Bacterial Type I Signal Peptidases as Antibiotic Targets

Table 1.  Comparison of the specific enzymatic activity of the SPases for different synthetic peptides and a preprotein substrate (topmost).

Substrate	Assay/detection method	k cat/KM (M−1s−1)	Substrate sequence†	Ref.
		Staphylococcus aureus SpsB	Escherichia coli LepB	Streptococcus pneumoniae Spi
A	SDS-PAGE/densitometry	16	1.1 × 107	–	Pro-OmpA-Nuclease A‡	[10,58]
B	FRET/spectrofluorometry	18.4	71.1	–	Y(NO2)FSASALA↓KIK(Abz)	[58,78]
C	FRET/spectrofluorometry	–	2.5 × 106	–	K(5)-L(10)- Y(NO2)FSASALA↓KIK(Abz)	[57]
D	FRET/spectrofluorometry	–	–	2.7 × 102	KLTFGTVK(Abz)PVQA↓IAGY(NO2)EWL	[79]
E	FRET/spectrofluorometry	4.6 × 104	–	–	A proprietary lipopeptide substrate	[58,102]
F	HPLC/UV detection	23.2 ± 1.8	–	–	Ac-A↓GPRPTRIAFGA-NH2	[80]
G	FRET/spectrofluorometry	2.3 × 106	4.2 × 105	–	Decanoyl-LTPTAKA↓ASKIDD-OH	[58]
H	FRET/spectrofluorometry	1.85 × 103	–	–	Dabcyl-ADHDAHA↓SET-EDANS	[38,81]

†Amino acids are abbreviated as single letter codes. The arrow indicates the point of cleavage and P1 and P3 residues are in bold.
‡A hybrid protein made up of signal peptide of outer membrane protein A from Escherichia coli and the nuclease A from Staphylococcus aureus.
Abz: 2-aminobenzoic acid; Ac: Acetylated; Dabcyl: 4-(4-dimethylaminophenylazo) benzoic acid; EDANS: 5-(2-aminoethyl)aminonaphthalene-1-sulfonic acid; FRET: Fluorescence resonance energy transfer; HPLC: High-performance liquid chromatography; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Y(NO2): 3-nitro-l-tyrosine.

Table 2.  Inhibitors of the bacterial type I signal peptidases with their in vitro and in vivo activities.

Inhibitor		SPase	IC50 (µM)†	Microorganism	MIC	Ref.
Peptide
1	(D)-KLKL6KLK-NH2	LepB	30	Escherichia coli E. coli ‡	16 µM 4.8 µM	[60]
				Staphylococcus aureus	8 µM
				Haemophilus influenzae	>64 µM
				Streptococcus pneumoniae	4.8 µM
β-lactam type
2	Allyl (5S,6S)-6-[(R)-acetoxyethyl] penem-3-carboxylate	LepB	0.38	E. coli	NK§	[16]
3	(5S,6S)-6-[(R)-hydroxyethyl] penem core with a C3 p-nitrobenzyl protected carboxylic acid			E. coli‡ S. aureus Staphylococcus epidermidis	≥200 µg/ml ≥200 µg/ml 50 µg/ml	[61]
4	Carbamate-derivatized penem			MRSA	100 µg/ml	[61]
5	(5S)-Tricyclic penem	LepB	0.2	E. coli	NK	[62]
		SpsB	5	MRSA	>128 µg/ml
Lipoglycopeptides
6	Natural lipoglycopeptides	LepB	0.11–0.19	E. coli	4–8 µM	[55,101]
		Spi	2.4–24.9	S. pneumoniae	8–64 µM
				S. aureus	32–64 µM
				H. influenzae	≥64 µM
Arylomycins (natural/synthetic)
7	Arylomycin A2	LepB	NK	E. coli	>128 µM	[65]
				E. coli ‡	128 µM
				S. aureus	>128 µM
				S. epidermidis	1 µM	[68,69]
8	Arylomycin C16 §			Streptococcus pyogenes Helicobacter pylori Chlamydia trachomatis Klebsiella pneumonia Yersinia pestis Clostridium perfringens Corynebacterium glutamicum Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus lugdunensis Staphylococcus saprophyticus	16 µg/ml 4 µg/ml 6 µg/ml >128 µg/ml 4 µg/ml >16 µg/ml 2 µg/ml 2 µg/ml 0.25 µg/ml 0.25 µg/ml >64 µg/ml
Lipopeptides (rationally designed)
9	Decanoyl-LTPTAKAPSKIDD-OH	SpsB	0.6	S. aureus	NK	[58]
10	Decanoyl-LTPTANA-α-ketoamide analogs	SpsB	0.1–16	S. aureus	NK	[58]
11	Decanoyl-PTANA-aldehyde	SpsB LepB	0.09 13.4	S. aureus E. coli	125 µg/ml >500 µg/ml	[72]

