Antibacterial therapeutics are of great medical importance and have saved countless human lives in past decades. Owing to the rise in drug resistance amongst major human pathogens in recent years, there is an urgent need for antibiotics with novel mechanisms of action (Barrett & Barrett, 2003). One promising target for the discovery of such new antibacterial therapeutics is the biosynthesis of fatty acids in bacteria (Heath et al., 2001; Payne, 2004). Fatty-acid biosynthesis is an essential and highly conserved enzymatic process. The growing fatty-acid chain is shuttled between the catalytic functions while bound to a small acyl carrier protein (ACP) via a phosphopantetheine linker. The high degree of conservation in fatty-acid biosynthesis creates the potential for a genuinely broad-spectrum antibiotic. On the other hand, significant differences between prokaryotic and eukaryotic (and in particular mammalian) fatty-acid biosynthesis will allow the necessary differentiation towards the prokaryotic target. In prokaryotes the series of catalytic steps for fatty-acid biosynthesis is performed by individual enzymes (type II fatty-acid biosynthesis; Rock & Jackowski, 2002), while in mammals all the required catalytic functions are united on a large single polypeptide chain (type I fatty-acid biosynthesis; Smith et al., 2003). The success of fatty-acid biosynthesis as an antibacterial target is exemplified by triclosan, a chlorinated bisphenol that selectively inhibits bacterial enoyl-ACP reductase. It has been in use for decades as an additive in many consumer products owing to its bactericidal properties (Bhargava & Leonard, 1996).
Another highly interesting target within the fatty-acid biosynthetic pathway is β-ketoacyl-ACP synthase (KAS; Khandekar et al., 2003). This enzyme catalyses the Claisen condensation reaction, i.e. carbon–carbon bond formation upon the addition of a new acetyl unit to the growing fatty-acid chain (Heath & Rock, 2002). The reaction can be divided into three distinct steps: (i) transfer of the acyl group from acyl-ACP to the active-site cysteine residue, resulting in a thioester, (ii) binding of malonyl-ACP and subsequent decarboxylation to form a carbanion and (iii) nucleophilic attack of the carbanion on the carbonyl carbon of the thioester to form the carbon–carbon bond. The functional oligomer of the KAS protein is the homodimer, with residues from both monomers being involved in each fatty-acid-binding pocket (Edwards et al., 1997; Huang et al., 1998; Olsen et al., 1999). Three related but distinct enzymes with KAS activity are found in Escherichia coli and some other bacteria (KAS I, II and III, also known as FabB, FabF and FabH, respectively; Heath & Rock, 2002). KAS I and KAS III are essential enzymes in E. coli, in contrast to KAS II (Rosenfeld et al., 1973; Garwin et al., 1980; Lai & Cronan, 2003).
Several novel inhibitors of bacterial KAS III protein have recently been reported (He & Reynolds, 2002; Daines et al., 2003; He et al., 2004; Nie et al., 2005), highlighting the ongoing efforts to target novel antibacterial drugs against KAS activity. KAS I is a particularly attractive target as it has already been validated through the action of two compounds from natural sources: cerulenin and thiolactomycin (Fig. 1). Both inhibit KAS I and KAS II but not KAS III activity (Price et al., 2001). Cerulenin (from Cephalosporium caerulens) has a reactive epoxy group that forms a covalent adduct with the active-site cysteine of KAS (Price et al., 2001; Vance et al., 1972; D'Agnolo et al., 1973; Moche et al., 1999). It is not a selective antibacterial, however, since it also inhibits the KAS function of the mammalian FAS multienzyme complex. The second compound, thiolactomycin, is a thiolactone from actinobacteria (Nocardia sp.) that reversibly inhibits bacterial KAS (Price et al., 2001; Nishida et al., 1986). It is active against a wide range of bacteria and other pathogens such as plasmodia and trypanosomes (Noto et al., 1982; Waller et al., 1998; Morita et al., 2000). It does not affect mammalian fatty-acid biosynthesis (Hayashi et al., 1983) and in mice it protects against infections without toxic side effects (Miyakawa et al., 1982). However, problems in synthesis and with stability have prevented its use as an antibiotic drug (Heath et al., 2001). Thiolactomycin-derived compounds with improved properties are currently being explored as inhibitors of bacterial and mammalian KAS (Sakya et al., 2001; Douglas et al., 2002; McFadden et al., 2005). Recently, a novel compound from a natural source, platensimycin, was discovered to be a potent inhibitor of bacterial KAS I and KAS II, with antibiotic properties in animal models (Wang et al., 2006). However, it remains challenging to develop such complex natural products into drug molecules with the required efficacy, synthetic access and pharmacological properties.
