Structural basis for E. coli Penicillin Binding Protein (PBP) 2 inhibition

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Structural basis for E. coli Penicillin Binding Protein (PBP) 2 inhibition, a platform for drug design Nicolas Levy, Jean-Michel Bruneau, Erwann Le Rouzic, Damien Bonnard, Frederic Le Strat, Audrey Caravano, Francis Chevreuil, Julien Barbion, Sophie Chasset, Benoît Ledoussal, François Moreau, and Marc Ruff J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00338 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Journal of Medicinal Chemistry

Structural basis for E. coli Penicillin Binding Protein (PBP) 2 inhibition, a platform for drug design Nicolas Levy1,2, Jean-Michel Bruneau1, Erwann Le Rouzic1, Damien Bonnard1, Frédéric Le Strat1, Audrey Caravano1, Francis Chevreuil1, Julien Barbion1, Sophie Chasset1, Benoît Ledoussal1, François Moreau1, Marc Ruff2* 1Mutabilis, 2IGBMC,

102 avenue Gaston Roussel, 93230 Romainville, France

1 rue Laurent Fries, 67404, Illkirch, France

*Corresponding author, E-mail: [email protected]

Penicillin-binding proteins (PBPs) are the targets of the β-lactams, the most successful class of antibiotics ever developed against bacterial infections. Unfortunately, the worldwide and rapid spread of large spectrum β-lactam resistance genes such as carbapenemases is detrimental to the use of antibiotics in this class. New potent PBP inhibitors are needed, especially compounds that resist β-lactamase hydrolysis. Here we describe the structure of the E. coli PBP2 in its Apo form and upon its reaction with 2 diazabicyclo derivatives, Avibactam and CPD4, a new potent PBP2 inhibitor. Examination of these structures shows that unlike Avibactam, CPD4 can perform a hydrophobic stacking on Trp370 in the active site of E. coli PBP2. This result, together with sequence analysis, homology modeling and SAR, allows us to propose CPD4 as potential starting scaffold to develop molecules active against a broad range of bacterial species at the top of the WHO priority list.

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Introduction Penicillin(s) and its successors β-lactam ring based antibacterials (penems, carbapenems, cephalosporins and monobactams) have been in use for 70 years and remain at the forefront of the armamentarium to combat bacterial infections. However, over the past several decades, the frequency of antimicrobial resistance to this molecular family has increased considerably. Antibacterial resistance is estimated to be responsible for 25,000 deaths per year in the European Union and 700,000 globally1, 2. A recent antimicrobial resistance benchmark3 emphasized that, although important products are being developed in companies involved in antibiotics R&D, there are too few to replace the antimicrobials now losing effectiveness and the current pipeline needs to be further strengthened. Therefore, there is an urgent need for new potent antibacterial compounds. In particular antibiotics able to fight the WHO priority pathogens4 are required. β-lactams target and inhibit the trans-peptidase activity of the essential bacterial cell wall synthesizing enzymes called penicillin-binding proteins (PBPs). These enzymes catalyze the terminal step of the synthesis of peptidoglycan, the major component of the cell wall of Gramnegative and Gram-positive bacteria, responsible for the maintenance of bacterial shape and for the resistance to internal turgor pressure. The rod shape in Gram-negative bacteria is maintained by a multi-protein “elongase” (or “elongasome”) complex involving, among other proteins, PBP25. PBP2 belongs to the High Molecular Mass class B PBP category6. It localizes in the lateral wall of bacteria and at the division site with a transient role in the initiation of cell division7. The main role of PBP2, however, is in the elongation process. The E. coli PBP2 enzyme is bound to different extents by the diverse β-lactam antibiotics. Imipenem and Meropenem8, 9, for instance, have a strong preference for PBP2 among all other PBPs and Mecillinam binds exclusively to PBP210, 11. In contrast, most cephalosporins such as Ceftazidime or Cefotaxime bind preferentially to PBP3 and are poor binders of PBP212. New


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non-β-lactam β-lactamase inhibitors were launched recently and appear to possess PBP2 specific binding activity. Avibactam, the first example in this series, has been approved in 2015 in combination with the 3rd generation cephalosporin Ceftazidime. Avibactam is a member of the diazabicyclooctane (DBO) class of β-lactamase inhibitors with broad activity against class A and class C β-lactamases, and activity against some class D enzymes. Although not active against class B (metallo) enzymes, this molecule is fairly resistant to β-lactamase degradation. The fact that several pharmaceutical and biotech companies are currently developing DBObased β-lactamase inhibitor combinations emphasizes the great interest that one can have for this series of compounds. Along with β-lactamase inhibition, Avibactam has a poor intrinsic antibacterial activity and is acting against a limited number of strains, due to moderate PBP2 inhibition13. Interestingly, this indicates that the DBO series may be improved to obtain ultimately potent PBP inhibitors14 which can be used as single therapeutic agent or combined to other antibiotics. In line with this, several DBO-based β-lactamase inhibitors with good activity against PBP2 are currently in development e.g. by Wockhardt15, Roche16 or Entasis17. We were able to generate the DBO compound CDP4, a potent PBP2-specific inhibitor, active against E. coli and P. aeruginosa strains. We describe here the first crystal structures of E. coli PBP2 in its apo-form or in complex with Avibactam or CPD4 (at 2.1, 2.4 and 2.0 Å resolution, respectively). These structures give rational insights into the mode of action of this highly potent molecule. The comparison of PBP2 sequences of several bacterial species, particularly those mentioned in the WHO priority list, combined with analysis of the crystal structure of PBP2 from Helicobacter pylori18, structures homology modeling and SAR allows us to propose that CPD4 could be a general starting scaffold to design new drugs active against a large range of bacterial species.



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Results Chemical synthesis Product CPD419 was synthetized in 13 steps from compound 120 (Figure 1). First racemic compound 1 underwent a Mitsunobu reaction with N-nosyl-protected O-allylhydroxylamine. The Boc group was then removed using zinc bromide in dichloromethane, the use of strong acids such as TFA and HCl leading to the cleavage of the tert-butyldimethylsilyl group. The nosyl protection was then removed by using thiophenol under basic conditions to provide diamine 3. The cyclization in urea was performed with diphosgene in the presence of trimethylamine in acetonitrile. Compound 4 was engaged in a Suzuki coupling to introduce the oxazolyl moiety. The presence of the fragile urea function susceptible to hydrolysis under aqueous basic conditions required to perform the Suzuki coupling in the absence of water. The use of cesium carbonate as base and Pd(PPh3)4 as catalyst in anhydrous THF at 60°C led in good yield to compound 5 which was engaged in the TBS removal step with TBAF. Hydroxyl group was transformed into an NHBoc function in a 4-step sequence. Compound 6 was first mesylated and substituted by sodium azide in DMF. Azide intermediate underwent a Staudinger reaction and was trapped with Boc-ON to provide compound 7 in 21% yield over 4 steps. Deallylation / sulfation was performed one-pot leading to compound 8. The Boc deprotection yielded CPD4 in its zwitterionic form after precipitation in methanol.


