New Results

A 2.8 Å structure of zoliflodacin in a DNA-cleavage complex with Staphylococcus aureus DNA gyrase

Harry Morgan, Magdalena Lipka-Lloyd, Anna J. Warren, Naomi Hughes, John Holmes, Nicolas P. Burton, Eshwar Mahenthiralingam, Ben D. Bax
doi: https://doi.org/10.1101/2022.11.30.518515

Abstract

Since 2000 some thirteen quinolones/fluoroquinolones have been developed and come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA-cleavage complexes with DNA gyrase and topo IV, the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here we report a 2.8 Å X-ray crystal structure which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA-cleavage site(s) as quinolones sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA) suggesting it may be more difficult for bacteria to develop target mediated resistance. We found that zoliflodacin had an MIC of 4 µg/mL against Acinetobacter baumannii, an improvement of 4-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, are likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

1. Introduction

Zoliflodacin is an oral spiropyrimidinetrione antibiotic currently in a phase III clinical trial for the treatment of gonorrhea, a sexually transmitted infection (STI) caused by the Gram-negative bacteria Neisseria gonorrhoeae [1-4]. Zoliflodacin (Figure 1a) was developed from QPT-1 (or PNU-286607) a compound discovered in Pharmacia by whole cell screening against Gram-negative (and Gram-positive) bacteria [5]. QPT-1 (Figure 1b), discovered for its antibacterial whole cell activity, was found to inhibit the bacterial type IIA topoisomerases; E. coli DNA gyrase (IC50 9 µM) and E. coli topoisomerase IV (IC50 30 µM) [5]. This method of discovery is reminiscent of the discovery of quinolone/fluoroquino-lone antibiotics, also initially discovered for whole cell activity and then found to be inhibitors of the bacterial type IIA topoisomerases [6].

Figure 1.
Figure 1.

Zoliflodacin, QPT-1 and S. aureus DNA gyrase DNA-cleavage complexes. (a) Chemical structure of zoliflodacin (oxygens red, nitrogens blue, fluorine green) (b) chemical structure of QPT-1 (c) A schematic of the central eight base-pairs of DNA, with two inhibitors (I) binding in the cleaved DNA and inhibiting DNA religation. Note that DNA-cleavage takes place between the -1 and +1 nucleotides on both the Watson and Crick strands (d) A schematic of the DNA-cleavage complex with two zoliflodacins S. aureus DNA gyrase and DNA presented in this paper. (e) The S. aureus DNA gyraseCORE construct used consists of residues B409-B644 from GyrB, fused to A2 to A491 from GyrA. The small greek key (GK) domain has been deleted from GyrB [13] (f) A simplified schematic of DNA gyrase, in which a G-DNA duplex (green) is cleaved by the enzyme and another DNA duplex (known as the T or transported DNA - red) is moved through the enzyme. The greek key domains are not involved in cleaving the gate (or G) -DNA segment [10, 13]. The C-terminal domains (CTD) are shown in pink (approx. positions as in full length E. coli structures [14]).

Topoisomerases are essential enzymes needed to relieve topological problems when the DNA double helix is unwound for both DNA replication and transcription [7]. Topoisomerases are divided into type I topoisomerases, that introduce single-stranded DNA breaks to modify DNA topology and type II topoisomerases that modify topology by introducing double-stranded DNA breaks [7-9]. Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV. While DNA gyrase can uniquely introduce negative supercoils into DNA, topo IV has good decatenase activity [9, 10]. A mechanism for topological changes introduced by DNA gyrase is shown in Figure 1.

The introduction of double-stranded breaks into DNA is potentially hazardous for the cell and the stabilization of DNA-cleavage complexes by quinolones is often bactericidal [11, 12].

The proposal that Gram-negative bacteria evolved a second cell wall to protect them from antibiotics produced by other micro-organisms [15] may partly explain the failure of new classes of antibiotics targeting Gram-negative bacteria, to date, in the 21st century [16-18]. Perhaps for Gram-negative bacteria the hardest task is to get into the cells and a whole cell screening approach followed by target identification of proven targets is more likely to be successful [19, 20]. Indeed, GlaxoSmithKline discovered and developed the NBTI gepotidacin, another new class of DNA gyrase targeting antibiotic currently in phase III clinical trials [21], from a hit compound active in a screen for whole cell antibacterial activity [13]. The chemical diversity of NBTIs such as gepotidacin, which stabilize single stranded DNA-cleavage complexes with bacterial type IIA topoisomerases, suggested that this class of compounds could not have a chemistry-based name [13, 22-27]. The name NBTI, although originally a pneumonic for novel bacterial topoisomerase inhibitor [13], could also be taken to stand for Non-DNA-cleavage pocket Binding on the Twofold-axis Inhibitor (as this describes the binding mode of the chemically diverse NBTIs [13, 22-27]).

The occurrence of antimicrobial resistance in hospital acquired ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) was a major cause for concern in 2009 [28]. New classes of antibiotics have now been developed for Gram-positive bacteria, such as the tiacumicin Fidaxomicin for Clostridioides difficile [29]. However, Gram-negative bacteria (the KAPE in ESKAPE) remain a major cause for concern. The popular quinolone/fluoroquinolone class of antibacterial agents were discovered over sixty years ago from a whole cell screening approach against Gram-negative bacteria [6]. Since then, chemistry has expanded quinolone activity to include such agents as delafloxacin, approved in 2017 for treating acute bacterial skin infections caused by the Gram-positive Staphylococcus aureus. Some thirteen out of thirty-eight new antibiotics introduced between 2000 and 2019 were quinolones [16-18, 30]. However, safety concerns about quinolone side effects have prompted regulatory recommendations to limit the use of quinolones to patients who do not have other treatment options in both Europe (https://www.ema.europa.eu/en/medicines/human/referrals/quinolone-fluoroquinolone-containing-medicinal-products) and the USA (https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-fda-advises-restricting-fluoroquino-lone-antibiotic-use-certain).