Structures of cerulenin, thiolactomycin and 2-phenylamino-4-methyl-5-acetylthiazole.
Here, we describe for the first time a small synthetic compound that binds to the KAS I active site. The interaction of this aminothiazole derivative with E. coli KAS I has an equilibrium dissociation constant of 25 µM. A high-resolution crystal structure of the protein–ligand complex at 1.35 Å could be obtained after optimization of the buffer conditions in order to improve the solubility of the ligand. The structure provides insights into the binding mode and suggests strategies for further optimization of this inhibitor.
2. Materials and methods
2.1. Binding assay and ligand solubility by analytical ultracentrifugation
Sedimentation-equilibrium runs were performed at a rotational speed of 16 000 rev min−1 at 293 K using a Beckman–Coulter Optima XL-I analytical ultracentrifuge. An eight-hole rotor fitted with six-channel Alu-filled Epon analytical cells was used. The reference channels were filled with 120 µl binding-assay buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 2 mM TCEP, 0.5 mM EDTA), whereas the sample channels were filled with 100 µl buffer solution containing 22.7 µM (1 mg ml−1) E. coli KAS I and 100 µM ligand. Cells with protein and ligand alone at the above concentrations were also spun as a control. After 20 h, a radial absorbance profile was recorded at 280 nm. A further recording taken 2 h later proved that equilibrium had been reached. Equilibrium absorbance profiles were then recorded at the specific wavelengths of the studied ligands.
Analysis of the profiles was performed using the program DISCREEQ (Schuck, 1994). Analysis of the optical density at the ligand characteristic wavelength yields, with the help of the absorption coefficient, the amount of bound ligand, the stoichiometry, the free ligand concentration through baseline analysis and ligand-induced protein precipitation if it occurs. The baseline was experimentally determined at 40 000 rev min−1. The ligand absorption coefficients were determined from absorption spectra recorded on an Uvikon 930 spectrophotometer. The absorption coefficient of the protein at 280 nm was calculated from the amino-acid sequence. For other wavelengths it was derived from a wavelength scan recorded at 3000 rev min−1 at the beginning of the equilibrium experiment.
The radial absorbance profiles of the low-molecular-weight aminothiazole compound were recorded at 380 nm. At this wavelength only the compound was detected, the protein absorption being negligible. In this case, visual inspection of the recorded absorbance profile readily revealed binding because at the chosen speed the profile for the ligand alone is flat (Fig. 2b).
(a) Fluorescence titration of the aminothiazole ligand against E. coli KAS I protein. The protein concentration was 4.9 µM in binding-assay buffer (20 mM Tris pH 7.5, 200 mM NaCl, 1 mM TCEP, 0.5 mM EDTA). The excitation and emission wavelengths were 280 and 340 nm, respectively. Owing to the high absorption coefficient of the compound at 340 nm, the efficient dipole–dipole interaction strongly quenched the emission of both tryptophan residues of the protein. Circles show the fluorescence intensity in arbitrary units after correction for filter effects; the line shows a sigmoidal fit resulting in an equilibrium dissociation constant Kd of 25 µM. (b) Sedimentation equilibrium of the aminothiazole compound in the absence and presence of E. coli KAS I. The absorbance profiles were recorded at 16 000 rev min−1 at 380 nm for 100 µM aminothiazole in the absence or presence of 22.7 µME. coli KAS I. At this wavelength the absorbance of the protein is negligible, so that only the ligand was detected. The ligand alone showed a flat radial profile for this rotational speed (triangles). In the presence of E. coli KAS I the radial concentration profile corresponds to the protein molar mass, demonstrating binding of the ligand (circles).
