In this report, we present a novel strategy for the design and synthesis of STAMPs with activity against the oral pathogen S. mutans. Successful design was achieved through a tunable, building-block approach that utilized various combinations of antimicrobial, targeting, and linker regions. Our results demonstrate that less-efficacious STAMPs can be improved when alternative killing regions are substituted in the design. This process resulted in Sm6(L1)B33, a STAMP that displayed killing kinetics consistent with oral therapeutic applications and selectivity for S. mutans in multispecies biofilms.
The data presented suggest that the activity of the PL-135 AMP may be inhibited by conjugation to other peptide subunits, as unmodified PL-135 displayed MIC activity against S. mutans
that was 2- to 8-fold better than that of progeny STAMPs, as shown in Table . Furthermore, library 1 STAMPs exhibited significantly reduced biofilm binding compared to library 2 conjugates with identical targeting regions, suggesting PL-135 interference in Sm6 activity as well. The unusually small size of PL-135 may impose a severe restriction on amino acid additions, especially when the mode of antimicrobial action depends on sequence-dependent self-association on the cell membrane or on binding to a discrete intracellular bacterial target (2
). It remains unclear why PL-135 should inhibit Sm6 targeting peptide function.
Our results suggest that the optimal arrangement of STAMP domains is likely AMP specific and depends on which of the domains least affects, or even enhances, the antimicrobial mechanism. For example, the Pseudomonas
-specific STAMPs G10KHc and G10KHn (oriented as target domain-killing domain and killing domain-target domain, respectively) both bind specifically to the target bacterium surface, but only G10KHc shows significant membrane disruption activity (5
). Further biochemical studies of pilot STAMP libraries of greater diversity are being conducted to fully evaluate whether correct pairings can be more accurately predicted.
Interestingly, Sm6 and Sm7 containing library 1 STAMPs were active against S. mutans
, whereas the constructs with any one of the other targeting peptides listed in Table were not. Targeting peptides Sm1 through Sm5 are strongly hydrophobic compared with Sm6 and Sm7 (8
), and it may be possible that this characteristic limits the dissociation of these molecules from the hydrophobic components of the S. mutans
cell wall, resulting in their inhibitory affect on AMPs when conjugated, in similarity to the results seen with some strong lipopolysaccharide (LPS)-binding AMPs (19
). However, the systematic design strategy employed here allowed us to generate a diverse array of STAMPs, including useful compounds such as Sm6(L1)B33, despite these stumbling blocks.
It remains to be seen whether the selectivity observed with the STAMPs described in this report can be maintained in the oral cavity during treatment. Typically, oral-care antimicrobials are applied at high doses, suggesting that any selectivity “window” would be overwhelmed by nonspecific STAMP activity at higher concentrations. However, there are up to a total of 1 × 108-9
CFU/ml of bacteria in the mouth, of which as many as 1 × 107
/ml can be S. mutans
). These bacterial burden levels are 10 to 100 times higher than those employed in the assays reported here, which suggests that typical oral therapeutic concentrations are necessary for activity and selectivity. Additionally, the typical 30 s to 2 min of treatment duration for oral rinse formulations may limit STAMP antimicrobial activity to targeted organisms, as seen in Fig. .
In conclusion, this report details the rational design of S. mutans-selective STAMPs with enhanced antimicrobial killing kinetics and selectivity compared to untargeted AMPs. The S. mutans-selective STAMPs were constructed using a tunable, combinatorial approach that generated a diverse number of STAMP sequences for antimicrobial evaluation and improvement, a process that may serve as an example for the systematic development of novel selective antimicrobial agents. We propose that these STAMPs could be useful in the design of therapeutics against oral or other mucosal pathogens, where the high diversity of “probiotic” beneficial microflora limits the effectiveness of broad-spectrum antimicrobial agents.