The determination of the specific DNA sequences cleaved by DNA gyrase or topoi-somerase IV [31, 32] was important in determining structures of quinolones in DNA-cleavage complexes. In particular, structural studies showed that quinolone antibiotics stabilize double-stranded DNA-cleavage complexes with the two bacterial topoisomerases, topo IV [33] and DNA gyrase [34, 35], by interacting with ParC or GyrA via a water-metal-ion bridge [12, 36]. Although the original DNA-sequences used in two papers describing structures showing the water metal-ion bridge [33, 35] were defined in 2005 [32] and were initially used in structures with S. pneumoniae topo IV [37, 38], they are asymmetric and in high enough resolution structures the DNA was clearly averaged around the twofold axis of the complex [35]. In this paper we used a two-fold symmetric 20-mer DNA duplex to avoid such problems [34, 39]. This 20-mer homoduplex DNA was previously used in determining structures with the progenitor of zoliflodacin, QPT-1 [34].

Herein we describe a 2.8 Å X-ray crystal structure of zoliflodacin in a DNA-cleavage complex with S. aureus DNA gyrase. The structure is compared with a structure with the quinolone, moxifloxacin, also in a DNA-cleavage complex with S. aureus DNA gyrase. We also show that zoliflodacin has reasonable activity against A. baumannii (MICs of 4 µg/mL - the A in ESKAPE).

2. Results

2.1. A 2.8 Å zoliflodacin DNA-cleavage complex with S. aureus DNA gyrase

Crystals of zoliflodacin in a complex with a 20-mer DNA homoduplex (20-447T) and S. aureus DNA gyrase were grown by a microbatch crystallization method and a 2.8 Å dataset was collected on beamline I24 at Diamond Light Source (see Materials and Methods for details). The structure was solved from a 2.5 Å QPT-1 complex with the same 20-447T DNA and S. aureus DNA gyrase in the same P61 space-group (PDB code: 5CDM; a=b = 93.9 Å, c = 412.5 Å) and refined (see Materials and methods for details – supplementary Figure 1 for electron density). The 20-447T DNA homoduplex contains 18 base-pairs and a G-T mismatch at either end of the DNA.

The structure showed two zoliflodacins binding in the cleaved DNA physically blocking religation (Figure 2). The DNA has been cleaved by and is covalently attached to tyrosine 123A from the GyrA subunit (and to the symmetry related tyrosine 123A’, from the second GyrA subunit in the complex). Catalytic metal ions (normally Mg2+ in bacteria) are required for DNA-cleavage, and in our structure, we see two Mn2+ ions occupying the ‘B-site’ in the GyrB and GyrB’ subunits [10].

Figure 2.
Figure 2. 2.8 Å zoliflodacin crystal structure with S. aureus DNA gyrase.

(a) View of 2.8 Å zoliflodacin crystal structure. The DNA (cartoon; green backbone and blue bases) has been cleaved by S. aureus gyrase (shown as backbone trace with semi-transparent surface). Tyr 123 (and Tyr 123’) have cleaved the DNA and are covalently attached. The compounds are shown as solid spheres (carbons orange, oxygens red, nitrogens blue). (b) An orthogonal (90º) view of the same complex. (c) An orthogonal (90º) view looking down the twofold axis of the complex. The two ends of the DNA duplex adopt different conformations due to crystal packing. Figure produced using ChimeraX [40, 41].

2.2. Zoliflodacin interacts with GyrB, whereas moxifloxacin interacts with GyrA

Figure 3 compares the binding sites of compounds in our 2.8 Å zoliflodacin structure with a 2.95 Å S. aureus DNA gyrase DNA-cleavage complex with the widely used quino-lone antibiotic, moxifloxacin (PDB code: 5CDQ [34]). Figure 3a shows the binding mode of zoliflodacin with the pyrimidinetrione (or barbituric acid moiety) of the compound making direct interactions with GyrB. In particular, the terminal oxygen of the pyrimidinetrione makes a hydrogen bond with the main-chain NH of aspartic acid B437.

Figure 3.
Figure 3. Equivalent views of zoliflodacin and moxifloxacin in S. aureus DNA gyrase DNA-cleavage complexes.

(a) A close-up view of a zoliflodacin binding site in the 2.8 Å structure. The pyrimidinetrione moiety of zoliflodacin interacts directly and indirectly (via a water) with Asp437 of GyrB (dotted green lines), in a similar manner to that described in the 2.5 Å QPT-1 structure. Y123’ has cleaved the DNA and forms a ‘phosphotyrosine’ type linkage with the cleaved DNA. The DNA-backbone is shown in fatter ‘stick’ representation with the bases drawn in thinner ‘line’ (base-pair H-bonds only shown for the +1, +4 base-pair). (b) In the 2.95 Å moxifloxacin (yellow carbons) structure the quinolone bound Mg2+ ion (green sphere) and coordinating waters (red spheres) make hydrogen bonds (dotted red lines) to S84, E88 and the bases either side of the DNA-cleavage site (at the +1 and -1 positions - see panel d). (c + d) Orthogonal (90º) views of the compound binding sites in zoliflodacin structure (c) and moxifloxacin structure (d). (e) Superposition of a and b. Figure produced using ChimeraX [40, 41].

This contrasts with moxifloxacin where the compound (Figure 3b and 3e) interacts with S84A and E88A from the GyrA subunit via the now well characterized water-metal ion (Mg2+) bridge [12, 33-35, 42, 43]. The lack of interactions with GyrA and the interactions with GyrB account for the much of the activity of zoliflodacin against quinolone resistant strains of bacteria (e.g. Table 4 in [3]; target mediated resistance is common in quinolone resistant bacteria [11]). The interactions of the quinolones with the GyrA (or ParC) subunit via the flexible water-metal ion bridge may account for some of the specificity of quinolones for DNA gyrase and topo IV (see sequence alignment in Figure 4) over the two human type IIA topoisomerases, Top2α and Top2β.