Ligand solubility in the buffer of interest was assessed by recording the sedimentation equilibrium in a further higher speed run (40 000 rev min−1) for a channel containing the ligand alone. The solubility was calculated from the area under the absorbance profile at the ligand specific wavelength, which was 380 nm for the aminothiazole (data not shown).
2.2. Fluorescence titration
Fluorescence titration was performed at 293 K in binding-assay buffer using an SLM-AMINCO 8100 double-grating spectrofluorometer. The protein concentration was 4.9 µM. Small aliquots of known ligand concentration were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall et al., 1983). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function, from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 protein–ligand complex (Eftink, 1997).
2.3. Cloning, expression, purification and crystallization
The cloning, expression and purification of E. coli KAS I was performed essentially as described previously (Edwards et al., 1997). Briefly, the gene for E. coli KAS I was amplified from genomic E. coli DNA and cloned into the vector pQE-80 via BamHI and SalI. The protein construct with an N-terminal His tag was expressed in E. coli M15 strain. The expression yield was high, with about 50% of the E. coli KAS I present in the soluble supernatant. Efficient purification of E. coli KAS I was achieved using immobilized metal-affinity chromatography (IMAC; Ni–NTA matrix) and size-exclusion chromatography (SEC; S75 matrix). Purification progress was assessed using SDS–PAGE. The purified protein was concentrated to 70 mg ml−1 in 20 mM HEPES pH 7.5, 200 mM NaCl, 2 mM TCEP, 0.5 mM EDTA, flash-frozen in liquid nitrogen in 200 µl aliqouts and stored at 193 K. E. coli KAS I was crystallized at room temperature in a hanging-drop experiment with 1.9 M ammonium sulfate, 3% PEG 400 and 0.1 M Tris–HCl pH 7.5 as precipitant (Olsen et al., 1995). The protein concentration was 70 mg ml−1 in 20 mM HEPES pH 7.5, 200 mM NaCl, 2 mM TCEP, 0.5 mM EDTA. The mixing ratio of buffer to protein was 2:1. Crystals appeared after 1–2 d and grew to full size after a few days. Crystals were soaked with thiolactomycin for 16–24 h after transfer into 2.3 M ammonium sulfate, 0.1 M Tris–HCl pH 7.5, 0.3 mM thiolactomycin, 3% DMSO. For soaking with the aminothiazole under PEG 8000 conditions, a 20 µl drop of 30% PEG 8000, 0.1 M ammonium sulfate, 0.1 M Tris–HCl pH 7.5, 5 mM TCEP was equilibrated overnight in a hanging-drop setup against 0.5 ml 2.3 M ammonium sulfate, 0.1 M Tris–HCl pH 7.5. The aminothiazole compound was added to the drop to 5 mM from a 100 mM DMSO stock (5% residual DMSO) and crystals were soaked in this drop for 16–24 h. For the apo structure from PEG 8000 conditions, DMSO without the aminothiazole was added to the drop to 5% and the TCEP was omitted from the drop. Crystals were frozen in 2.3 M ammonium sulfate, 0.1 M Tris–HCl pH 7.5, 25% glycerol for the ammonium sulfate conditions and directly from the drop for the PEG 8000 conditions.
2.4. Data collection, processing and refinement
Diffraction data for all data sets were collected at 120 K at the Swiss Light Source (SLS), Villigen, Switzerland. The diffraction images were processed using XDS and scaled with XSCALE (Kabsch, 1993). No significant difference in unit-cell parameters was observed between crystals soaked in the ammonium sulfate and PEG 8000 conditions (Table 1). The structure was solved by molecular replacement using E. coli KAS I (PDB code 1ek4 ; Olsen et al., 2001) as a model. Rigid-body refinement of the four monomers followed by restrained refinement in REFMAC (Murshudov et al., 1999; Collaborative Computational Project, Number 4, 1994) and ARP/wARP v.5.0 (Perrakis et al., 1999) was used for refinement and to add water molecules to the model. Ligand building in the Fo − Fc difference electron-density maps and manual rebuilding were performed with MOLOC (Gerber, 1992). The high resolution of the data sets (1.35–1.60 Å) enabled the refinement of alternative side-chain conformations for several side chains. Final refinement was performed without noncrystallographic symmetry (NCS) restraints. Individual isotropic B factors were refined in REFMAC for all data sets, with the exception of the structure with aminothiazole ligand bound. Here, individual anisotropic B factors were introduced in the final round of refinement, resulting in a decrease in Rfree from 0.179 to 0.151. The refinement statistics are reported in Table 1. Figures were prepared using PyMOL (DeLano, 2002).
