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

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.

introducing double-stranded DNA breaks [7][8][9]. Most bacteria possess two type IIA topoi-45 somerases, DNA gyrase and topo IV. While DNA gyrase can uniquely introduce negative 46 supercoils into DNA, topo IV has good decatenase activity [9,10]. A mechanism for top-47 ological changes introduced by DNA gyrase is shown in Figure 1. 48 The introduction of double-stranded breaks into DNA is potentially hazardous for 49 the cell and the stabilization of DNA-cleavage complexes by quinolones is often bacteri-50 cidal [11,12]. structure of zoliflodacin (oxygens red, nitrogens blue, fluorine green) (b) chemical structure of QPT-54 1 (c) A schematic of the central eight base-pairs of DNA, with two inhibitors (I) binding in the 55 cleaved DNA and inhibiting DNA religation. Note that DNA-cleavage takes place between the -1 56 and +1 nucleotides on both the Watson and Crick strands (d) A schematic of the DNA-cleavage 57 complex with two zoliflodacins S. aureus DNA gyrase and DNA presented in this paper. (e) The S. 58 aureus DNA gyrase CORE construct used consists of residues B409-B644 from GyrB, fused to A2 to 59 A491 from GyrA. The small greek key (GK) domain has been deleted from GyrB [13] (f) A simplified 60 schematic of DNA gyrase, in which a G-DNA duplex (green) is cleaved by the enzyme and another 61 DNA duplex (known as the T or transported DNA -red) is moved through the enzyme. The greek 62 key domains are not involved in cleaving the gate (or G) -DNA segment [10,13]. The C-terminal 63 domains (CTD) are shown in pink (approx. positions as in full length E. coli structures [14]). 64 The proposal that Gram-negative bacteria evolved a second cell wall to protect them 65 from antibiotics produced by other micro-organisms [15] may partly explain the failure of 66 new classes of antibiotics targeting Gram-negative bacteria, to date, in the 21st century 67 [16][17][18]. Perhaps for Gram-negative bacteria the hardest task is to get into the cells and a 68 whole cell screening approach followed by target identification of proven targets is more 69 likely to be successful [19,20]. Indeed, GlaxoSmithKline discovered and developed the 70 NBTI gepotidacin, another new class of DNA gyrase targeting antibiotic currently in 71 phase III clinical trials [21], from a hit compound active in a screen for whole cell antibac-72 terial activity [13]. The chemical diversity of NBTIs such as gepotidacin, which stabilize 73 single stranded DNA-cleavage complexes with bacterial type IIA topoisomerases, sug-74 gested that this class of compounds could not have a chemistry-based name [13,[22][23][24][25][26][27]. 75 The name NBTI, although originally a pneumonic for novel bacterial topoisomerase in-76 hibitor [13], could also be taken to stand for Non-DNA-cleavage pocket Binding on the 77 Twofold-axis Inhibitor (as this describes the binding mode of the chemically diverse 78 NBTIs [13,[22][23][24][25][26][27]).

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The occurrence of antimicrobial resistance in hospital acquired ESKAPE pathogens 80 (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, 81 Pseudomonas aeruginosa, and Enterobacter species) was a major cause for concern in 2009 82 [28]. New classes of antibiotics have now been developed for Gram-positive bacteria, such 83 as the tiacumicin Fidaxomicin for Clostridioides difficile [29]. However, Gram-negative bac-84 teria (the KAPE in ESKAPE) remain a major cause for concern. The popular quino-85 lone/fluoroquinolone class of antibacterial agents were discovered over sixty years ago 86 from a whole cell screening approach against Gram-negative bacteria [6]. Since then, 87 chemistry has expanded quinolone activity to include such agents as delafloxacin, ap-88 proved in 2017 for treating acute bacterial skin infections caused by the Gram-positive 89 Staphylococcus aureus. Some thirteen out of thirty-eight new antibiotics introduced be-90 tween 2000 and 2019 were quinolones [16][17][18]30]. However, safety concerns about quino-91 lone side effects have prompted regulatory recommendations to limit the use of quin-92 olones to patients who do not have other treatment options in both Europe 93 (https://www.ema.europa.eu/en/medicines/human/referrals/quinolone-fluoroquinolone-94 containing-medicinal-products) and the USA (https://www.fda.gov/drugs/drug-safety-95 and-availability/fda-drug-safety-communication-fda-advises-restricting-fluoroquino-96 lone-antibiotic-use-certain).