Figure 4.
Figure 4. Sequence alignment highlighting residues contacting zoliflodacin, moxifloxacin, gepotidacin or etoposide.

An alignment of resides in TOPRIM, Greek Key and WHD (N-terminal part of) domains of: S. aureus GyrB/GyrA (SaGyrBA), A. baumannii ParE/ParC (AbParEC), E. coli. GyrB/GyrA (EcGyrBA) and human Top2β (HuTOP2B). Residues in ‘Conser.’ are conserved in eight aligned sequences in supplementary Figure 5 from Chan et al., 2015 [34] (five bacterial topo2As and the three eucaryotic topo2As; note the central R in PLRGK can be conservatively varied to K). Amino acids are highlighted in sequences if they contact (< 3.8 Å) compounds in S. aureus DNA gyrase structures with compounds. Contacts mapped onto the S. aureus DNA gyrase sequence are Zoli. = contacts in the 2.8 Å zoliflodacin structure (PDB code: 8BP2). The contacts mapped onto A. baumannii topo IV (ParE/ParC) sequence are Moxi. = contacts from the 2.95 Å moxifloxacin complex (PDB code: 5CDQ: note for moxifloxacin the Mg2+ ion and waters of the water metal-ion bridge are taken as part of the compound. Contacts in a 3.25 Å A. Baumannii topo IV moxifloxacin complex structure, 2XKK, are nearly identical to those shown). The contacts mapped onto E. coli Gyrase sequence are Gepo. = from the 2.37 Å gepotidacin with uncleaved DNA (PDB code: 6QTP; note contacts in the 2.31 Å structure 6QTK, and in the full-length E. Coli CryoEM gepotidacin structures. e.g PDB code: 6RKS are very similar). The contacts mapped onto the HuTOP2B structure are from the 2.16 Å etoposide complex with human Top2β (PDB code: 3QX3 - but are similar in S. aureus etoposide crystal structures: 5CDP and 5CDN). The secondary structural elements in a 3.5 Å S. aureus gyrase complex with DNA and GSK299423 (2XCR) are shown above the alignment - note deletion of the greek key domain gave a GSK299423 structure at 2.1 Å (2XCS). The * above the sequence alignment indicates positions of catalytic residues (Glu B435, AspB508, Asp B510, Arg A122, Tyr A123 in S. aureus DNA gyrase). The quinolone resistance-determining region (QRDR), defined as 426-447 in E. coli GyrB 67-106 in E. coli GyrA is underlined in italics on the EcGyrB/A sequences [11].

However, the residues on GyrB which partly form the DNA gyrase-zoliflodacin binding interactions, are conserved not only in the bacterial type IIA topoisomerases but also in the human enzymes. As shown in the sequence alignment in Figure 4, and in Figure 3a, zoliflodacin recognizes and interacts with the GD from the conserved EGDSA motif and the RG from the PLRGK. While E435 at the start of the EGDSA motif is a catalytic residue the other residues from the EGDSA are not catalytic, neither are the PLRGK motif residues. In QPT-1 only the GD and RG residues from GyrB contacts the compound The specificity of spiropyrimidinetriones, such as zoliflodacin, towards bacterial type IIA topoisomerases such as human topoisomerases is believed to be because such compounds can be squeezed out of the pocket when the DNA-gate closes in human topoisomerases [34]. Conformational flexibility in spiropyrimidinetrione ligands, such as QPT1 and zoliflodacin, may be important in allowing ligands to maintain favorable interactions within the binding sites as the DNA wriggles the protein [34, 44, 45]. In addition to the multiple tautomeric forms that the pyrimidinetrione moiety can adopt (only one of which is chemically called a ‘pyrimidinetrione’), and the conformational flexibility of the anilino-nitrogen [34], the methyl-oxazolidine-2-one may also be able to adopt more than one conformation. Multiple high-resolution structures will be required to fully discern how the compound wriggles (when its binding pocket changes shape) as the enzyme is moved around by its substrate DNA [45]. However, from this initial 2.8 Å zoliflodacin structure it is clear that the major protein interactions made by zoliflodacin are clearly with the GD and the RG from the highly conserved EGDSA and PLRGK motifs (Figures 3 and 4).

In the 2.1 Å crystal structure of the NBTI GSK299423 with the S. aureus gyraseCORE and DNA (PDB code: 2XCS; [13]), a Y123F mutant was used so the DNA could not be cleaved. In this 2.1 Å GSK299423 structure the +1:+4 base-pair (Figure 1c) occupies a similar space to the inhibitors in the zoliflodacin and moxifloxacin structures. Some reasons for the conservation of the EGDSA and PLRGK motifs (Figure 4) may be discerned from this 2.1 Å structure. While the side-chain of E435 (the first residue of the EGDSA motif) coordinates the catalytic metal (at the ‘A’ position - poised to cleave the DNA) both the main-chain NH and side-chain hydroxyl of serine 438 are within hydrogen-bonding distance of the phosphate between nucleotides 1 and 2. The main-chain C=O of Arg 458 and the main-chain NH of Lys 460 (from the PLRGK motif), accept and donate hydrogen bonds to the -1 guanine base helping to hold it firmly in place. NBTIs can stabilize complexes with one strand cleaved or with no DNA-cleavage [13, 24, 46], however experimental nucleotide preferences for NBTI-cleavage have not yet, to the best of our knowledge, been determined [47].