See Figure 5 for chemical structures of 2, 4, 5, 6, 7, 8 and 11.
†Half-maximal (50%) inhibitory concentration. ‡Polymyxin B nonapeptide permeabilized cells.
§For the detailed list of bacteria tested against arylomycin, see the articles cited. MIC: Minimum inhibitory concentration; NK: Not known.

Authors and Disclosures

Smitha Rao CV¹ & Jozef Anné<sup>†1

1</sup>Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium

† Author for correspondence
+ 32 1633 7371 Fax: 32 1633 7340 jozef.anne@rega.kuleuven.be

Sidebar

Executive Summary

There is an Urgent Need for Novel Antibacterials With a Novel Action Mechanism

• The remarkable adaptability of bacteria together with uncontrolled and improper use of antibiotics have led to the development of drug-resistant and multidrug-resistant bacterial strains.

• The simultaneous downtrend in the development of new antibiotics has created an urgent need for antibacterial agents with a unique action mechanism to avoid cross-resistance with existing antibacterial drugs.

Type I Signal Peptidase, an Important Enzyme in the Preprotein Translocation Pathway, Represents an Attractive Antibacterial Target

• Signal peptidases (SPases) cleave off signal peptides, releasing the mature protein, during or shortly after translocation across the membrane.

• Bacterial SPases are: ubiquitous, essential for viability, required for virulence and differ from their human counterpart. The catalytic domain on the outer side of the membrane is relatively easily accessible to potential inhibitors.

SPases Have Been Characterized in vitro in a Number of Pathogens While Escherichia coli LepB Remains by Far the Most Widely Studied

• Crystal structure of a catalytically active fragment of E. coli LepB, designated as Δ2-75 SPase, solved in apoform and in complex with three different SPase inhibitors, has been valuable in understanding the active site topology and druggable regions.

• SPases from Gram-positive and Gram-negative bacteria share a common protein fold despite differences, such as the overall size and substrate specificity.

• Solution NMR can, in principle, be used for structural analysis of the full-length SPase, an integral membrane protein, but calls for optimization.

• The Ala-X-Ala substrate specificity of the SPase has been explained by the crystal structure. The residues contributing to substrate specificity and the high fidelity of the enzyme were identified by site-directed mutagenesis.

• The contact surface of SPase upon binding to a signal peptide, as revealed by NMR, confirms the involvement of residues previously identified by structural and biochemical studies.

SPase Activity Can be Easily Measured Both in vitro & in vivo

• In vivo assays involve measurement of preprotein (e.g., β-lactamase) processing by the SPase in whole cells.

• Synthetic substrates with the key features of native SPase substrates, namely a hydrophobic stretch, AXA cassette and a turn-inducing motif (Pro at P5) are the most efficient for in vitro assays.

A Few Potent SPase Inhibitors Have Been Identified & Characterized

• Peptides that resemble natural signal sequence (e.g., an antimicrobial peptide of the flesh fruit fly) are effective SPase inhibitors with modest antibacterial activity.

• Among the β-lactam analogs, 5S,6S penems and tricyclic penems are potent irreversible inhibitors of SPase but their poor antibacterial activity renders them unsuitable at present.

• Arylomycins and lipoglycopeptide natural products are potent inhibitors of SPase in vitro with antibacterial activity ranging from very high as seen in case of S. epidermidis to not significant in some other species. A proven broad-spectrum antibiotic, arylomycin, needs to be modified in order to overcome resistance caused by a point mutation that is naturally occurring or can be acquired. There is ample scope for rational modification due to the knowledge of synthesis and co-crystal structure.

• Substrate-based lipopeptide inhibitors are effective in vitro but lack significant antibacterial activity. The amino terminal fatty acid in SPase substrates and inhibitors appear to be critical for potency.

Future Perspective

• The 3D structure of the full-length SPase and structural data of SPase from Gram-positive bacteria is needed to facilitate the search for drugs based on SPase targets.

• A good combination of in vitro and in vivo assays and a diverse library of natural and synthetic products are required for HTS.

• Parallel or complementary approaches for finding SPase inhibitors include fragment-based screening, virtual screening and structure-based drug design.