, when compared to growth 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, n.s. *p* > 0.05, Dunnett's test).

**Table 1.** Percentage of growth inhibition of different marine bacteria with the addition of napyradiomycins (**1**), (**4**), and (**8** and **11**). Shown are the average values of the percentage of growth inhibition of three replicates with the standard error of the mean (SEM). N.I.—not inhibited.


Napyradiomycin (**1**) drastically inhibited (>90%) the growth of *M. luteus* at a concentration of 3.91 μg/mL. At the lowest tested concentration, 0.98 μg/mL, (**1**) significantly inhibited the growth of *M. luteus* (42.9 ± 0.4%). For *C. marina,* (**1**) was active at higher concentrations, albeit with a lower growth inhibition percentage (5.5 ± 0.9% for 3.91 μg/mL; 18.2 ± 0.7% for 7.81 μg/mL, and 15.3 ± 0.7% for 15.60 μg/mL). The growth of *P. batsensis* was also significantly inhibited by this napyradiomycin at the concentration of 7.81 μg/mL (47.4 ± 0.3%) and 15.60 μg/mL (62.7 ± 0.6%).

Napyradiomycin (**4**) was able to significantly inhibit approximately 50% of the growth of *M. luteus* at all tested concentrations.

Napyradiomycins (**8** and **11**) drastically inhibited (>90%) the growth of *M. luteus* at a concentration of 3.91 μg/mL. At the lowest concentration, 0.98 μg/mL, (**8** and **11**) still inhibited bacterial growth significantly (70.5 ± 1.9%). The growth of *C. marina was* also significantly inhibited by (**8** and **11**) at concentrations of 7.81 μg/mL (16.0 ± 1.2%), and 15.60 μg/mL (17.4 ± 0.5%). Although (**8** and **11**) inhibited the bacterial growth of *P. inhibens* and *P. batsensis* at a concentration of 31.25 μg/mL their growth was not inhibited at the lower tested concentrations (15.60 μg/mL to 0.98 μg/mL).

CuSO4 (5 μM), a potent antifouling agen<sup>t</sup> used in antifouling paints, was used as reference. The growth of *P. inhibens*, *P. batsensis*, and *M. hydrocarbonoclasticus* was inhibited by CuSO4 (66.2 ± 5.6%, 44.8 ± 6.3%, and 35.8 ± 0.8%). *M. luteus* and *C. marina* growth was not inhibited.