2.3 Target mediated resistance to Zoliflodacin in Neisseria gonorrhoeae

The binding of zoliflodacin to the conserved motifs on GyrB correlates well with the low prevalence of target-mediated resistance; only one of some 12,493 N. gonorrhoeae genomes from the PathogenWatch database has a predicted first level resistance mutation [48]. Assessing the probability of developing resistance is an important step in development of any new antibiotic. The development of zoliflodacin (AZD0914) for gonorrhea followed from a 2015 paper assessing the likelihood of developing resistance in N. gonorrhoeae [49]. This paper showed that higher MICs (resistance) was associated with target mutations in three amino acids in N. gonorrhoeae DNA gyrase, namely GyrB: D429N, K450T or S467N [49]. These mutations were identified by in vitro selection of resistance and can give a 4-fold to 16-fold increase in the MIC of zoliflodacin [49, 50]. Interestingly these N. gonorrhoeae GyrB mutations correspond to D437, R458 and N475 in S. aureus DNA gyrase. The D429N mutation is associated with slower growth of bacteria [51]. All three regions are close to the compound (see Figure 3a). In the D437N mutant (S. aureus DNA gyrase) the asparagine side chain may have its NH2 group pointing towards the compound (because if the sidechain was in the opposite orientation the hydrogens on the NH2 would clash with hydrogens on proline 56 - the P in PLRGK).

Zoliflodacin has an extra methyl-oxazolidine-2-one ring, which QPT-1 does not possess (Figure 1a, b) and this extra ring makes van der Waals contacts with residues N476 and E477. While there is clear electron density for the both the additional fluorine and the extra ring (which are not in the QPT-1 structure - Figure 5), the 2.8 Å electron density map is not able to clearly define all water structure or totally unambiguously define the orientation of the extra five-membered ring (see supplementary Figure 1). N475 is equivalent to the third mutated residue in N. gonorrhoeae GyrB, Ser 467 [49]. Mutation of this residue, which is adjacent to residues contacting the compound, presumably effects their conformations. A similar effect is perhaps seen in the S. aureus ParC V67A, found in a strain of S. aureus resistant to gepotidacin [52]. In high resolution S. aureus DNA gyrase NBTI crystal structures three residues (A, G and M) from the GyrA motif 68-ARIVGDVM-75 are within Van Der Waals distance of compounds [13, 22, 24]. ParC V67A is the first V in the equivalent ParC sequence AKTVGDVI, i.e Val 67 is adjacent to an amino acid making direct Van Der Waals contacts with compounds.

Figure 5.
Figure 5. Difference map density from refined QPT-1 structure against zoliflodacin data.

(a) An Fo-Fc map from refined QPT-1 coordinates is contoured at 3 σ shows extra features in zoliflodacin structure not in QPT-1 starting coordinates. The arrow points to extra density modelled as a water – which could be a metal ion (it is close to the oxygen of a phosphate from the DNA backbone and also an oxygen from the methyl-oxazolidine-2-one) (b) refined QPT-1 coordinates are also shown with yellow carbons.

2.5 Improved activity of zoliflodacin against A. baumannii compared to QPT-1

The MIC of zoliflodacin against two carbapenem resistant outbreak strains of Acinetobacter baumannii [53] was determined (see Materials and methods for details) as 4 µg/mL (Table 1). This suggests that, although its activity has been optimized against other Gram-negative bacteria, that the potency of zoliflodacin against A. baumannii is better than that of QPT-1 from which it was developed (the activity of QPT-1 against A. baumannii is from Supplementary Table 4 in Chan et al., (2015); but note QPT-1 is considerably more active against an efflux knock-out strain: A. baumannii BM4454 (βadeABC βadeIJK) [54]).

View this table:
Table 1.

MIC of zoliflodacin for A. baumannii.

3. Discussion

Some Gram-negative bacteria are difficult to kill with antibiotics. Not only do they have two cell walls but they also have export pumps that can rapidly pump antibiotics out of the bacteria [54]. Such bacterial export pumps can play a role in antimicrobial resistance [55]. There is much interest in compounds that can inhibit antibiotic efflux pumps [56], as there is clearly a potential for combination therapies. If the MICs of a compound such as zoliflodacin could be lowered, the dose might be lowered, and the therapeutic window increased.

However, the success of whole cell screening, including early counter-screening of human cells for safety seems to have been effective in discovering two new classes of Gram-negative targeting antibiotics [5, 13]. A similar approach, but starting with a natural product, has recently lead to the discovery of evybactin a new class on M. tuberculosis DNA gyrase targeting compound [57]; this compound appears to work in a similar manner to the thiophene inhibitors [58] compounds that allosterically stabilize DNA-cleavage complexes [59] by binding to a ‘third-site’; a hinge pocket [10].

Interestingly two of the mutations in N. gonorrhoeae GyrB that give rise to resistance to zoliflodacin (AZD0914) are Asp429Asn and Lys450Thr, correspond in S. aureus crystal structures to residues involved in making up the binding pocket of the compound (Figure 3). Namely Asp 437 (=Asp429) is the D from the conserved EGDSA motif and Arg 458 (=Lys450) is from the conserved PLRGK motif.

In a previous paper describing crystals structures of QPT-1, moxifloxacin and etoposide in DNA-cleavage complexes with S. aureus DNA gyrase [34], the DNA-gate of DNA-gyrase was proposed to act like a pair of swing doors, through which the T-segment could be pushed (Figure 1f) but that would then swing close. Such a model might partly account for why in N. gonorrhoeae mutations are only seen in GyrB not in ParE [49]. The swinging closed of the DNA-gate in DNA gyrase might be predicted to give slower ‘off’ rates for zoliflodacin compared to topoisomerase IV; note it was also proposed that zoliflodacin would be squeezed out of a slightly larger equivalent pocket in human topo2s [34]. Much work remains to be done; for example, one current model suggests that before the C- (or exit) gate can be opened the small greek key domain senses the presence of the T-DNA segment (once it has passed through the G-gate) and then moves the catalytic metal away from the active site (see supplementary discussion and supplementary Figures 12 and 13 in [34]). This model allows the DNA to be religated by the lysine residue from the highly conserved YKGLG motif at the C-terminus of the greek key domain (see Figure 4), while not allowing DNA-cleavage by the catalytic metal when the exit gate is opened, and not allowing exit-gate opening while the gate-DNA is cleaved. In this model this is a ‘safety feature’ of type IIA topoisomerases - allowing DNA religation by the YKGLG lysine but inhibiting DNA-cleavage by the catalytic metal. Interestingly it has also been shown that DNA-gyrase can catalyze supercoiling by introducing a single nick in the DNA [60], per-haps this mechanism is also a safe way of introducing negative supercoils into DNA without opening the C-gate.

