X-ray Crystallographic Structure of a Teixobactin Analogue Reveals Key Interactions of the Teixobactin Pharmacophore

Abstract

The X-ray crystallographic structure of a truncated teixobactin analogue reveals hydrogen-bonding and hydrophobic interactions and a cavity that binds a chloride anion. Minimum inhibitory concentration (MIC) assays against Gram-positive bacteria correlate the observed structure with antibiotic activity.

The antibiotic teixobactin—first reported in 2015—kills Gram-positive bacteria without detectable resistance and offers promise against rising resistance in pathogens such as methicillin-resistant Staphylococcus aureus (MRSA).^1,2 In reflection of this promise, the initial report has been cited more than 500 times. Teixobactin is a non-ribosomal cyclic undecadepsipeptide and contains the rare amino acid allo-enduracididine at position 10.³ Teixobactin inhibits cell wall formation in Gram-positive bacteria by binding to lipid II and related peptidoglycan precursors.

Since the initial publication, multiple research groups have worked to synthesize teixobactin and to elucidate its pharmacophore. Two reports of the total synthesis of teixobactin have been published,^4,5 as well as a third describing the synthesis of the cyclic depsipeptide ring.⁶ A 10-step synthesis of allo-enduracididine suitable for preparing gram-quantities has also been reported.⁷ Several research groups have reported structure-activity relationship studies of Arg₁₀-teixobactin (Fig. 1) and related homologues in which arginine is used as a surrogate for allo-enduracididine.^8–13 Very recently, Singh et al. reported NMR-based structures and structure-activity-relationships of Arg₁₀-teixobactin and its diastereomers at positions 1, 4, 5, and 8.¹⁴

Fig. 1.

Structures of teixobactin and homologues.

We recently reported the elucidation of the teixobactin pharmacophore, describing syntheses and structure-activity studies of a variety of teixobactin homologues.¹⁰ On the basis of these data, we proposed a model in which the amide NH groups of the cyclic depsipeptide ring bind to the pyrophosphate group of lipid II through hydrogen-bonding interactions, in a fashion similar to the binding of nisin to lipid II (PDB 1WCO).¹⁵ We further proposed that the hydrophobic residues N-Me-D-Phe, Ile, and D-allo-Ile at positions 1, 2, and 5 help anchor teixobactin to the plasma membrane and demonstrated that residues 1–5 could be replaced with a lipid group. The resulting homologue lipobactin 1 (dodecanoyl-Δ_1–5-Arg₁₀-teixobactin) is only 2–4 times less active than Arg₁₀-teixobactin (Fig. 1).

In the current study, we report the X-ray crystallographic structure of a truncated version of lipobactin 1 in which the dodecanoyl group is replaced with an acetyl group, Ac-Δ_1–5-Arg₁₀-teixobactin (Fig. 1). In attempting to crystallize this homologue with inorganic pyrophosphate anions, we instead obtained a complex with chloride anion and observe that the chloride anion coordinates to three amide NH groups of the cyclic depsipeptide ring, the amide NH group of Ser₇, and the guanidinium group of Arg₁₀. Here we describe the X-ray crystallographic structure of Ac-Δ_1–5-Arg₁₀-teixobactin as the hydrochloride salt and relate the observed structure to changes in activity upon mutation of Arg₁₀-teixobactin.

We began our efforts to crystallize the teixobactin pharmacophore by screening Arg₁₀-teixobactin in 864 conditions in a 96-well plate format using crystallization kits from Hampton Research (PEG/Ion, Index, and Crystal Screen). Initial efforts to screen Arg₁₀-teixobactin for crystallization were thwarted by the propensity of the peptide to form a gel at concentrations as low as 5 mg/mL used for screening. Truncation by removal of residues 1–5 (Δ_1–5-Arg₁₀-teixobactin) eliminated the propensity to form a gel but afforded no crystals. We postulated that a monocationic homologue would better crystallize than the dicationic homologue and were gratified that Ac-Δ_1–5-Arg₁₀-teixobactin afforded crystals suitable for X-ray crystallography. Only conditions containing chloride anion afforded suitable crystals. Attempts to crystallize with inorganic pyrophosphate anions, with HCl being used to vary the pH of the pyrophosphate buffer, still afforded the chloride salt. The X-ray crystallographic structure shows Ac-Δ_1–5-Arg₁₀-teixobactin as the hydrochloride salt (Fig. 2).^‡

Fig. 2.

X-ray crystallographic structure of Ac-Δ_1–5-Arg₁₀-teixobactin as the hydrochloride salt. (A) Top view. (B) Side view. (C) Rotated side view, in which the side chains of Ile₆ and Ile₁₁ are shown as spheres. Hydrogens attached to carbons that are shown as sticks are omitted for clarity. Water of crystallization (1.5 H₂O per molecule of peptide) is not shown.

In the X-ray crystallographic structure, the carbonyl groups of D-Thr₈, Ala₉, Arg₁₀, and Ile₁₁ in the cyclic depsipeptide ring point upward, while the amide NH groups of Ala₉, Arg₁₀, and Ile₁₁ point downward (Fig. 2B). The α-amino group of D-Thr₈ and the attached residues (Ser₇ and Ile₆), run downward at almost a right angle to the cyclic depsipeptide ring. The side chain of Arg₁₀ also runs downward. The side chains of Ala₉ and Ile₁₁, as well as the methyl group of D-Thr₈ point outward from the cyclic depsipeptide ring (Fig. 2A). The amide NH group of Ala₉ hydrogen bonds to the oxygen atom of the hydroxy group of Ser₇. The side chains of Ile₆ and Ile₁₁ are in loose contact, suggesting a hydrophobic interaction (Fig. 2C). The methyl group of D-Thr₈ sits near the Ile₆ and Ile₁₁ side chains, creating a hydrophobic patch.

