Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis. Using conserved catalytic nucleophiles at their N-and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH 3 ) 2 Cl 2 ], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N-and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.Inteins, self-splicing protein elements that invade a host gene, have been of great interest to the field of biotechnology. The unique ability of an intein to break and form peptide linkages has led to their use in applications (1, 2) ranging from sensors of small molecules (3, 4) and environmental conditions (5) to single step protein purification platforms (6, 7). However, there are still underexplored reactions involving native inteins, notably their susceptibility as targets for antimicrobial drugs (8). In nature, inteins reside in key host proteins across several bacterial and fungal pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Cryptococcus neoformans (9).These splicing elements divide the host protein into two parts, termed N-and C-exteins on the corresponding ends of the intein. In the precursor form, the host protein is usually non-functional because of the tendency of an intein to insert into highly conserved functional regions of the protein. In M. tuberculosis, inteins occur in three genes, recA, sufB, and dnaB (9). The functionality of these proteins is key to the survival of the bacterium and relies upon the splicing of the intein from the respective host proteins. Because inteins do not occur in multicellular organisms, prevention of protein splicing provides a promising strategy for the development of novel antimicrobial therapeutics.Canonical intein splicing occurs by a multistep process (10). First, a nucleophilic attack is initiated by the N-terminal cysteine residue (C1) of the intein on the preceding peptide bond, resulting in thioester formation (Fig. 1A, step 1). A second nucleophilic a...

c Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of "database filtering" bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 M) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created.

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Edited by George CarmanAs pathogenic bacteria become resistant to traditional antibiotics, alternate approaches such as designing and testing new potent selective antimicrobial peptides (AMP) are increasingly attractive. However, whereas much is known regarding the relationship between the AMP sequence and potency, less research has focused on developing links between AMP properties, such as design and structure, with mechanisms. Here we use four natural AMPs of varying known secondary structures and mechanisms of lipid bilayer disruption as controls to determine the mechanisms of four rationally designed AMPs with similar secondary structures and rearranged amino acid sequences. Using a Quartz Crystal Microbalance with Dissipation, we were able to differentiate between molecular models of AMP actions such as barrel-stave pore formation, toroidal pore formation, and peptide insertion mechanisms by quantifying differential frequencies throughout an oscillating supported lipid bilayer. Barrel-stave pores were identified by uniform frequency modulation, whereas toroidal pores possessed characteristic changes in oscillation frequency throughout the bilayer. The resulting modes of action demonstrate that rearrangement of an amino acid sequence of the AMP resulted in identical overall mechanisms, and that a given secondary structure did not necessarily predict mechanism. Also, increased mass addition to Gram-positive mimetic membranes from AMP disruption corresponded with lower minimum inhibitory concentrations against the Gram-positive Staphylococcus aureus.All multicellular organisms such as animals and plants protect themselves against pathogenic microbes by producing antimicrobial peptides (AMPs) 2 that selectively disrupt bacterial cell membranes (1). Although they are enormously diverse, AMPs are mainly comprised of hydrophobic and cationic amino acids that are spatially organized along the molecule. Because bacteria depend on the integrity of their anionic cell membrane, disrupting their membrane with cationic peptides could offer an alternate strategy to conventional antibiotics for killing pathogenic bacteria (2). As these pathogens become resistant to traditional antibiotics, alternate approaches such as designing and testing new potent selective antimicrobial peptides are increasingly attractive.The proper composition of amino acids, their sequential arrangement, and peptide length are essential for effective action of AMPs (3). It has been demonstrated that AMP activity is more closely tied to amino acid composition than amino acid sequence or AMP structure (4 -8). However, it has recently been shown that for the ␣-helical class of AMPs, ordering amino acids during AMP design into an imperfect amphipathic ␣-helix, a helix barrel-stave with one hydrophobic and one hydrophilic face where the hydrophobic face is disrupted by one hydrophilic amino acid, is beneficial for increasing AMP activity (9). Understanding the mechanism behind how these peptides disrupt cell membranes could benefit future designs of p...