Safety and the size of the therapeutic window are clearly important in antibacterial drug discovery. It will be interesting to see if the new spiropyrimidinetrione class of compounds, such as zoliflodacin, can be developed to be safer and more efficacious medicines, with less of a tendency for target-mediated antibiotic resistance, than the quinolones.

4. Materials and Methods

4.1 Protein purification and crystallization of a zoliflodacin DNA-cleavage complex

The S. aureus DNA gyrase fusion truncate GyrB27:A56 (GKdel) (Mw 78,020) was expressed in E. coli and purified based on the procedure of Bax et al., [13] modified as described [25]. The purified protein (at 10 mg/mL = 0.128 mM) was in 20 mM HEPES pH 7.0, 5 mM MnCl2 and 100 mM NaSO4. The DNA oligonucleotide used in crystallizations, 20-447T, was custom ordered from Eurogentec (Seraing, Belgium). Received in lyophilized form, the DNA was resuspended in nuclease-free water and annealed from 86 to 21°C over 45 minutes to give the duplex DNA at a concentration of 2 mM. Zoliflodacin was purchased from Med-ChemExpress (New Jersey, USA) as a solid and was dissolved in 100% DMSO forming a 100 mM stock solution.

Crystallization complexes were formed by mixing protein, HEPES buffer, DNA and compound and incubating on ice for 1 hour 15 minutes. Crystals of S. aureus GyrB27:A56 (GKdel)-zoliflodacin-20-447T were grown by the microbatch under oil method [39], with streak seeding being implemented for subsequent plates after the first plate gave crystals. Following established protocols, a crystallization screen consisting of Bis-Tris buffer pH 6.3 - 6.0 (90, 150 mM) and PEG 5kMME (13 – 7%) was used. For a single drop, 1 µL of complex mixture was mixed with 1 µL of crystallization buffer in a 72-well Terasaki microbatch plate, prior to covering with paraffin oil. The plates were incubated at 20°C and crystal growth was observed between 5 and 30 days. Seed solution was prepared by crushing several previously grown hexagonal rod-shaped S. aureus GyrB27:A56 (GKdel)-zoliflodacin-20-447T crystals in 20 µL of crystallization buffer. The crystal which gave a 2.8 Å dataset was grown in a crystallization plate where 1 µL complex mixture (0.066 mM GyrB27:A56 dimer, 0.171 mM 20-447T DNA duplex, 5.714 mM zoliflodacin, 2.571 mM MnCl2 and 342.9 mM HEPES pH 7.2) was mixed with 1 µL crystallization buffer (90 mM Bis-Tris pH 6.3, 9% PEG 5 kMME). A large single crystal (Figure 1) was transferred to a cryobuffer (15% glycerol, 19% PEG 5kMME, 1 mM zoliflodacin, 5% DMSO, 81 mM Bis-Tris pH 6.3) before flash-cooling in liquid nitrogen for data collection.

4.2 Data collection, structure determination and refinement

Data were collected (3600 × 0.lº degree images) on beamline I24 at Diamond Light Source. Data were processed and merged with dials [61-63] as shown in Table 2. A low-resolution cutoff of 25 Å was applied when manually reprocessing the data with dials to avoid problems with the backstop shadow. The high-resolution cutoff was determined by having a CC1/2 > 0.30 [64]. The structure was refined starting from the 2.5 Å complex with the same DNA and the related compound QPT-1 (PDB code: 5CDM) [34, 39]. The data, which are not twinned and are in space-group P61, were reindexed (H=k, K=h, L=-l) to be in the same hand and on the same origin as other liganded structures in the same space-group (e.g PDB codes: 2XCS, 4BUL, 5IWI, 5IWM, 5NPP, 6QTK, 6QX1 and 5CDM). Initial rigid body refinement of 5cdm-BA-x.pdb (P61 cell: a=b=93.88Å, c=412.48Å), reduced the R-factor (R-free) from 0.3900 (0.3899) to 0.2684 (0.2743). Further refinement with phenix.refine [65, 66] and refmac [67, 68] gave the final structure (Table 3), which had reasonable geometry. Restraints for zoliflodacin were generated in Acedrg [69]. As we are interested in structures with ligands/inhibitors we use the standard BA-x numbering scheme throughout this paper [10]. This means zoliflodacin inhibitors in sites 1 and 1’ have CHAINID I, and residue numbers 1 and 201 (see Figure 3 in ref. [10]). Electron density maps for the inhibitors are shown in supplementary Figure 1. The water structure near the inhibitors was based on that in the 2.5 Å structure with QPT-1 (PDB code: 5CDM). Water and glycerol structure was based on electron density maps and higher resolution structures (the 1.98 Å S. aureus complex PDB code: 5IWI, which contains over 940 waters, was superposed).

View this table:
Table 2.

Data collection statistics.

View this table:
Table 3.

Refinement statistics.

At each DNA-cleavage site a single catalytic Mn2+ ion is seen at the B-site [10]. Electron density on His C 391, was interpreted as being due to a Mn2+ ion coordinated by a Bis-Tris buffer molecule, which mediates a crystal contact with one end of the DNA. This interpretation of the electron density was confirmed by re-refining the original 2.1 Å structure of GSK299423 with the S. aureus gyraseCORE structure [13]; originally this electron density had been misinterpreted as being due to DNA. The new interpretation explains why both Bis-Tris and Mn2+ ions are needed in the crystallization buffer, when growing P61 crystals of S. aureus gyraseCORE with ligands.

4.3 Structural analysis

Van der Waals contacts with ligands (defined as 3.8 Å or less) were calculated with contact from the CCP4 suite of programs [71]. Structures were superposed using coot [72] or with limited sets of defined Cαs using LsqKab from CCP4 suite [71].