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Figure 1: Synthesis of CPD4 from racemic alcohol 1, reagents and conditions : (a) DIAD, PPh3, N-allyloxy-2-nitro-benzenesulfonamide, toluene, rt (93%); (b) ZnBr2; CH2Cl2, rt; (c) PhSH, K2CO3, CH3CN, rt; (d) Diphosgene, Et3N; CH3CN, rt; (e) 5-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)oxazole, Pd(PPh3)4, Cs2CO3, THF, 60°C; (f) n-Bu4NF, THF, rt; (g) MeSO2Cl, pyridine, 0°C; (h) NaN3, DMF, 65°C; (i) PMe3, THF/Toluene, rt ; (j) Boc-ON; (k) Pd(PPh3)4, AcOH, CH2Cl2, rt; (l) SO3.pyridine complex, pyridine, rt; (m) TFA, CH2Cl2, 0°C. Protein purification and crystallization Preliminary experiments indicated that production of the periplasmic part [44-633] of E. coli PBP2 was rendered difficult due to low expression levels and low solubility. Different constructs were generated with different fusion proteins and His6-NusA was finally chosen as the best solubilizing partner. To obtain a minimal fragment able to crystallize, we conducted limited proteolysis experiments on the His6-tag purified protein and generated a smaller, soluble, active, and homogeneous form of the protein. Among the several proteases tested (AspN, Arg-C, Trypsin, Glu-C), Glu-C allowed us to produce a 56 kDa (Figure S1A) fragment that could be purified to homogeneity. Enzymatic integrity of this fragment was assessed by Bocillin-FL binding assay (Figure S1A) and thermal stability and CPD4 binding capacity were confirmed by thermal shift assay (Figure S2). For crystallization, protease processed and purified PBP2 (PBP2 Eco) was incubated or not with 50 µM ligand CPD4 for 30 minutes at room temperature before concentration to OD 0.6-0.9. Crystals of CPD4 bound or Apo protein were obtained, and the X-ray structures were solved allowing us to define the limits of the proteolyzed fragment [57-615]. This led us to re-clone the protein with the new limits and PBP2 [57-615] was expressed, purified, and enzymatic integrity of this new fragment was assessed by Bocillin-FL binding assay (Figure S1B). Recombinant PBP2 [57-615] (PBP2 Eco [57-615])



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samples were crystallized after binding to Avibactam following the same procedure, except for the crystallization temperature that was raised to 27°C. Overall Structure description E. coli PBP2 displays the characteristic fold of class B PBPs. It is composed of an elongated tripartite N terminus and a large transpeptidase (TP) domain at its C terminus, that bears the catalytic activity of the protein. The root mean square deviation (rmsd) of a global superposition of the Apo, Avibactam and CPD4 structures shows that the three structures are quasi identical. (PBP2 apo / PBP2 Avibactam or PBP2 apo / PBP2 CPD4: rmsd = 0.27 Å; PBP2 Avibactam / PBP2 CPD4: rmsd = 0.22 Å, for C-alpha atoms). The structures encompass residues 57 to 613, with an unmodeled loop spanning from 550 to 568. This loop is highly flexible, and the corresponding density could not be seen either in PBP2 Eco obtained from proteolysis or PBP2 Eco [57-615] structures (Figure 2A). The N terminus is divided into three sub-domains: the anchor domain, the linker domain and the head domain (Figure 2B, C, D).


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Figure 2: Topology and domain organization of E. coli PBP2. (A) Primary structure of E. coli PBP2. Red: Anchor domain, green: TP domain, blue: head domain, yellow: linker domain, white: flexible loop (residues 550 to 568). (B) Surface representation of the crystallographic structure. The active site is circled in red. (C) Ribbon representation. (D) Secondary structure representation showing the topology of the protein. The anchor domain is composed mainly of three long β strands and a short 310 helix. In the linear sequence, the β1 strand is separated from the rest of the anchor domain by the linker and head domains, as well as part of the TP domain. The linker domain is composed of four alphahelices, two 310 helices, two β strands and is in close contact with the TP domain. The head domain protrudes on two long β strands and contains a third small β strand as well as three alpha helices and two 310 helices. The TP domain is the largest of the protein. It is structured around three β sheets: one 4 strand sheet which contacts the N terminus (β2, β10, β17, β18), the main five strand β sheet (β11, β12, β19, β20, β21) in the center of the domain, involved in the active site, and a small 4 strand β hairpin at the extremity of the protein (β13, β14, β15, β16). The other structured parts of the domain are composed of 15 helices (13 alpha helices and 2 310 helices). Protein interface analysis Anchor and head domains of PBPs are known to promote protein-protein interactions, with other PBPs21 or with other partners18, 22, and are annotated as dimerization domains in protein domain databases (Interpro IPR005311). We examined possible significant interaction interfaces in PBP2 Eco crystals using PISA interface analysis software23. Interestingly, the only physiologically relevant predicted interface (P-value 0.1027; -15.5 kcal/mol binding energy) corresponds to a dimer formed by interactions of both anchor and head domains between two PBP2 monomers (Figure S3 and Table S1). This could reflect an unanticipated dimerization



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potential for PBP2 which could be important in some synthesis mechanisms as it has been described for PBP1a, PBP1b or PBP324-26. Active site structure The active site of E. coli PBP2 forms a deep solvent-exposed groove in the side of the TP domain (Figure 2B). The three conserved motifs common to β-lactamases are found here and constitute the scaffold of the active site. The S-X-X-K (Ser330 - Thr331 – Val332 – Lys333) motif that contains the nucleophilic serine 330 is located at the junction of helices η6 and α12 and constitutes the inner surface of the cleft. The S-X-N/D (Ser387 – Ala388 – Asp389) motif is found in the linker region between helices α13 and α14 at the top of the pocket. The third motif K-T/S-G-T (Lys544 – Ser545 – Gly546 – Thr547) is part of the β19 strand and makes the floor of the cleft. A fourth major component of the binding pocket is Trp370, part of a conserved sequence R-D/N-W-K/N located between strands β15 and β16, which forms a large protruding surface at the ceiling of the active site (Figure 3A). Finally, Ile453, part of a fifth conserved motif I-G-Q-G (Ile453 – Gly454 – Gln455 – Gly456), constitutes one side of the ligand binding pocket.