2.5. Molecular modelling
ROCS v.2.1.1 (Rapid Overlay of Chemical Structures, Openeye Scientific Software) was applied in order to investigate the shape and chemical similarity of the aminothiazole compound and thiolactomycin. The conformation of thiolactomycin was taken from the E. coli KAS I–thiolactomycin crystal structure. For the aminothiazole compound, OMEGA v.1.8.1 (Openeye Scientific Software) was used to generate multiple low-energy conformations. Default settings were used in the ROCS and OMEGA calculations, except for turning on the ImplicitMillsDean flag for the colour score in ROCS. The final scores derived were a shape Tanimoto score for shape similarity (between 0 and 1) and a scaled colour score for chemical similarity (between 0 and 1). FRED v.1.2.10 (Fast Rigid Exhaustive Docking, Openeye Scientific Software) was used as a docking tool to investigate the binding mode of the aminothiazole compound to E. coli KAS I. Docking was performed using the structure of the apoprotein with water molecules removed. The binding site was defined as a box around the positions of thiolactomycin and cerulenin and increased by 2 Å. ChemScore was used as a scoring function.
3. Results and discussion
3.1. Identification of novel molecules for the inhibition of E. coli KAS I
For the E. coli KAS I protein, structural information is available for the apoprotein and for several protein–inhibitor complexes (Olsen et al., 1999, 2001; Price et al., 2001). Based on this knowledge, an in silico screening was performed with the goal of identifying novel hits for exploration in a drug-discovery program. About 100 hits were selected based on compound availability, synthetic feasibility and drug-likeness to be tested experimentally for binding to E. coli KAS I by analytical ultracentrifugation. This method is independent of the biochemistry of the target, requires essentially no target-specific assay development, has no requirements for labelling or immobilization methods and has a moderate throughput of 20–30 compounds per day. For one of the compounds, an aminothiazole derivative (2-phenylamino-4-methyl-5-acetylthiazole; Fig. 1), a clear sign of co-sedimentation with the protein was detected. The binding affinity of this novel ligand to E. coli KAS I was further evaluated by fluorescence titration. Upon addition of the aminothiazole ligand to the protein in solution, the inherent tryptophan fluorescence of the protein was quenched, reflecting population of the ligand–protein complex (Fig. 2a). The titration curve yields an equilibrium dissociation constant (Kd) of 25 µM for binding of the novel aminothiazole ligand to the E. coli KAS I protein. The affinity of this compound is similar to those of the known reference inhibitors thiolactomycin (25 µM) and cerulenin (3 µM) (Price et al., 2001). Further characterization of the complex was achieved by analytical ultracentrifugation experiments. Comparison of the sedimentation equilibrium of the ligand and of a mixture of ligand and protein shows a clear indication of complex formation (Fig. 2b). In addition, the solubility of the aminothiazole derivative was determined to be at least 100 µM in the buffer used for the binding assay.
3.2. Crystallization of E. coli KAS I with bound aminothiazole ligand
Crystallographic structure determination of E. coli KAS I with bound aminothiazole ligand was used to obtain molecular details of the interactions with its target. E. coli KAS I was prepared and crystallized as described previously (Olsen et al., 1999). A cocrystallization method was reported in the structure determination of E. coli KAS I with inhibitors (Price et al., 2001), but crystal soaking allows more efficient screening of a large number of compounds. Consequently, we established a soaking procedure for the crystals of E. coli KAS I with the reference inhibitor thiolactomycin. The resulting Fo − Fc electron-density map from a data set at 1.5 Å (Table 1) clearly confirmed binding of thiolactomycin at the active site.
However, when crystal soaking was performed with the aminothiazole compound, the Fo
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