4.4 Minimum Inhibitory Concentration Assay

The MIC of zoliflodacin against two carbapenem resistant outbreak strains of A. baumannii (BCC 807, BCC 810) [53] was determined using the modified broth microdilution reference method ISO 20776-1:2019 [73] as recommended by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) [74]. The concentration range tested was between 40 – 0.313 µg/mL in two-fold serial dilutions.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Difference density (Fo-Fc) for the two zoliflodacins.

Author Contributions

“Conceptualization, B.D.B. and H.M.; protein purification J.H. and N.P.B; complex prepara-tion and crystallization H.M and M.L.-L.; data-collection and processing, H.M. and A.W.; structure determination and analysis H.M and B.D.B.; microbiology and MIC determination N.H. and E.M.; writing—original draft preparation, B.D.B. and H.M.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.”

Funding

“This research received no external funding”.

Data Availability Statement

The 2.8 Å zoliflodacin structure has been deposited with the protein databank (the PDB) with the code: 8BP2. The PDB nomenclature for compounds and metal ions is inconsistent between different structures. So sensibly named PDB files, following BA-X nomenclature [10] are made available from a table of structures on the ‘Research’ tab on Ben Bax’s website at Cardiff (https://www.cardiff.ac.uk/people/view/1141625-bax-ben).

Conflicts of Interest

“The authors declare no obvious conflict of interest. Ben Bax worked for GlaxoSmithKline from 2000-2016 and is a GlaxoSmithKline shareholder. “

Acknowledgments

We thank Professor Simon Ward and the Medicines Discovery Institute (MDI) in Cardiff for supporting this work. We thank Diamond Light Source (DLS) for beamtime and general support. HM is a joint student between the MDI and DLS.