Figure 3: Active site structure of the APO protein. (A) The side chains of the conserved residues are shown. (B) Network of hydrogens bonds in the active site in the absence of ligand.


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The catalytic Ser330 is exposed to solvent and positioned favorably for acylation. It is linked to other residues of the active site through a network of direct hydrogen bonds or indirectly through several water molecules (Figure 3B). Both Avibactam and CPD4 were found highly structured in active site, and fully defined in electron density (Figure 4A and D, respectively). Avibactam binds to PBP2 through covalent bonding with Ser330 and through several hydrogen bonds with its environment. It is bound to Asp389 via its amide group (3.1Å), to Ser387 via nitrogen bound to sulfate (3.4 Å) and to Ser545 and Thr547 via its sulfate group (2.2 and 2.3 Å respectively) (Figure 4A, B, C).



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Figure 4: Active site structure in complex with (A, B, C) Avibactam and (D, E, F) CPD4. A and D are representations of ligands electron densities as calculated from Polder OMIT maps contoured at 6σ (green mesh). B and E are surface representation (grey surface) of the ligand binding pocket and of the hydrogen bonds between ligands and surrounding residues or solvent molecules (yellow dashed lines). C and F are Ligplot+ representations of protein/ligands interactions. CPD4 binds to PBP2 through covalent bonding with Ser330 and through several hydrogen bonds with its environment. It is bound to Asp389 via its amine group (3.0 / 3.4Å), to Ser387 via nitrogen bound to sulfate (3.2 Å) and to Ser545 and Thr547 via its sulfate group (2.7/3.2 and 2.6/3.4 Å respectively). Moreover, a significant stacking is observed between the oxazole cycle of CPD4 and Trp370 with distances as close as 3.2 Å (circled in green in Figure 4 D, E, F). Ligand design and potency analysis On the way to the discovery of novel direct-acting DBOs with PBP inhibition properties, we discovered a very potent PBP2 specific inhibitor structurally different from those currently in development such as Nacubactam, Zidebactam, ETX-1317 and ETX-2514 (Figure 5).

Figure 5. Gram negative active DBOs in development with specific PBP2 inhibition. 10 ACS Paragon Plus Environment

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As presented in Figure 6, starting from the unsubstituted DBO CPD1 which is fully inactive against PBP2 of E. coli (IC50 > 200µM), the introduction of an endocyclic double bond led to a strong improvement of this inhibition (CPD2, IC50 PBP2 = 0.57 µM), thus reaching the level of Avibactam with a carboxamide group. CPD2 was therefore a good starting point for further SAR development in areas so far poorly explored.

Figure 6: IC50 and MIC of Avibactam and several CPD intermediates on Escherichia coli and Pseudomonas aeruginosa. Our initial studies were focused on modulating the substitution of the double bond to see the impact on PBP2 and to improve the antibacterial activity of DBOs. We decided to explore broadly 5 and 6-membered heterocycles in the northeast position of the double bond. The synthesis of these analogs is described in detail in the patent27 and the results are briefly summarized in Table S2 with different oxazole and pyridine heterocycles. For most analogs, the addition of heterocycles in northeast position allowed to improve PBP2 inhibition by a factor of 2 to 33 compared to CPD2 with a naked endocyclic double bond. The 2-pyridine analog CPD7 inhibited PBP2 less potently than the isomers CPD5 and CPD6. Substitution of northeast position with different oxazoles and isoxazoles (CPD8-9-3) (Table S2) led to PBP2 inhibition culminating at 17nM with CPD8. None of these compounds inhibited significantly other PBPs of E. coli (1a, 1b, 3 – data not shown). From these derivatives, the best MIC values 11 ACS Paragon Plus Environment


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against E. coli isolates were obtained with CPD3. This compound is a strong PBP2 inhibitor and is effectively transported into the periplasm presumably thanks to its adequate heteroatom positions in the oxazole ring. At comparable PBP2 IC50, the pyridines, less hydrophilic, are also apparently less permeant. None of CPD3-5-6-7-8-9 did show any antibacterial activity against P. aeruginosa (MICs >32 mg/L). As pointed out by Nikaido28, the zwiterrionic character of compounds favors their permeation through the porins into Gram-negative bacteria. Novexel29, 30 described DBO-based zwitterions with unusual antipseudomonal activity. The zwitterion CPD4, which combines CPD3 with an aminomethyl side chain, not only shows an improved PBP2 inhibition compared to CPD3 but also leads to very potent MICs against E. coli and P. aeruginosa, attesting for an excellent permeation (Figure 6). Interestingly, as mentioned by Nikaido, despite a higher distribution coefficient (cLogD) for the zwitterion CPD4 compared to CPD3, CPD4 permeates more efficiently into the bacteria probably thanks to the optimal positioning of the positive and the negative charges for porin translocation31. The MIC profile of CPD4 was tested against a panel of 58 resistant E. coli clinical isolates (ESBLs, MBL and OXA genes families) and 62 P. aeruginosa isolates with MIC50 / MIC90 of Ceftazidime of > 32 / > 32 mg/L. MIC50 of CPD4 on this panel was 0.031 and 1 mg/L on E. coli (Table 1A) and P. aeruginosa (Table 1B) respectively (MIC90 >32 and 16 mg/L). These results are indicative of a good antibacterial potential for this series. It is interesting to note that CPD4 outperformed Zidebactam activity and was also superior to Meropenem on these selections of clinical isolates.


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MIC

32

Number of isolates

MIC50

MIC90

A: MIC against E. coli (mg/L - 58 isolates) CPD4

39

0

1

0

0

0

0

1

17

0.031

>32

Zidebactam

0

0

2

7

11

6

1

5

26

32

>32

Avibactam

0

0

0

0

0

3

19

12

24

32

>32

Ceftazidime

0

4

1

5

2

6

2

2

36

>32

>32

Meropenem

28

4

3

5

3

2

2

1

10

0.5

>32

B: MIC against P. aeruginosa (mg/L - 62 isolates) CPD4

7

11

15

18

1

0

4

1

5

1

16

Zidebactam

0

0

0

0

15

21

22

1

3

8

16

Avibactam

0

0

0

0

0

0

0

0

62

>32

>32

Ceftazidime

0

0

0

6

9

3

1

7

36

>32

>32

Meropenem

1

2

5

11

7

7

6

6

17

8

>32

Table 1: MIC profile of CPD4 against E. coli (A) and P. aeruginosa (B) clinical isolates Ligand affinity analysis. PBP2 reaction with β-lactam antibacterial relies on hydrolysis of the β-lactam ring by its catalytic Ser330. This results in a transient covalent protein-ligand complex. Schematic representation of initial and protein-bound states of both Avibactam and CPD4 ligands are displayed in Figure 7.



