Bacterial Type I Signal Peptidases as Antibiotic Targets

Smitha Rao CV; Jozef Anné

Disclosures

Future Microbiol. 2011;6(11):1279-1296. 

In This Article

Inhibitors of SPase & their Antibacterial Activity

Finding potent SPase I inhibitors with effective antibacterial activity is considered a challenging task. Early studies proved that SPases are insensitive to classical protease inhibitors of serine, cysteine, aspartic and metalloproteases,[17] that very few SPase molecules per cell need to be fully active for E. coli to grow and multiply (at least at 30°C in a nonstressed environment[24]) and that E. coli SPase activity is adversely affected by high concentration of sodium chloride (>160 mM), magnesium chloride (1 mM) and dinitrophenol.[59] Nevertheless, a few effective inhibitors have been identified.

Peptide & Protein Inhibitors

Similar to proteases in general, SPases are competitively inhibited by SPs in vitro, and in vivo by preproteins or synthetic peptide substrates with Pro at the +1 position, rendering an impaired growth of cells.[17] However, attempts to find effective peptide inhibitors using the classical approach were not successful.[16] Interestingly, an antimicrobial peptide from the fruit fly, resembling a natural signal sequence, was found to inhibit SPase competitively.[60] The protease resistant peptide D-KLKL6KLK has an IC50 of 50 µM against LepB and an MIC of 16 µM against E. coli, and also shows bactericidal activity against Gram-positive pathogens (Table 2).

β-lactam-type Inhibitors

The β-lactam analogs, monocyclic azetidinones, were the first nonpeptide inhibitors reported to inhibit E. coli LepB in a pH- and time-dependent manner, although only at 500 µM concentrations.[39] 5S-Penem sterioisomers were identified as potent irreversible inhibitors along with a few other weak inhibitors including clavam and clavem systems.[16] One of the compounds, allyl (5S,6S)-6-[(R)-acetoxyethyl]) penem-3-carboxylate (Figure 5) inhibits the activity of both E. coli LepB (IC50 = 0.38 µM) as well as S. aureus SpsB.[9] It completely blocks the processing of β-lactamase protein by the leader peptidase of an E. coli ESS strain, which has a leaky outer membrane.

Figure 5.

Selected SPase inhibitors summarized in Table 2.

This penem inhibitor was used for co-crystallization of the Δ2–75 SPase.[43] The co-crystal structure revealed that the side chain methyl group (C16) of the penem, previously reported to be essential for the effectiveness of the inhibitor, was located in the SPase substrate-binding pocket (S1), probably mimicking the P1 (-1) (Ala) side chain of the substrate. The structure showed that the inhibitor acts via acylation of the active site Ser.

Unfortunately, penem inhibitors have poor antibacterial activity against some of the more clinically relevant strains, which might be explained by low compound penetration across the cell wall and compound instability.[16] Recently, the synthesis of the 5S penem was described and as with the parent penem, no significant antibacterial activity was observed against S. aureus and E. coli.[61] Interestingly, Harris et al. observed moderate activity against S. epidermidis.[61] In addition, carbamate-derivatized penems (Figure 5) moderately inhibited MRSA (Table 2).

Furthermore, (5S) tricyclic penems (Figure 5) with a third heterocyclic ring fused to the C2 and C3 positions of the (5S,6S)-6-[(R)-hydroxyethyl]-penem core have a higher potency with IC50 values of 0.2 µM and 5 µM against E. coli and MRSA SPases, respectively.[62] This higher in vitro potency is thought to be due to additional binding features, such as the extension of the inhibitor into the S3 binding pocket of the SPase I, as observed by molecular modeling.[62] Again, the antibacterial activity of the tricyclic penems is poor (Table 2), possibly owing to compound instability.

Arylomycins & Lipoglycopeptide Inhibitors

Structure Arylomycins were first identified as new biaryl-bridged lipopeptide antibiotics produced by Streptomyces sp. Tü 6075,[63] with their antibiotic property subsequently attributed to their ability to inhibit SPase.[44] The lipoglycopeptide inhibitors of SPase were identified independently, also from other Streptomyces spp..[55,101] Arylomycins and lipoglycopeptides are classified as secondary metabolites synthesized by nonribosomal peptide synthesis and share a common core structure (Figure 5) consisting of a biaryl-linked, N-methylated peptide macrocycle attached to a lipopeptide tail. Lipoglycopeptides contain a 2-deoxymannose sugar unit in the macrocycle and have a longer (up to five carbon) fatty acid tail in comparison with arylomycins. Arylomycins are grouped into two related series called the A and the B series, which are colorless and yellow colored, respectively. Arylomycins are lipohexapeptides with the sequence D-N-MeSer-D-Ala-Gly-L-N-MeHpg-L-Ala-L-Tyr (Figure 5), cyclized by a [3,3] biaryl bond between MeHpg and Tyr (in A series, or a nitro-substituted Tyr in B series) and with a C11–15 branched fatty acid attached via an amide bond to the amino terminus.[64]

Antibiotic activity, together with its mode of action revealed by the SPase co-structure,[44] led to the efforts in synthesis of the inhibitor. The total synthesis of arylomycin A2[65,66] and B2[67] has been accomplished using the intramolecular Suzuki–Miyaura reaction, as a key step for macrocyclization.