REFERENCES

  1. 1.↵
    Jacobsson, S., et al., Pharmacodynamic Evaluation of Dosing, Bacterial Kill, and Resistance Suppression for Zoliflodacin Against Neisseria gonorrhoeae in a Dynamic Hollow Fiber Infection Model. Front Pharmacol, 2021. 12: p. 682135.
    OpenUrlPubMed
  2. 2.
    Unemo, M., et al., High susceptibility to zoliflodacin and conserved target (GyrB) for zoliflodacin among 1209 consecutive clinical Neisseria gonorrhoeae isolates from 25 European countries, 2018. J Antimicrob Chemother, 2021. 76(5): p. 1221–1228.
    OpenUrlCrossRef
  3. 3.↵
    Bradford, P.A., et al., Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae, Including Multi-Drug-Resistant Isolates. ACS Infect Dis, 2020. 6(6): p. 1332–1345.
    OpenUrl
  4. 4.↵
    Jacobsson, S., et al., High in vitro susceptibility to the first-in-class spiropyrimidinetrione zoliflodacin among consecutive clinical Neisseria gonorrhoeae isolates from Thailand (2018) and South Africa (2015-2017). Antimicrob Agents Chemother, 2019.
  5. 5.↵
    Miller, A.A., et al., Discovery and characterization of QPT-1, the progenitor of a new class of bacterial topoisomerase inhibitors. Antimicrob. Agents Chemother, 2008. 52(8): p. 2806–2812.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Bisacchi, G.S., Origins of the quinolone class of antibacterials: an expanded “discovery story” miniperspective. Journal of medicinal chemistry, 2015. 58(12): p. 4874–4882.
    OpenUrlCrossRef
  7. 7.↵
    Schoeffler, A.J. and J.M. Berger, DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q. Rev. Biophys, 2008. 41(1): p. 41–101.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.
    Pommier, Y., et al., Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat Rev Mol Cell Biol, 2016. 17(11): p. 703–721.
    OpenUrlCrossRefPubMed
  9. 9.↵
    Bates, A.D. and A. Maxwell, DNA topology. 2005: Oxford University Press, USA.
  10. 10.↵
    Bax, B.D., et al., DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion. J Mol Biol, 2019. 431(18): p. 3427–3449.
    OpenUrl
  11. 11.↵
    Bush, N.G., et al., Quinolones: mechanism, lethality and their contributions to antibiotic resistance. Molecules, 2020. 25(23): p. 5662.
    OpenUrl
  12. 12.↵
    Aldred, K.J., R.J. Kerns, and N. Osheroff, Mechanism of quinolone action and resistance. Biochemistry, 2014. 53(10): p. 1565–1574.
    OpenUrlCrossRefPubMed
  13. 13.↵
    Bax, B.D., et al., Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature, 2010. 466(7309): p. 935–940.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    Vanden Broeck, A., et al., Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex. Nat Commun, 2019. 10(1): p. 4935.
    OpenUrlCrossRefPubMed
  15. 15.↵
    Gupta, R.S., Origin of diderm (Gram-negative) bacteria: antibiotic selection pressure rather than endosymbiosis likely led to the evolution of bacterial cells with two membranes. Antonie Van Leeuwenhoek, 2011. 100(2): p. 171–182.
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    Butler, M.S. and D.L. Paterson, Antibiotics in the clinical pipeline in October 2019. J Antibiot (Tokyo), 2020. 73(6): p. 329–364.
    OpenUrlCrossRef
  17. 17.
    Butler, M.S., M.A. Blaskovich, and M.A. Cooper, Antibiotics in the clinical pipeline in 2013. J Antibiot (Tokyo), 2013. 66(10): p. 571–91.
    OpenUrlCrossRefPubMed
  18. 18.↵
    Butler, M.S. and M.A. Cooper, Antibiotics in the clinical pipeline in 2011. J Antibiot (Tokyo), 2011. 64(6): p. 413–25.
    OpenUrlCrossRefPubMed
  19. 19.↵
    Payne, D.J., et al., Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov, 2007. 6(1): p. 29–40.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Tommasi, R., et al., ESKAPEing the labyrinth of antibacterial discovery. Nature reviews Drug discovery, 2015. 14(8): p. 529–542.
    OpenUrlCrossRefPubMed
  21. 21.↵
    Scangarella-Oman, N.E., et al., Dose Selection for Phase III Clinical Evaluation of Gepotidacin (GSK2140944) in the Treatment of Uncomplicated Urinary Tract Infections. Antimicrob Agents Chemother, 2022: p. AAC0149221.
  22. 22.↵
    Miles, T.J., et al., Novel tricyclics (eg, GSK945237) as potent inhibitors of bacterial type IIA topoisomerases. Bioorganic & medicinal chemistry letters, 2016. 26(10): p. 2464–2469.
    OpenUrl
  23. 23.
    Miles, T.J., et al., Novel hydroxyl tricyclics (eg, GSK966587) as potent inhibitors of bacterial type IIA topoisomerases. Bioorganic & medicinal chemistry letters, 2013. 23(19): p. 5437–5441.
    OpenUrl
  24. 24.↵
    Gibson, E.G., et al., Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase. ACS Infect Dis, 2019. 5(4): p. 570–581.
    OpenUrl
  25. 25.↵
    Kolaric, A., et al., Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds. Nat Commun, 2021. 12(1): p. 150.
    OpenUrl
  26. 26.
    Singh, S.B., et al., Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2). Bioorg. Med. Chem. Lett, 2015. 25(9): p. 1831–1835.
    OpenUrl
  27. 27.↵
    Singh, S.B., et al., Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents. ACS Med. Chem. Lett, 2014. 5(5): p. 609–614.
    OpenUrlCrossRefPubMed
  28. 28.↵
    Boucher, H.W., et al., Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis, 2009. 48(1): p. 1–12.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Tashiro, S., et al., Oral fidaxomicin versus vancomycin for the treatment of Clostridioides difficile infection: A systematic review and meta-analysis of randomized controlled trials. Journal of Infection and Chemotherapy, 2022.
  30. 30.
    Butler, M.S., M.A. Blaskovich, and M.A. Cooper, Antibiotics in the clinical pipeline at the end of 2015. J Antibiot (Tokyo), 2017. 70(1): p. 3–24.
    OpenUrlCrossRefPubMed
  31. 31.↵
    Lockshon, D. and D.R. Morris, Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization. Journal of molecular biology, 1985. 181(1): p. 63–74.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    Leo, E., et al., Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site. Journal of Biological Chemistry, 2005. 280(14): p. 14252–14263.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Wohlkonig, A., et al., Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance. Nat. Struct. Mol. Biol, 2010. 17(9): p. 1152–1153.
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    Chan, P.F., et al., Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin. Nat. Commun, 2015. 6: p. 10048.
    OpenUrlCrossRefPubMed
  35. 35.↵
    Blower, T.R., et al., Crystal structure and stability of gyrase-fluoroquinolone cleaved complexes from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A, 2016. 113(7): p. 1706–1713.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    Wohlkonig, A., et al., Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance. Nature structural & molecular biology, 2010. 17(9): p. 1152.
    OpenUrl
  37. 37.↵
    Laponogov, I., et al., Structural insight into the quinolone-DNA cleavage complex of type IIA topoisomerases. Nat. Struct. Mol. Biol, 2009. 16(6): p. 667–669.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Laponogov, I., et al., Structural basis of gate-DNA breakage and resealing by type II topoisomerases. PLoS. One, 2010. 5(6): p. e11338.
    OpenUrlCrossRefPubMed
  39. 39.↵
    Srikannathasan, V., et al., Crystallization and preliminary X-ray crystallographic analysis of covalent DNA cleavage complexes of Staphyloccocus Aureus DNA Gyrase with QPT-1, Moxilfloxacin and Etoposide. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun, 2015. 71: p. 1242–1246.