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Figure 7: Avibactam and CPD4 reaction and rearrangement with PBP2 catalytic Ser330 To understand the differences in potency between Avibactam and CPD4, we calculated the binding energy (using Autodock Vina32) of the two ligands after the covalent bond formation with Ser330 and rearrangement (without considering the covalent bond). After the covalent bond formation and ligand rearrangement the affinities of Avibactam and CPD4 for E. coli PBP2 are 1.56 and -0.12 kcal/mol respectively. This indicates that the final position of the ligand in the pocket is not energetically favorable for Avibactam if not covalently linked. On the contrary, the final structure for CPD4 is more stable, a fact that could explain the improvement of CDP4 potency. This overall higher binding stability of CPD4 compared to Avibactam can be confirmed in the structures by observing respective atomic displacement parameters of the ligands. Average B factor for Avibactam is 87% higher than for CPD4 while average B factor for the protein alone is 50% higher only (Table S3).


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Comparison with PBP2 from the WHO priority pathogen list. Sequence alignment of PBP2 from the WHO priority pathogen list was performed for organisms displaying at least 30% sequence identity with E. coli PBP2. The results are summarized in Table 2 and Figure S4. Residues in the binding pocket important for ligand binding and final state stabilization (Trp370, Ser387, Ala329, Ile453, Asp389, Ser330, Lys333, Gly546, Thr547, Ser545) are conserved in the 9 species (Figure 8).

Figure 8: Alignment close-up on conserved motifs forming PBP2 active site: Important conserved residues are highlighted in blue boxes. E. coli and Helicobacter pylori PBP2 sequences are the most distant examples displayed in the alignment (Table 2), yet superposition of these two Apo structures lead to an overall rmsd of 1.5 Å showing a highly conserved global structure. Moreover, local superposition in PyMOL of the conserved motifs forming the ligand binding pocket (Figure 3) leads to a rmsd of 1.36 Å for all atoms and 0.59 Å for C-alphas showing an even closer structural conservation at the level of the catalytic site.



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Figure 9: Local superposition of the active site structures of E. coli and H. pylori PBP2: Active site structures of E. coli (blue) and H. pylori (orange) PBP2 (A), corresponding to conserved residues described in table (B), were superimposed in PyMOL. Root mean square deviation between these residues is 0.59 Å for C-alphas and 1.36 Å for all atoms. Structure modeling. PBP2 homology modeling of the seven species present in the WHO priority list was performed using the structures of E. coli and H. pylori PBP2. The rmsd of the structures using E. coli PBP2 as a reference are summarized in Table 2.

Sequence number

Organism

%identity

WHO Priority

Structure

RMSD (Å)

Seq 1

Escherichia coli

100

Critical

Yes (this paper)

0.00

Seq 2

Shigella spp.

74

Medium

Model (this paper)

0.64

Seq 3

Salmonella bongori

96

High

Model (this paper)

0.70

Seq 4

Enterobacter spp.

93

Critical

Model (this paper)

0.65

Seq 5

Haemophilus influenzae

57

Medium

Model (this paper)

0.82

Seq 6

Pseudomonas aeruginosa

44

Critical

Model (this paper)

1.26

Seq 7

Acinetobacter baumannii

39

Critical

Model (this paper)

1.25

Seq 8

Campylobacter spp.

32

High

Model (this paper)

1.80

Seq 9

Helicobacter pylori

30

High

Yes (Contreras-Martell 2017)

1.93

SAR (MIC) (mg/L) AVI

CPD4

16-32

0.016

>32

2.0


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Table 2: Percentage of identity and structures rmsd relative to E. coli of PBP2 from the WHO priority list. SAR (MIC) data are reported for P. aeruginosa and E. coli. There is a strong improvement of the potency with CPD4 compared to Avibactam. The active site structures are well conserved whereas there is more variability in the anchor and head domain (Figure 10). This structure conservation, together with the strong improvement of the MIC between Avibactam and CDP4 for E. coli and P. aeruginosa indicates that CPD4 is a good starting point to discover new potent inhibitors of PBP2 of other bacterial species.

Figure 10: Superposition of the 2 X-ray structures and 7 homology models of PBP2. 17 ACS Paragon Plus Environment


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Discussion and conclusion In this work we describe the crystallographic structures of E. coli PBP2 in the Apo form and in complex with two diazabicyclooctane inhibitors, Avibactam and CPD4. Analysis of the structures provides insight into the binding mode of a highly potent PBP2 inhibitor, CPD4. The catalytic site shows a remarkably stable structure with all-atom rmsd as low as 0.27 Å between the Apo protein and the Avibactam or CPD4 complexes, indicating that there is no structural rearrangement of PBP2 upon ligand binding, covalent bond formation and ligand rearrangement. Residues of the active site show only subtle side-chain movement upon ligand binding which reflects easy ligand access and allows straightforward drug design. In comparison to Avibactam, CPD4 possesses a better final affinity for the protein, and the stacking of its pyrazole cycle with the hydrophobic tryptophan of the active site provides a strong positioning effect. CPD4 (IC50 = 0.01 µM) and CPD3 (IC50 = 0.025 µM) display similar activity against PBP2 but CPD4 is 250-fold more potent against E. coli UFR39 (MIC = 0.016 mg/L) and has a strong increase in potency against P. aeruginosa (MIC = 2mg/L). This is possibly due to an improved transport into the periplasm. Such small polar compounds are expected to reach the periplasm via the porins which generally favor the transport of zwitterions versus anions, and thus favor CPD4 versus CPD3. CPD4 inhibition potential for other PBPs was tested against recombinant E. coli and P. aeruginosa PBP1a, PBP1b, and PBP3: no inhibition was observed up to 200 µM against these enzymes (Data not shown). CPD4 is therefore a DBO-based PBP2-specific inhibitor like Zidebactam and Nacubactam. CPD4 was found to be a moderate inhibitor (sub µM level) of class A β-lactamases (TEM-1, SHV-1, CTX-M-15, KPC-2), low inhibitor of class C enzymes (P99 Amp-C, CMY-37) and good inhibitor (low nM level) of some class D enzymes (OXA-15, OXA-48) (data not shown).