Biological Activity Initially, the antibacterial activity of arylomycins was tested only against a very small number of bacterial species and gave a general idea that it is effective against some, but not against others including E. coli.[63] On the other hand, lipoglycopeptides were shown to be potent inhibitors of SPases of E. coli and S. pneumoniae and with modest antibacterial activity against a panel of pathogens (Table 2).[55] These inhibitors blocked protein secretion in vivo, as demonstrated using the β-lactamase secretion assay in S. aureus. Subsequently, the synthesis of arylomycin A2 and a few derivatives enabled the study of the effect imparted by the three main modifications: N-methylation, glycosylation and lipidation (common to arylomycins and the related lipoglycopeptides) on biological activity.[65] The data suggested that while glycosylation is less critical, fatty acid chain length and N-methylation make important contributions to biological activity, but then it varies depending on the species. Notably, an arylomycin derivative, designated arylomycin C16, possessing the longer iso-C16 fatty acid (characteristic of the lipoglycopeptides) but lacking the sugar moiety (as in the case of natural arylomycin A2) (Figure 5), was found to have inhibitory activity similar to that of the lipoglycopeptides against S. aureus and polymyxin B nonapeptide permeabilized E. coli (MIC of 16 µM).[65] In comparison, natural arylomycin A2 does not inhibit S. aureus (up to 128 µM) and only weakly inhibits permeabilized E. coli (MIC of 128 µM). Interestingly, arylomycin C16 and the natural arylomycin A2 are highly potent against S. epidermidis (MIC of 0.5 and 1 µM, respectively), comparable to the antibiotics currently prescribed for its treatment.[65] This also applies for a few other important species of coagulase-negative staphylococci, such as Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus hominis, in which arylomycin C16 exhibits antibacterial activity equal to, or greater than, vancomycin, the antibiotic most commonly used for treatment of coagulase-negative staphylococci infections (Table 2).[68] It has been demonstrated that the application of arylomycin A2 on S. epidermidis biofilms results in a dose-dependent reduction in cell viability.[25] Further work is needed to establish the use of SPase inhibitors in the treatment of biofilm-associated infections.

When challenged with arylomycin C16, S. epidermidis evolved resistance via a point mutation in a less conserved region of the SPase with which arylomycin interacts.[69] The resistance conferring mutation of Ser29 to Pro29 (position 29, in one of the two active SPases of S. epidermidis, SpsIB) reduces the affinity of arylomycin to the SPase. Interestingly, analogous mutations were found to be responsible for natural resistance to arylomycin observed in E. coli, P. aeruginosa and in some strains of S. aureus.[69] The results indicated that the absence of Pro29 is often predictive of arylomycin sensitivity, but varies across species. Importantly, it was demonstrated that arylomycin C16 has a broad-spectrum of activity that includes several clinical isolates (Table 2) and it does not have adverse effects on human cells, up to 20 µg/ml.[69] Although the mutation currently limits therapeutic application of arylomycins, it should be possible to modify these natural products to overcome the resistance mechanism. This could be done, for instance, by introducing additional interactions with the target site or with the neighboring regions, thereby increasing the affinity or by introducing an additional mode of action into the compound. A recent structure–activity relationship study, focused on the lipopeptide tail part of arylomycin, revealed that both the methylation state and the length of straight chain fatty acid of the arylomycins are already optimized for activity, while phenyl-modified derivatives are likely to be better scaffolds for arylomycin optimization than the natural, saturated fatty acid chains.[70]

Co-crystal Structure of Δ2-75 SPase/arylomycin A2 The co-crystal structure of Δ2-75 SPase in complex with arylomycin A2 revealed the mode of action and the contact surface of the noncovalently bound inhibitor at the active site.[44,45] The structure shows the hexapeptide of arylomycin with its C-terminal biaryl-bridged end pointing toward the active site, and making β-sheet type hydrogen bonding interactions with the SPase binding cleft, similar to the interactions seen in serine protease substrate complexes. The C-terminal carboxylated oxygen atom (O45) interacts with three catalytic residues including the nucleophile Ser90 Oγ, the general base Lys145 Nζ and the oxyanion hole Ser88 Oγ. Its carbonyl oxygen atom O44 is hydrogen-bonded to Ile144N and the general base Lys 145 Nζ. The L-Ala methyl side chain (C30) of arylomycin A2 sits in the proposed S3 binding pocket and the D-Ala methyl side chain (C9) of the inhibitor pointed into a shallow pocket that possibly could be the S5 binding pocket of the enzyme. The methylene groups of the 12-carbon fatty acid of arylomycin A2 make van der Waals interactions with the predicted membrane association surface of the SPase.[45] This could explain the significance of the amino-terminal fatty acid shown to be important for the effectiveness of both SPase inhibitors as well as substrates (see the preceding section on lipopeptide inhibitors).