The amide NH groups of Arg₁₀ and Ile₁₁ in the cyclic depsipeptide ring, as well as the amide NH groups of Ser₇ and D-Thr₈ and the guanidinium group of Arg₁₀, hydrogen bond to the chloride anion (Fig. 2B). This mode of interaction is similar to that of nisin with the pyrophosphate group of lipid II.¹⁵ We envision that the binding cavity of teixobactin and its analogues may be able to adjust to accommodate larger anions, including the pyrophosphate group of lipid II and other related peptidoglycan precursors.

To explore the roles of the hydrophobic residues at positions 6, 9, and 11, we mutated each of these residues to lysine and compared the activity of the resulting homologues to that of Arg₁₀-teixobactin in minimum inhibitory concentration (MIC) assays in four types of Gram-positive bacteria. Mutation of either Ile₆ or Ile₁₁ to lysine results in loss of activity, while mutation of Ala₉ to lysine does not (Table 1).^§ These data suggest that the hydrophobicity of Ile₆ and Ile₁₁ is important in teixobactin activity, while that of Ala₉ is not. The outward pointing geometry of the Ala₉ side chain, coupled with the activity of Lys₉,Arg₁₀-teixobactin, suggest that the 9-position should allow functionalization to provide other modified homologous of teixobactin that are active.

Table 1.

MIC values of teixobactin homologues in μg/mL.^a

	Staphylococcus epidermidis ATCC 14990	Streptococcus salivarius ATCC 13419	Enterococcus durans ATCC 6056	Bacillus subtilis ATCC 6051	Escherichia coli ATCC 10798
Arg10-teixobactin	1	1	4	2	>32
lipobactin 1	4	4	8	4	>32
Ac-Δ1–5-Arg10-teixobactin	>32	>32	>32	>32	>32
Lys6,Arg10-teixobactin	>32	>32	>32	>32	>32
Arg10,Lys11-teixobactin	>32	>32	>32	>32	>32
Lys9,Arg10-teixobactin	1	1	4	1	>32
Chg6,Arg10,Chg11-teixobactin	1	0.5	2	1	>32
Ala7,Arg10-teixobactin	32	16	>32	32	>32
D-Dap8,Arg10-teixobactin	2	1	4	1	>32
vancomycin	0.5	0.5	0.5	1	>32
teixobactin	0.06	0.03	0.5	0.06	>32

^aAll teixobactin homologues were prepared and studied as the trifluoroacetate salts. The Staphylococcus, Streptococcus, Enterococcus, and Bacillus species are non-pathogenic (BSL-1) Gram-positive bacteria. The E. coli serves as a Gram-negative control. Vancomycin and teixobactin serve as positive controls.

To further explore the role of hydrophobicity at positions 6 and 11 and the contact between the Ile₆ and Ile₁₁ side chains, we mutated both of these residues to cyclohexylglycine (Chg). Cyclohexylglycine may be thought of as a homologue of isoleucine, in which two carbons have been added to the sec-butyl side chain to form a cyclohexane ring. The resulting homologue, Chg₆,Arg₁₀,Chg₁₁-teixobactin, has slightly greater activity than Arg₁₀-teixobactin, with three of the four measured MIC values in the Gram-positive bacteria lower by a factor of two (Table 1). This finding suggests that hydrophobicity or hydrophobic contact at positions 6 and 11 is important in the activity of teixobactin.

To explore the hydrogen bond between the amide NH group of Ala₉ and side chain of Ser₇, we mutated Ser₇ to alanine. The resulting homologue, Ala₇,Arg₁₀-teixobactin, shows greatly diminished activity (Table 1).^§§ This finding supports the importance of this hydrogen bond in the activity of teixobactin.

The hydrogen bonding of the depsipeptide ring to the chloride anion (Fig. 2) suggests the possibility of increasing the activity of teixobactin homologues by strengthening the complexation with the pyrophosphate group of lipid II. To explore this idea, we mutated D-Thr₈ to D-diaminopropionic acid (D-Dap). The mutation of D-Thr₈ to D-Dap replaces the lactone oxygen atom with an amide NH group, but also results in the loss of the threonine methyl group. The resulting homologue, D-Dap₈,Arg₁₀-teixobactin, shows comparable activity to Arg₁₀-teixobactin (Table 1). Direct comparison of these two homologues is hampered, because two factors are changed at one time in making this mutation. A reasonable interpretation of this observation is that enhanced activity from replacing the lactone oxygen atom with an NH group is offset by the increased conformational flexibility of the ring associated with removal of the D-Thr₈ methyl group.

The studies described here demonstrate how the X-ray crystallographic structure of a truncated teixobactin analogue can reveal key interactions of teixobactin. The structure reveals a 13-membered cyclic depsipeptide ring in which the amide groups and ester group of residues 8–11 align. The amide NH groups of residues 10 and 11, in conjunction with those of residues 7 and 8 and the guanidinium side chain of residue 10, create a cavity that can bind an anion. The hydrophobic side chains at positions 6 and 11 are required for activity, whereas that at position 9 is not. The hydrogen bond between Ser₇ and Ala₉ is also important for activity. The teixobactin pharmacophore tolerates the amide substitution of lactone oxygen in the ring. Fig. 3 summarizes these findings. We are now using the X-ray crystallographic structure and structure-activity relationships that we have observed to design teixobactin homologues with better pharmacological properties.

Fig. 3.

Summary of key findings.