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Similarly to other PBP2-specific inhibitors such as Mecillinam33, spontaneous resistance frequency (SRF) of E. coli treated with CPD4 was expected to be high due to triggering of the bacterial stringent response that increases the intracellular levels of the signal mediator guanosine- 3′,5′-bisdiphosphate (ppGpp) which renders PBP2 dispensable to the bacteria. The mutation frequency measured in several Enterobacteriaceae and P. aeruginosa isolates was indeed high even at more than 8-fold the MIC (> 10-5 – Data not shown). A use of CPD4 in combination would therefore be more appropriate in order to reduce the risk of resistance selection. Combining CPD4 with another β-lactam (e.g. a good PBP3 inhibitor such as a monobactam or a cephalosporin) was not considered due to the poor β-lactamase inhibition profile of this compound. Such a combination would certainly take advantage of the combined PBP2 and PBP3 inhibition with expected improvements on bactericide and frequency of resistance but would not properly address the protection of the β-lactam versus the widespread dissemination of ESBLs (Extended-Spectrum Beta-Lactamases). One could rather consider a combination of CPD4 with Fosfomycin, especially against severe infections caused by MDR P. aeruginosa. Initial results on this combination are promising and warrant further exploration. E. coli PBP2 is the first enterobacterial and the second Gram-negative PBP2 structure known with H. pylori18. Although both proteins are the most distant in our sequence alignment, they display remarkable structural similarities, especially at the level of the active site. This strong structural conservation allowed us to perform homology modeling to construct models for the unknown structures from the WHO priority list with a percentage of identity greater than 30% with the E. coli PBP2. The strong structural conservation of the active sites together with the strong improve in potency of CPD4 versus Avibactam for E. coli and P. aeruginosa, validate CPD4, the structures (X-ray) and models to be used for drug design experiments.



Page 20 of 35

Overall, the structures and models described in this work in complex with CPD4 and Avibactam together with the structure of Apo PBP2 from H. pylori and SAR data provide a strong framework for the design of inhibitors based on CPD4 and can be used as a platform for to decipher molecules that would display a large spectrum of activity against the WHO priority list organisms.

Experimental section Chemical synthesis. General Methods, Reagents and Material (Figure 1). All reagents were purchased from commercial sources and used without further purification unless stated otherwise. The progress of all reactions was monitored by UPLC/MS. The instrument was a Waters Acquity UPLC-H-Class/SQD with a DAD detector (190-400 nm) and a Waters BEH C18 column (1.7 µM 2.1x50mm). A Waters HSS T3 column (1.8 µM 2.1x50mm) was used for the monitoring of sulfated compounds. Purifications by chromatography were performed on a Biotage Isolera chromatography system equipped with KP-SIL SNAP cartridges or Ultra SNAP cartridges with the solvent mixtures specified in the corresponding experiment. Thin-layer chromatography (TLC) was carried out on Merck precoated silica gel 60 F254 plates with an UV−visible lamp. Proton (1H) NMR spectra were recorded on Bruker NMR instrument (300 MHz or 400 MHz) using CDCl3 or DMSO-d6 as solvents. Chemical shifts are given in parts per million (ppm) ( relative to residual solvent peak). Tert-butyl trans-3-[allyloxy-(2-nitrophenyl) sulfonyl-amino]-6-[[tert-butyl(dimethyl)silyl] oxy-methyl]-4-iodo-3,6-dihydro-2H-pyridine-1-carboxylate (2). To a solution of tert-butyl cis6-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-4-iodo-3,6-dihydro-2H-pyridine-1carboxylate (1)20


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(12.05 g, 25.67 mmol) in toluene (170 mL) at rt was added triphenylphosphine (8.08 g, 30.80 mmol), N-(allyloxy)-2-nitrobenzenesulfonamide (6.63 g, 25.67 mmol) and diisopropyl azodicarboxylate (6.06 mL, 30.80 mmol). The reaction mixture was stirred at rt overnight and concentrated in vacuo. The crude was purified by flash chromatography on silica gel (cyclohexane/ethyl acetate 100/0 to 85/15) to give compound (2) (17.0 g, 23.95 mmol, 93%).1H NMR (300 MHz, CDCl3):  8.06-8.19 (m, 1H), 7.69-7.84 (m, 2H), 7.54-7.66 (m, 1H), 6.73 (d, J = 4.1 Hz, 1H), 5.68-5.89 (m, 1H), 5.12-5.38 (m, 2H), 3.93-4.78 (m, 5H), 3.16-3.75 (m, 3H), 1.35 (s, 9H), 0.88 (s, 9H), 0.03 (s, 6H). trans-N-allyloxy-6-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-iodo-1,2,3,6-tetrahydropyridin3-amine (3). To a solution of compound (2) (17.0 g, 23.95 mmol) in CH2Cl2 (177 mL) was added zinc bromide (16.2 g, 71.86 mmol). The reaction mixture was stirred at rt overnight then diluted with dichloromethane and washed with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The crude was diluted with acetonitrile (177 mL). K2CO3 (16.6 g, 119.77 mmol) was added, followed by thiophenol (12.3 mL, 119.77 mmol). The reaction mixture was stirred at rt for 1 h and concentrated in vacuo. Dichloromethane was added and the resulting solids were removed by filtration. The filtrate was concentrated in vacuo.

The

crude

was

purified

by

flash

chromatography

on

silica

gel

(dichloromethane/methanol 100/0 to 90/10) to provide diamine (3) (7.99 g, 18.83 mmol, 78%). MS (ESI): 425 [M+H]+. 1H NMR (300 MHz, CDCl3) :  6.53-6.56 (m, 1H), 5.88-5.35 (m, 2H), 5.25-5.35 (m, 1H), 5.18-5.25 (m, 1H), 4.22 (dq, J = 6.0, 1.2 Hz, 2H), 3.53-3.60 (m, 2H), 3.37-3.45 (m, 2H), 3.21 (dd, J = 12.6, 3.9 Hz, 1H), 3.14 (dd, J = 12.6, 5.1 Hz, 1H), 1.82 (bs, 1H), 0.89 (s, 9H), 0.06 (s, 6H). Trans-6-allyloxy-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-iodo-1,6diazabicyclo[3.2.1]oct-3-en-7-one (4). To a solution of diamine 3 (7.99 g, 18.83 mmol) in anhydrous acetonitrile (980 mL) at 0°C under inert atmosphere was added triethylamine (10.56