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The determination of the specific DNA sequences cleaved by DNA gyrase or topoi-98 somerase IV [31, 32] was important in determining structures of quinolones in DNA-99 cleavage complexes. In particular, structural studies showed that quinolone antibiotics 100 stabilize double-stranded DNA-cleavage complexes with the two bacterial topoisomer-101 ases, topo IV [33] and DNA gyrase [34,35], by interacting with ParC or GyrA via a water-102 metal-ion bridge [12,36]. Although the original DNA-sequences used in two papers de-103 scribing structures showing the water metal-ion bridge [33,35] were defined in 2005 [32] 104 and were initially used in structures with S. pneumoniae topo IV [37,38], they are asym-105 metric and in high enough resolution structures the DNA was clearly averaged around 106 the twofold axis of the complex [35]. In this paper we used a two-fold symmetric 20-mer 107 DNA duplex to avoid such problems [34,39]. This 20-mer homoduplex DNA was previ-108 ously used in determining structures with the progenitor of zoliflodacin, QPT-1 [34].

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Herein we describe a 2.8 Å X-ray crystal structure of zoliflodacin in a DNA-cleavage 110 complex with S. aureus DNA gyrase. The structure is compared with a structure with the 111 quinolone, moxifloxacin, also in a DNA-cleavage complex with S. aureus DNA gyrase. We 112 also show that zoliflodacin has reasonable activity against A. baumannii (MICs of 4 µg/mL 113 -the A in ESKAPE).  Crystals of zoliflodacin in a complex with a 20-mer DNA homoduplex (20-447T) and 118 S. aureus DNA gyrase were grown by a microbatch crystallization method and a 2.8 Å 119 dataset was collected on beamline I24 at Diamond Light Source (see Materials and Meth-120 ods for details). The structure was solved from a 2.5 Å QPT-1 complex with the same 20-121 447T DNA and S. aureus DNA gyrase in the same P61 space-group (PDB code: 5CDM; a=b 122 = 93.9 Å, c = 412.5 Å) and refined (see Materials and methods for details -supplementary 123 Figure 1 for electron density). The 20-447T DNA homoduplex contains 18 base-pairs and 124 a G-T mismatch at either end of the DNA.

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The structure showed two zoliflodacins binding in the cleaved DNA physically 126 blocking religation ( Figure 2). The DNA has been cleaved by and is covalently attached to 127 tyrosine 123A from the GyrA subunit (and to the symmetry related tyrosine 123A', from 128 the second GyrA subunit in the complex). Catalytic metal ions (normally Mg 2+ in bacteria) 129 are required for DNA-cleavage, and in our structure, we see two Mn 2+ ions occupying the 130 'B-site' in the GyrB and GyrB' subunits [10].   Figure 3a shows the binding mode of zoliflodacin with the pyrimidinetrione (or barbituric acid moiety) of the compound 160 making direct interactions with GyrB. In particular, the terminal oxygen of the pyrim-161 idinetrione makes a hydrogen bond with the main-chain NH of aspartic acid B437.

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This contrasts with moxifloxacin where the compound (Figure 3b and 3e) interacts 163 with S84A and E88A from the GyrA subunit via the now well characterized water-metal 164 ion (Mg 2+ ) bridge [12, 33-35, 42, 43]. The lack of interactions with GyrA and the interactions 165 with GyrB account for the much of the activity of zoliflodacin against quinolone resistant 166 strains of bacteria (e.g. Table 4 in [3]; target mediated resistance is common in quinolone 167 resistant bacteria [11]). The interactions of the quinolones with the GyrA (or ParC) subunit 168 via the flexible water-metal ion bridge may account for some of the specificity of quin-169 olones for DNA gyrase and topo IV (see sequence alignment in Figure 4) over the two 170 human type IIA topoisomerases, Top2a and Top2b.