    OpenUrl
  40. 40.↵
    Pettersen, E.F., et al., UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Science, 2021. 30(1): p. 70–82.
    OpenUrlCrossRef
  41. 41.↵
    Goddard, T.D., et al., UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Science, 2018. 27(1): p. 14–25.
    OpenUrlCrossRefPubMed
  42. 42.
    Aldred, K.J., et al., Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance. Nucleic Acids Res, 2013. 41(8): p. 4628–4639.
    OpenUrlCrossRefPubMed
  43. 43.
    Aldred, K.J., et al., Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance. Biochemistry, 2012. 51(1): p. 370–381.
    OpenUrlCrossRefPubMedWeb of Science
  44. 44.↵
    Bax, B., C.W. Chung, and C. Edge, Getting the chemistry right: protonation, tautomers and the importance of H atoms in biological chemistry. Acta Crystallogr D Struct Biol, 2017. 73(Pt 2): p. 131–140.
    OpenUrl
  45. 45.↵
    Fogg, J.M., et al., Bullied no more: when and how DNA shoves proteins around. Q. Rev. Biophys, 2012. 45(3): p. 257–299.
    OpenUrlCrossRefPubMed
  46. 46.↵
    Gibson, E.G., et al., Bimodal actions of a naphthyridone/aminopiperidine-based antibacterial that targets gyrase and topoisomerase IV. Biochemistry, 2019. 58(44): p. 4447–4455.
    OpenUrlCrossRef
  47. 47.↵
    Sutormin, D., et al., Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome. Nucleic Acids Res, 2019. 47(3): p. 1601.
    OpenUrl
  48. 48.↵
    Adamson, P.C., et al., Using a public database of Neisseria gonorrhoeae genomes to detect mutations associated with zoliflodacin resistance. J Antimicrob Chemother, 2021. 76(11): p. 2847–2849.
    OpenUrlCrossRef
  49. 49.↵
    Alm, R.A., et al., Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae. Antimicrob. Agents Chemother, 2015. 59(3): p. 1478–1486.
    OpenUrlAbstract/FREE Full Text
  50. 50.↵
    Foerster, S., et al., In vitro antimicrobial combination testing of and evolution of resistance to the first-in-class spiropyrimidinetrione zoliflodacin combined with six therapeutically relevant antimicrobials for Neisseria gonorrhoeae. J Antimicrob Chemother, 2019. 74(12): p. 3521–3529.
    OpenUrl
  51. 51.↵
    Foerster, S., et al., Genetic resistance determinants, in vitro time-kill curve analysis and pharmacodynamic functions for the novel topoisomerase II inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae. Frontiers in microbiology, 2015. 6: p. 1377.
    OpenUrl
  52. 52.↵
    Scangarella-Oman, N.E., et al., In Vitro Activity and Microbiological Efficacy of Gepotidacin from a Phase 2, Randomized, Multicenter, Dose-Ranging Study in Patients with Acute Bacterial Skin and Skin Structure Infections. Antimicrob Agents Chemother, 2020. 64(3).
  53. 53.↵
    Coelho, J.M., et al., Occurrence of carbapenem-resistant Acinetobacter baumannii clones at multiple hospitals in London and Southeast England. Journal of clinical microbiology, 2006. 44(10): p. 3623–3627.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    Damier-Piolle, L., et al., AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrobial agents and chemotherapy, 2008. 52(2): p. 557–562.
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    Colclough, A.L., et al., RND efflux pumps in Gram-negative bacteria; regulation, structure and role in antibiotic resistance. Future Microbiology, 2020. 15(2): p. 143–157.
    OpenUrl
  56. 56.↵
    Lowrence, R.C., et al., Tackling drug resistance with efflux pump inhibitors: from bacteria to cancerous cells. Critical Reviews in Microbiology, 2019. 45(3): p. 334–353.
    OpenUrl
  57. 57.↵
    Imai, Y., et al., Evybactin is a DNA gyrase inhibitor that selectively kills Mycobacterium tuberculosis. Nature Chemical Biology, 2022: p. 1–9.
  58. 58.↵
    Chan, P.F., et al., Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase. Proceedings of the National Academy of Sciences, 2017. 114(22): p. E4492–E4500.
    OpenUrlAbstract/FREE Full Text
  59. 59.↵
    Thalji, R.K., et al., Structure-guided design of antibacterials that allosterically inhibit DNA gyrase. Bioorg Med Chem Lett, 2019. 29(11): p. 1407–1412.
    OpenUrl
  60. 60.↵
    Gubaev, A., D. Weidlich, and D. Klostermeier, DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism. Nucleic acids research, 2016. 44(21): p. 10354–10366.
    OpenUrlCrossRefPubMed
  61. 61.↵
    Winter, G., et al., DIALS as a toolkit. Protein Science, 2022. 31(1): p. 232–250.
    OpenUrlCrossRef
  62. 62.
    Beilsten-Edmands, J., et al., Scaling diffraction data in the DIALS software package: algorithms and new approaches for multi-crystal scaling. Acta Crystallographica Section D: Structural Biology, 2020. 76(4): p. 385–399.
    OpenUrl
  63. 63.↵
    Winter, G., et al., DIALS: implementation and evaluation of a new integration package. Acta Crystallogr D Struct Biol, 2018. 74(Pt 2): p. 85–97.
    OpenUrlCrossRefPubMed
  64. 64.↵
    Evans, P.R. and G.N. Murshudov, How good are my data and what is the resolution? Acta Crystallogr. D. Biol. Crystallogr, 2013. 69(Pt 7): p. 1204–1214.
    OpenUrlCrossRefPubMedWeb of Science
  65. 65.↵
    Afonine, P.V., et al., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol, 2018. 74(Pt 6): p. 531–544.
    OpenUrlCrossRef
  66. 66.↵
    Afonine, P.V., et al., Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr, 2012. 68(Pt 4): p. 352–67.
    OpenUrlCrossRefPubMedWeb of Science
  67. 67.↵
    Kovalevskiy, O., et al., Overview of refinement procedures within REFMAC5: utilizing data from different sources. Acta Crystallogr D Struct Biol, 2018. 74(Pt 3): p. 215–227.
    OpenUrlCrossRefPubMed
  68. 68.↵
    Murshudov, G.N., et al., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr, 2011. 67(Pt 4): p. 355–67.
    OpenUrlCrossRefPubMedWeb of Science
  69. 69.↵
    Long, F., et al., AceDRG: a stereochemical description generator for ligands. Acta Crystallogr D Struct Biol, 2017. 73(Pt 2): p. 112–122.
    OpenUrlCrossRefPubMed
  70. 70.
    Kumar, K.S.D., et al., Online_DPI: a web server to calculate the diffraction precision index for a protein structure. Journal of Applied Crystallography, 2015. 48(3): p. 939–942.
    OpenUrlCrossRef
  71. 71.↵
    Winn, M.D., et al., Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr, 2011. 67(Pt 4): p. 235–242.
    OpenUrlCrossRefPubMedWeb of Science
  72. 72.↵
    Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 12 Pt 1): p. 2126–32.
    OpenUrlCrossRefPubMedWeb of Science
  73. 73.↵
    Standardization, I.O.f., Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices—part 1: broth micro-dilution reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. 2019, International Organization for Standardization Geneva, Switzerland.
  74. 74.↵
    EUCAST. MIC determination of non-fastidious and fastidious organisms 2022 [cited 2022 9 NOVEMBER]; Available from: https://www.eucast.org/ast_of_bacteria/mic_determination.
Back to top
Posted November 30, 2022.
Download PDF
Email
A 2.8 Å structure of zoliflodacin in a DNA-cleavage complex with Staphylococcus aureus DNA gyrase
Harry Morgan, Magdalena Lipka-Lloyd, Anna J. Warren, Naomi Hughes, John Holmes, Nicolas P. Burton, Eshwar Mahenthiralingam, Ben D. Bax
bioRxiv 2022.11.30.518515; doi: https://doi.org/10.1101/2022.11.30.518515
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area