Page 22 of 35

mL, 75.31 mmol). A solution of diphosgene (1.14 mL, 9.41 mmol) in anhydrous acetonitrile (20 mL) was dropwise added over 5 h. Once the addition finished, the reaction mixture is allowed to reach rt and stirred for 3 days. Water was added and the mixture was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The crude was purified by flash chromatography on silica gel (cyclohexane/ethyl acetate 100/0 to 80/20) to give urea (4) (7.25 g, 16.10 mmol, 85%). MS (ESI): 451 [M+H]+. 1H NMR (300 MHz, CDCl3) :  6.37-6.41 (m, 1H), 5.97-6.12 (m, 1H), 5.34-5.43 (m, 1H), 5.28-5.34 (m, 1H), 4.35-4.53 (m, 2H), 4.05-4.08 (m, 1H), 3.80-3.90 (m, 3H), 3.57 (d, J = 11.1 Hz, 1H), 3.19 (dd, J = 11.1, 3.0 Hz, 1H), 0.88 (s, 9H), 0.06 (s, 6H). Trans-6-allyloxy-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-oxazol-5-yl-1,6-diazabicyclo [3.2.1]oct-3-en-7-one (5). In a sealed flask, a mixture of iodide (4) (5.20 g, 11.55 mmol), 5(4,4,5,5-tetramethyl-1,2,3-dioxaborolan-2-yl)oxazole (2.70 g, 13.86 mmol) and cesium carbonate (7.52 g, 23.09 mmol) in anhydrous THF (100 mL) was degassed under argon for 5 min and Pd(PPh3)4 (400 mg, 0.35 mmol) was added. The mixture was heated at 60°C overnight. Water was added and the mixture was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by flash chromatography on silica gel (cyclohexane/ethyl acetate 100/0 to 70/30) to provide coupling product (5) (3.64 g, 9.30 mmol, 80%). MS (ESI): 392 [M+H]+. 1H NMR (300 MHz, CDCl3) :  7.83 (s, 1H), 7.03 (s, 1H), 6.15 (d, J = 3.0 Hz, 1H), 5.92-6.08 (m, 1H), 5.27-5.40 (m, 2H), 4.33-4.50 (m, 2H), 4.11-4.14 (m, 1H), 3.87-4.06 (m, 3H), 3.54 (d, J = 11.0 Hz, 1H), 3.37 (dd, J = 11.0, 3.0 Hz, 1H), 0.88 (s, 9H), 0.07 (s, 6H). Trans-6-allyloxy-2-(hydroxymethyl)-4-oxazol-5-yl-1,6-diazabicyclo[3.2.1]oct-3-en-7-one (6). To a solution of compound (5) (3.64 g, 9.30 mmol) in THF (45 mL) at 0°C was added tetrabutylammonium fluoride 1M in THF (13.9 mL, 13.94 mmol). The reaction mixture was stirred at 0°C for 1 h and concentrated in vacuo. The crude was purified by flash


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chromatography on silica gel (100% ethyl acetate) to provide alcohol (6) (1.53 g, 5.52 mmol, 57%). MS (ESI): 278 [M+H]+. 1H NMR (400 MHz, CDCl3) :  7.83 (s, 1H), 7.06 (s, 1H), 5.94-6.06 (m, 2H), 5.28-5.39 (m, 3H), 4.36-4.50 (m, 2H), 4.12-4.19 (m, 2H), 3.69-3.87 (m, 2H), 3.40 (dd, J = 11.2, 2.9 Hz, 1H), 3.32 (d, J = 11.2 Hz, 1H). Tert-butyl

N-[[trans-6-allyloxy-4-oxazol-5-yl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-2-yl]

methyl]carbamate (7). A solution of alcohol (6) (1.53 g, 5.52 mmol) in pyridine (17 mL) was cooled to 0°C. Methanesulfonyl chloride (0.67 mL, 8.61 mmol) was added and the reaction mixture was stirred at the same temperature for 2 h. After concentrating in vacuo, the crude was dissolved in dichloromethane and successively washed with a 1N HCl solution and brine. The organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The crude was dissolved in DMF (29 mL) and sodium azide (1.79 g, 27.59 mmol) was added. The reaction mixture was heated at 65°C overnight and concentrated in vacuo. Water was added to the crude, which was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude was dissolved in a mixture of THF (16.7 mL) and toluene (16.7 mL) and a solution of trimethylphosphine 1M in THF (8.28 mL, 8.28 mmol) was added at 0°C. After 1 h stirring at rt, the mixture was cooled to 0°C and a solution of 2-(Boc-oxyimino)-2-phenylacetonitrile (2.04 g, 8.28 mmol) in THF (11 mL) was dropwise added. The mixture was stirred at rt for 1 h and concentrated in vacuo. The crude was purified by flash chromatography on silica gel (cyclohexane/ethyl acetate 95/5 to 0/100) to provide carbamate (7) (440 mg, 1.17 mmol, 21%). MS (ESI): 392 [M+H]+. 1H NMR (300 MHz, CDCl3) :  7.83 (s, 1H), 7.05 (s, 1H), 5.92-6.07 (m, 2H), 5.28-5.41 (m, 2H), 4.99-5.12 (m, 1H), 4.33-4.50 (m, 2H), 4.15 (d, J = 2.9 Hz, 1H), 3.98-4.07 (m, 1H), 3.53-3.67 (m, 1H), 3.37 (dd, J = 11.3, 2.9 Hz, 1H), 3.11-3.30 (m, 1H), 1.45 (s, 9H). Triphenyl-[(E)-prop-1-enyl] phosphonium [trans-2-[(tert-butoxycarbonylamino)methyl]-4oxazol-5-yl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl] sulfate (8). To a solution of carbamate



Page 24 of 35

(7) (440 mg, 1.17 mmol) and glacial acetic acid (134 µL, 2.34 mmol) in anhydrous dichloromethane (13 mL) was added in one portion Pd(PPh3)4 (675 mg, 0.58 mmol). After stirring for 2 h, a solution of sulfur trioxide pyridine complex (753 mg, 4.73 mmol) in anhydrous pyridine (15 mL) was added and the mixture was stirred overnight at rt. The reaction mixture was concentrated in vacuo, diluted with dichloromethane and filtered. The filtrate was concentrated in vacuo. The crude was purified by flash chromatography on silica gel (dichloromethane/acetone 97/3 to 20/80) to sulfate (8) (560 mg, 0.78 mmol, 67%). MS (ESI): 415 [M]-. 1H NMR (400 MHz, CDCl3)  7.82 – 7.57 (m, 16H), 7.47 (s, 1H), 7.23 – 7.08 (m, 1H), 6.71 – 6.50 (m, 1H), 5.85 (d, J = 2.6 Hz, 1H), 5.14 (d, J = 6.5 Hz, 1H), 4.78 (d, J = 2.8 Hz, 1H), 3.95 (dt, J = 10.5, 3.9 Hz, 1H), 3.65 – 3.55 (m, 1H), 3.52 (dd, J = 11.3, 3.0 Hz, 1H), 3.21 (d, J = 11.3 Hz, 1H), 3.19 – 3.07 (m, 1H), 2.32 – 2.22 (m, 3H), 1.44 (s, 9H). Trans 2-(aminomethyl)-4-oxazol-5-yl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl] hydrogen sulfate (CPD4). To a solution of sulfate (8) (1.16 g, 1.63 mmol) in anhydrous dichloromethane (3 mL) at 0°C was dropwise added trifluoroacetic acid (3 mL). The mixture was stirred at 0°C for 30 min and poured in cold methanol (30 mL) at 0°C. The mixture was stirred at rt for 1h. The precipitate was filtered, rinsed with methanol and dried in vacuo to provide sulfate CPD4 (438 mg, 1.38 mmol, 85%). MS (ESI): 315 [M-H]-. 1H NMR (300 MHz, DMSO-d6)  8.42 (s, 1H), 8.00 (bs, 3H), 7.31 (s, 1H), 5.99 (d, J = 3.3 Hz, 1H), 4.60 (d, J = 2.2 Hz, 1H), 4.13 – 3.99 (m, 1H), 3.46 (d, J = 11.7 Hz, 1H), 3.42 – 3.17 (m, 3H). Protein production and purification. Preliminary experiments indicated that production of the periplasmic part of E. coli PBP2 was rendered difficult due to low expression levels and poor solubility. Therefore, different constructs were initially made using different fusion partners and NusA was finally chosen as the best partner. After cloning of the NusA DNA sequence in frame after the His6 tag in pET28a and insertion of the E. coli K12 pbpA sequence coding for the periplasmic domain [44-633] of PBP2, the fusion protein was expressed in BL21