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However, the residues on GyrB which partly form the DNA gyrase-zoliflodacin bind-172 ing interactions, are conserved not only in the bacterial type IIA topoisomerases but also 173 in the human enzymes. As shown in the sequence alignment in Figure 4, and in Figure 3a, 174 zoliflodacin recognizes and interacts with the GD from the conserved EGDSA motif and 175 the RG from the PLRGK. While E435 at the start of the EGDSA motif is a catalytic residue 176 the other residues from the EGDSA are not catalytic, neither are the PLRGK motif resi-177 dues. In QPT-1 only the GD and RG residues from GyrB contacts the compound 178 179 The specificity of spiropyrimidinetriones, such as zoliflodacin, towards bacterial type IIA 204 topoisomerases such as human topoisomerases is believed to be because such compounds 205 can be squeezed out of the pocket when the DNA-gate closes in human topoisomerases 206 [34]. Conformational flexibility in spiropyrimidinetrione ligands, such as QPT1 and zoli-207 flodacin, may be important in allowing ligands to maintain favorable interactions within 208 the binding sites as the DNA wriggles the protein [34,44,45]. In addition to the multiple 209 tautomeric forms that the pyrimidinetrione moiety can adopt (only one of which is chem-210 ically called a 'pyrimidinetrione'), and the conformational flexibility of the anilino-nitro-211 gen [34], the methyl-oxazolidine-2-one may also be able to adopt more than one confor-212 mation. 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 223 around by its substrate DNA [45]. However, from this initial 2.8 Å zoliflodacin structure 224 it is clear that the major protein interactions made by zoliflodacin are clearly with the GD 225 and the RG from the highly conserved EGDSA and PLRGK motifs (Figures 3 and 4).

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In the 2.1 Å crystal structure of the NBTI GSK299423 with the S. aureus gyrase CORE 227 and DNA (PDB code: 2XCS; [13]), a Y123F mutant was used so the DNA could not be 228 cleaved. In this 2.1 Å GSK299423 structure the +1:+4 base-pair ( Figure 1c) occupies a sim-229 ilar space to the inhibitors in the zoliflodacin and moxifloxacin structures. Some reasons 230 for the conservation of the EGDSA and PLRGK motifs (Figure 4) may be discerned from 231 this 2.1 Å structure. While the side-chain of E435 (the first residue of the EGDSA motif) 232 coordinates the catalytic metal (at the 'A' position -poised to cleave the DNA) both the 233 main-chain NH and side-chain hydroxyl of serine 438 are within hydrogen-bonding dis-234 tance of the phosphate between nucleotides 1 and 2. The main-chain C=O of Arg 458 and 235 the main-chain NH of Lys 460 (from the PLRGK motif), accept and donate hydrogen 236 bonds to the -1 guanine base helping to hold it firmly in place. NBTIs can stabilize com-237 plexes with one strand cleaved or with no DNA-cleavage [13,24,46], however experi-238 mental nucleotide preferences for NBTI-cleavage have not yet, to the best of our 239 knowledge, been determined [47]. The binding of zoliflodacin to the conserved motifs on GyrB correlates well with the 244 low prevalence of target-mediated resistance; only one of some 12,493 N. gonorrhoeae 245 genomes from the PathogenWatch database has a predicted first level resistance mutation 246 [48]. Assessing the probability of developing resistance is an important step in develop-247 ment of any new antibiotic. The development of zoliflodacin (AZD0914) for gonorrhea 248 followed from a 2015 paper assessing the likelihood of developing resistance in N. gonor-249 rhoeae [49]. This paper showed that higher MICs (resistance) was associated with target 250 mutations in three amino acids in N. gonorrhoeae DNA gyrase, namely GyrB: D429N, 251 K450T or S467N [49]. These mutations were identified by in vitro selection of resistance 252 and can give a 4-fold to 16-fold increase in the MIC of zoliflodacin [49, 50]. Interestingly 253 these N. gonorrhoeae GyrB mutations correspond to D437, R458 and N475 in S. aureus DNA 254 gyrase. The D429N mutation is associated with slower growth of bacteria [51]. All three 255 regions are close to the compound (see Figure 3a). In the D437N mutant (S. aureus DNA 256 gyrase) the asparagine side chain may have its NH2 group pointing towards the com-257 pound (because if the sidechain was in the opposite orientation the hydrogens on the NH2 258 would clash with hydrogens on proline 56 -the P in PLRGK).