In an interesting development, the structure of Δ2-75 SPase in a ternary complex with arylomycin A2 and a morpholino-β-sultam derivative (BAL0019193) revealed SPase inhibition by the two compounds at the same time via binding to nonoverlapping subsites near the catalytic center.[45] β-sultams are reactive sulfonyl analogs of β-lactams, which are weak inhibitors of SPase, in contrast to arylomycin A2 (with an IC50 of 610 ± 18 µM and 1 ± 0.2 µM against Δ2-75 SPase). The β-sultams inactivate serine enzymes by sulfonylation of active site Ser,[71] but then the β-sultam in the ternary complex was bound noncovalently above the active site of the SPase, taking a parallel orientation relative to the biaryl ring moiety of arylomycin A2. The β-sultam inhibitor possibly acts by displacing a water molecule that is co-ordinated to the catalytic Lys145, proposed to function as the deacylating water in SPase catalysis.[45] Overall, the structural data by Paetzel and co-workers[43–45] provides an excellent template for inhibitor design. In particular, it can be applied for suitably extending the interactions of arylomycin A2 such that the molecule occupies the novel space defined by the β-sultam inhibitor in the active site of the SPase.

Rationally Designed Lipopeptides

Compounds designed on the basis of preprotein sequence data in S. aureus, incorporating key structural features of a signal sequence, namely a hydrophobic stretch, a helix terminus or turn motif and the AXA cassette, led to the identification of highly effective synthetic substrates and inhibitors of SpsB.[58] It was further demonstrated that SpsB could be inhibited in a classical manner by either incorporating proline at P1 or by introducing a serine trap (α-ketoamides) at the cleavage site of a lipopeptide substrate to obtain competitive or time-dependent inhibitors, respectively (Table 2). A competitive inhibitor described by Bruton et al.[58] was used as a lead compound to optimize the minimum length required for effective inhibition and to confirm that a fatty acid chain is essential.[72] Reducing the length of the core lipopeptide by eliminating amino acids from both termini and conversion of the C-terminal carboxylic acid of the peptide to an aldehyde resulted in an improved potency against S. aureus and E. coli SPases (Table 2). However, the lipopeptide aldehyde (Figure 5) had no significant antibacterial activity at the concentration tested.

The amino terminal fatty acid in the inhibitor appears to be critical for potency for both the in vitro and in vivo activity against S. aureus SpsB and E. coli LepB. A direct association between the fatty acid and the membrane association surface of the SPase was provided by the SPase co-structure with arylomycin A2.[45] Previous attempts at developing peptide inhibitors using the classical approach were not effective, most likely owing to the absence of the fatty acid. Although the reason for this is still unclear, it is presumed that the amino-terminal fatty acid might mimic the hydrophobic region of the SP of preproteins by appropriately aiding in orientating the inhibitor/substrate in the binding cleft of the enzyme. Another hypothesis is that the fatty acid in the inhibitor probably helps in partitioning into the hydrophobic part of the detergent milieu on which the SPase is likely to be present[45] as even the soluble derivatives of both SpsB[38] and LepB[40] are detergent dependent. With regard to their potential use as drugs, however, this requirement might give them a slight disadvantage over small molecule inhibitors.

Overall data on SPase inhibitors (summarized in Table 2) shows that the potency of each inhibitor varies not only across but also within the bacterial species (e.g., S. aureus and S. epidermidis). The reason for this variation possibly includes factors such as differences in the wall structure or composition and subtle differences between the SPases from different bacteria. In addition, a varying degree of disparity is observed between inhibitor potency in vitro and antibacterial activities in vivo in each bacterium tested. This is most evident in the case of 5S-penems and the lipopeptide aldehyde, both of which are highly potent inhibitors but lack significant antibacterial activity. Although the exact reason for this is not known, in vivo stability of the compound could be an issue. Another question that remains is whether the SPase that acts as a part of a membrane-bound complex restricts accessibility of the catalytic center, thereby giving a kinetic advantage to a substrate over an exogenous inhibitor.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as:

processing....