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(DE3) E. coli cells co-transformed with the pG-Tf2 plasmid that encodes the GroEL, GroES and Tig protein chaperones. To obtain a minimal form of PBP2 more prone to crystallize, we conducted limited proteolysis experiments to generate a smaller, soluble, active, and homogeneous protein. Among the different proteases used (Asp-N, Arg-C, Trypsin, Glu-C) the latter was found the most suitable, generating a 56 kDa fragment (PBP2 Eco) still able to bind Bocillin-FL, indicating that this fragment was correctly folded (Figure S1A). Solving the crystal structure of the previous fragment generated by limited proteolysis allowed us to define construct limits. The PBP2 Eco [57-615] fragment was produced and purified by means of IMAC and IEX chromatography, prior and after, respectively, cleavage of the His6-NusA tag and resulted in a pure and active protein (Figure S1B). Detailed protocols are in Supplementary methods. Thermal shift assay. Thermal stability of the protease processed sample PBP2 Eco and binding of CPD4 were assessed by thermal shift assay34. Monitoring of fluorescence change in a real-time PCR machine during thermal unfolding in presence of SYPRO Orange dye allowed us to determine a melting temperature (Tm) of the sample at the inflexion point of the denaturation curve. Apo PBP2 Eco is characterized by a denaturation temperature of 47°C (+/0.5 °C). Binding of CPD4 in a protein / ligand ratio of 1 / 10 resulted in a shift of the Tm to 55.5°C (+/- 0.5°C). This strong shift indicates efficient stabilization of the protein by CPD4 (Figure S2). E. coli PBP2 inhibition and Minimum inhibitory concentration (MIC) assay. PBP inhibition was measured in a fluorescent penicillin binding fluorescence anisotropy assay adapted from published methods35, 36. Antibacterial activity of compounds was assessed against carbapenem-resistant genotyped bacterial strains of E. coli and P. aeruginosa. MICs were determined by the broth microdilution method according to the Clinical Laboratory Standards Institute (CLSI – M7-A7). Detailed protocols are in Supplementary methods.



Page 26 of 35

Crystallization assays. PBP2 from mild proteolysis: Extensive screening was performed with both Apo and CPD4-complexed protein. For the latter, protein samples after purification were incubated with 50 μM CPD4 for 30 minutes at room temperature prior to the setting of crystallization drops. Sample concentration during equilibration was forced by equilibrating crystallization drops against 1.5 to 2.0 M NaCl reservoirs. The Morpheus screen condition G11 was optimized and provided good quality crystals (50 μm size) in two months. Two different crystal forms were obtained together in these drops, differing by the size of the crystal lattice but belonging to the same space group (Table S4). Recombinant PBP2 Eco [57-615] was crystallized in the same conditions. Less than two weeks were needed to grow crystals of 100 μM that appeared to be of the same type as the form 1 described earlier. For the complex with Avibactam, the protein sample was incubated with 50 μM of Avibactam for 30 minutes at room temperature prior to setting of crystallization drops. Detailed protocols are in Supplementary methods. Structure determination and refinement. Data collection was performed either at the Swiss Light Source (SLS, Villigen, Switzerland) on a Dectris Pilatus 2M detector or in house on a Dectris Eiger R 4M detector. X-ray diffraction images were indexed and scaled with XDS37, 38. A 2.0 Å resolution dataset from a PBP2 obtained from mild proteolysis + CPD4 crystal allowed us to get a first partial model as an output from the online automated molecular replacement pipeline MoRDa39. Several models were tested by the software and the best result was obtained using a Pseudomonas aeruginosa PBP3 structure (PDB entry 4WEK). After model improvement using Phenix Morph Model40, 41, several rounds of manual building with Coot42 and automatic building with Phenix Autobuild43 allowed us to correct the model and build the missing parts. Residues Asn57 to Leu613 could be built except for the Val550-Leu568 loop. This structure was refined using Phenix Refine44 and was subsequently used as a molecular replacement model to solve the structures of Apo PBP2 from mild proteolysis and PBP2 [57-


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615] – Avibactam with Phenix Phaser MR45. Ligand structures were refined as covalently modified serine. These modified residues were constructed in Accelrys Discovery Studio client and restraint files were generated using the JLigand tool in the CCP4 suite46,

47.

Electron

densities for ligands were calculated as Polder OMIT maps48 in Phenix and represented at a 6.0 sigma cutoff. Data collection and refinement statistics are summarized in Table S3. Crystallographic structures were deposited in PDB under the identification numbers 6G9S (PBP2 from mild proteolysis + CPD4), 6G9P (Apo PBP2 from mild proteolysis) and 6G9F (PBP2 [57-615] – Avibactam). Sequence alignment, Structure analysis, modeling and binding energy calculation experiments. Sequence alignment was done using MSAProbs49 through Jalview50, a multiple sequence alignment utility. Visualization of the alignment and secondary structure features was done using ESPript351. Protein interface analysis was performed using PISA interface analysis software23 through the Coot structure visualization program42. To analyze and compare the binding of CPD4 and avibactam, the energies of binding were calculated with Autodock Vina and ADT software32, 52 without taking into account the covalent bond using the structures of the final state. The unknown structures where obtained by homology modeling with Modeller53 through PyMOL54 and PyMod55 using the 2 known apo PBP2 structures (6G9P (this publication) and 5LP418). Structures rmsd were calculated with Coot. All figures of structures were generated in PyMOL or CHIMERA56. 2D view of the binding pocket in the presence of Avibactam or CPD4 were generated by Ligplot+57