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Zoliflodacin has an extra methyl-oxazolidine-2-one ring, which QPT-1 does not pos-260 sess (Figure 1a, b) and this extra ring makes van der Waals contacts with residues N476 261 and E477. While there is clear electron density for the both the additional fluorine and the 262 extra ring (which are not in the QPT-1 structure - Figure 5), the 2.8 Å electron density map 263 is not able to clearly define all water structure or totally unambiguously define the orien-264 tation of the extra five-membered ring (see supplementary Figure 1). N475 is equivalent 265 to the third mutated residue in N. gonorrhoeae GyrB, Ser 467 [49]. Mutation of this residue, 266 which is adjacent to residues contacting the compound, presumably effects their confor-267 mations. A similar effect is perhaps seen in the S. aureus ParC V67A, found in a strain of 268 S. aureus resistant to gepotidacin [52]. In high resolution S. aureus DNA gyrase NBTI crys-269 tal structures three residues (A, G and M) from the GyrA motif 68-ARIVGDVM-75 are 270 within Van Der Waals distance of compounds [13,22,24]. ParC V67A is the first V in the 271 equivalent ParC sequence AKTVGDVI, i.e Val 67 is adjacent to an amino acid making 272 direct Van Der Waals contacts with compounds. 273 2.5 Improved activity of zoliflodacin against A. baumannii compared to QPT-1. 274 The MIC of zoliflodacin against two carbapenem resistant outbreak strains of Acinetobacter 275 baumannii [53] was determined (see Materials and methods for details) as 4 µg/mL (Table 276 1). This suggests that, although its activity has been optimized against other Gram-nega-277 tive bacteria, that the potency of zoliflodacin against A. baumannii is better than that of 278 QPT-1 from which it was developed (the activity of QPT-1 against A. baumannii is from 279 Supplementary Some Gram-negative bacteria are difficult to kill with antibiotics. Not only do they 287 have two cell walls but they also have export pumps that can rapidly pump antibiotics 288 out of the bacteria [54]. Such bacterial export pumps can play a role in antimicrobial re-289 sistance [55]. There is much interest in compounds that can inhibit antibiotic efflux pumps 290 [56], as there is clearly a potential for combination therapies. If the MICs of a compound 291 such as zoliflodacin could be lowered, the dose might be lowered, and the therapeutic 292 window increased.

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However, the success of whole cell screening, including early counter-screening of 294 human cells for safety seems to have been effective in discovering two new classes of 295 Gram-negative targeting antibiotics [5,13]. A similar approach, but starting with a natural 296 product, has recently lead to the discovery of evybactin a new class on M. tuberculosis 297 DNA gyrase targeting compound [57]; this compound appears to work in a similar man-298 ner to the thiophene inhibitors [58] compounds that allosterically stabilize DNA-cleavage 299 complexes [59] by binding to a 'third-site'; a hinge pocket [10].

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Interestingly two of the mutations in N. gonorrhoeae GyrB that give rise to resistance 301 to zoliflodacin (AZD0914) are Asp429Asn and Lys450Thr, correspond in S. aureus crystal 302 structures to residues involved in making up the binding pocket of the compound (Figure 303 3). Namely Asp 437 (=Asp429) is the D from the conserved EGDSA motif and Arg 458 304 (=Lys450) is from the conserved PLRGK motif.

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In a previous paper describing crystals structures of QPT-1, moxifloxacin and etopo-306 side in DNA-cleavage complexes with S. aureus DNA gyrase [34], the DNA-gate of DNA-307 gyrase was proposed to act like a pair of swing doors, through which the T-segment could 308 be pushed (Figure 1f) but that would then swing close. Such a model might partly account 309 for why in N. gonorrhoeae mutations are only seen in GyrB not in ParE [49]. The swinging 310 closed of the DNA-gate in DNA gyrase might be predicted to give slower 'off' rates for 311 zoliflodacin compared to topoisomerase IV; note it was also proposed that zoliflodacin 312 would be squeezed out of a slightly larger equivalent pocket in human topo2s [34]. Much 313 work remains to be done; for example, one current model suggests that before the C-(or 314 exit) gate can be opened the small greek key domain senses the presence of the T-DNA 315 segment (once it has passed through the G-gate) and then moves the catalytic metal away 316 from the active site (see supplementary discussion and supplementary Figures 12 and 13 317  in [34]). This model allows the DNA to be religated by the lysine residue from the highly 318 conserved YKGLG motif at the C-terminus of the greek key domain (see Figure 4), while 319 not allowing DNA-cleavage by the catalytic metal when the exit gate is opened, and not 320 allowing exit-gate opening while the gate-DNA is cleaved. In this model this is a 'safety 321 feature' of type IIA topoisomerases -allowing DNA religation by the YKGLG lysine but 322 inhibiting DNA-cleavage by the catalytic metal. Interestingly it has also been shown that 323 DNA-gyrase can catalyze supercoiling by introducing a single nick in the DNA [60], per-324 haps this mechanism is also a safe way of introducing negative supercoils into DNA with-325 out opening the C-gate.

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Safety and the size of the therapeutic window are clearly important in antibacterial 327 drug discovery. It will be interesting to see if the new spiropyrimidinetrione class of com-328 pounds, such as zoliflodacin, can be developed to be safer and more efficacious medicines, 329 with less of a tendency for target-mediated antibiotic resistance, than the quinolones.  The S. aureus DNA gyrase fusion truncate GyrB27:A56 (GKdel) (Mw 78,020) was expressed 333 in E. coli and purified based on the procedure of Bax et al., [13] modified as described [25]. 334 The purified protein (at 10 mg/mL = 0.128 mM) was in 20 mM HEPES pH 7.0, 5 mM MnCl2 335 and 100 mM NaSO4. The DNA oligonucleotide used in crystallizations, 20-447T, was cus-336 tom ordered from Eurogentec (Seraing, Belgium). Received in lyophilized form, the DNA 337 was resuspended in nuclease-free water and annealed from 86 to 21°C over 45 minutes to 338 give the duplex DNA at a concentration of 2 mM. Zoliflodacin was purchased from Med-339 ChemExpress (New Jersey, USA) as a solid and was dissolved in 100% DMSO forming a 340 100 mM stock solution.

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Crystallization complexes were formed by mixing protein, HEPES buffer, DNA and com-342 pound and incubating on ice for 1 hour 15 minutes. Crystals of S. aureus GyrB27:A56 343 (GKdel)-zoliflodacin-20-447T were grown by the microbatch under oil method [39], with 344 streak seeding being implemented for subsequent plates after the first plate gave crystals. 345 Following established protocols, a crystallization screen consisting of Bis-Tris buffer pH 346 6.3 -6.0 (90, 150 mM) and PEG 5kMME (13 -7%) was used. For a single drop, 1 µL of 347 complex mixture was mixed with 1 µL of crystallization buffer in a 72-well Terasaki mi-348 crobatch plate, prior to covering with paraffin oil. The plates were incubated at 20°C and 349 crystal growth was observed between 5 and 30 days. Bis-Tris pH 6.3, 9% PEG 5 kMME). A large single crystal (Figure 1) was transferred to a 356 cryobuffer (15% glycerol, 19% PEG 5kMME, 1 mM zoliflodacin, 5% DMSO, 81 mM Bis-Tris 357 pH 6.3) before flash-cooling in liquid nitrogen for data collection.  Table 2. A low-resolution 361 cutoff of 25 Å was applied when manually reprocessing the data with dials to avoid prob-362 lems with the backstop shadow. The high-resolution cutoff was determined by having a 363  (Table 3), which had reasonable geometry. 393 Restraints for zoliflodacin were generated in Acedrg [69]. As we are interested in struc-394 tures with ligands/inhibitors we use the standard BA-x numbering scheme throughout 395 this paper [10]. This means zoliflodacin inhibitors in sites 1 and 1' have CHAINID I, and 396 residue numbers 1 and 201 (see Figure 3 in ref. [10]). Electron density maps for the inhib-397 itors are shown in supplementary Figure 1. The water structure near the inhibitors was 398 based on that in the 2.5 Å structure with QPT-1 (PDB code: 5CDM). Water and glycerol 399 structure was based on electron density maps and higher resolution structures (the 1.98 Å 400 S. aureus complex PDB code: 5IWI, which contains over 940 waters, was superposed). At each DNA-cleavage site a single catalytic Mn 2+ ion is seen at the B-site [10]. Electron 427 density on His C 391, was interpreted as being due to a Mn 2+ ion coordinated by a Bis-Tris 428 buffer molecule, which mediates a crystal contact with one end of the DNA. This inter-429 pretation of the electron density was confirmed by re-refining the original 2.1 Å structure 430 of GSK299423 with the S. aureus gyrase CORE structure [13]; originally this electron density 431 had been misinterpreted as being due to DNA. Antimicrobial Susceptibility Testing) [74]. The concentration range tested was between 40 445 -0.313 µg/mL in two-fold serial dilutions.