Page 28 of 35

Supporting Information Supplementary Methods: DNA manipulations and plasmid construction, Purification of E. coli PBP2 [44-633] from a His6-NusA-PBP2 fusion protein, Purification of E. coli PBP2 [57615] from a His6-NusA-P3C-PBP2 fusion protein, Thermal shift assay, Assessment of penicillin-binding activity and protein purity by SDS-PAGE, E. coli PBP2 inhibition assay, Minimum inhibitory concentration (MIC) assays, PBP2 crystallization and data acquisition. Supplementary figures: Figure S1: SDS-PAGE analysis of the purified E. coli PBP2 transpeptidase containing fragment, Figure S2: Thermal stability and ligand binding assay on protease processed sample, Figure S3: PISA dimerization interface, Figure S4: full sequence alignment of PBPs from the WHO priority list with a cutoff at 30 % identity. Supplementary tables: Table S1: PISA predicted physiologically relevant interface data. Table S2: Effect of different oxazole and pyridine heterocycles addition on CPD2 in the northeast position of the double bond. Table S3: Data collection and refinement statistics for the three structures described in this study. Table S4: Crystal lattice parameters of the two crystal forms observed. Abbreviations used: PBP, Penicillin Binding Protein; WHO, World Health Organization; R&D, research and development; DBO, diazabicyclooctane; SAR, structure-activity relationship; His6, polyhistidine-tag; TP domain, transpeptidase domain; MIC, minimal inhibitory concentration; SRF, spontaneous resistance frequency; cLogD, calculated distribution coefficient at pH 7.4; IMAC, immobilized metal affinity chromatography; IEX, ion exchange chromatography. Homology models coordinates in PDB format: 2Shigella-sp-HomologyModel.pdb,


Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3Salmonella-bongori-HomologyModel.pdb, 4Enterobacter-sp-HomologyModel.pdb, 5Haemophilus-influenzae-HomologyModel.pdb, 6Pseudomonas-aeruginosa-HomologyModel.pdb, 7Acinetobacter-baumannii-HomologyModel.pdb, 8Campylobacter-sp-HomologyModel.pdb. Author contributions: ELR and DB generated the plasmid constructs used in this study. JMB performed protein expression, proteolysis assays and sample purification. FLS, SC, AC, FC, JB and BL designed and conducted compound synthesis. DB performed PBP inhibition experiments. ELR and DB conducted MIC experiments. NL performed thermal shift experiments, crystallization assays, data collection and structure resolution. NL and MR performed structure analysis. MR performed molecular modelling and docking experiments. NL, JMB, MR and FM designed the study. NL, JMB, SC, FLS, ELR, DB, FM and MR drafted the manuscript. All authors read and approved the final manuscript. PDB ID Codes: 6G9S: E. coli PBP2 from mild proteolysis + CPD4 (ET5, C10 H14 N4 O7 S), (3~{R},6~{S})6-(AMINOMETHYL)-4-(1,3-OXAZOL-5-YL)-3-(SULFOOXYAMINO)-3,6-DIHYDRO2~{H}-PYRIDINE-1-ET5 CARBOXYLIC ACID 6G9P: E. coli Apo PBP2 from mild proteolysis 6G9F: E. coli PBP2 [57-615] + Avibactam (NXL, C7 H13 N3 O6 S), (2S,5R)-1-FORMYL [(SULFOOXY) AMINO]PIPERIDINE-2-CARBOXAMIDE Authors will release the atomic coordinates and experimental data upon article publication.



Page 30 of 35

Molecular Formula Strings: The molecular formula strings of the compounds described in this manuscript are joined in the following file: SmilesMolecularFormulaStrings.csv Corresponding Author Information: Marc Ruff, Institut de Génétique, de Biologie Moléculaire et Cellulaire, Integrated Structural Biology Department, 1 rue Laurent Fries, 67404 Illkirch, France. Email: [email protected] Acknowledgments The authors thank Stéphanie Floquet, Elodie Drocourt and Juliette Nguyen for assistance in biochemistry and microbiology as well as Sophie Vomscheid, Christophe Simon and Sébastien Richard in chemistry. They thank Alastair McEwen, Pierre Poussin and all the members of the IGBMC Structural Biology and Genomics platform and the members of the IGBMC common services for their contribution. The authors wish to thank Robert Drillien (IGBMC) for suggestions about the manuscript. The authors thank Vincent Olieric and the staff of the Swiss Light Source synchrotron for help with data collection. The authors acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC. The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115583, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in-kind contribution. The ENABLE project is also financially supported by contributions from Academic and SME partners.


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Conflict of interests: NL, JMB, ELR, DB, FLS, AC, JB, FC, SC, BL and FM declare that they are full time employees of Mutabilis. MR declares that he has no competing interests.

References (1) Carlet, J.; Rambaud, C.; Pulcini, C. Save antibiotics: a call for action of the World Alliance Against Antibiotic Resistance (WAAAR). BMC Infect Dis 2014, 14, 436. (2) O'Neill. Antimicrobial resistance: tackling a crisis for the health and wealth of nations. AMR-Review [online] 2014, page 4. https://amrreview.org/sites/default/files/AMR%20Review%20Paper%20%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations_ 1.pdf (accessed Jun 15, 2018) (3) Access-to-Medicine-Foundation. Antimicrobial Resistance Benchmark 2018: Methodology. Naritaweg, CB Amsterdam, The Netherlands. 2018, 1-41. (4) WHO. List of Bacteria for which New Antibiotics are Urgently Needed. http://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-whichnew-antibiotics-are-urgently-needed. (accessed June 10, 2018). (5) Den Blaauwen, T.; de Pedro, M. A.; Nguyen-Disteche, M.; Ayala, J. A. Morphogenesis of rod-shaped sacculi. FEMS Microbiol Rev 2008, 32, 321-344. (6) Ghuysen, J. M. Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol 1991, 45, 37-67. (7) Den Blaauwen, T.; Aarsman, M. E.; Vischer, N. O.; Nanninga, N. Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole. Mol Microbiol 2003, 47, 539-547. (8) Davies, T. A.; Page, M. G.; Shang, W.; Andrew, T.; Kania, M.; Bush, K. Binding of Ceftobiprole and comparators to the penicillin-binding proteins of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Antimicrob Agents Chemother 2007, 51, 2621-2624. (9) Davies, T. A.; Shang, W.; Bush, K.; Flamm, R. K. Affinity of doripenem and comparators to penicillin-binding proteins in Escherichia coli and Pseudomonas aeruginosa. Antimicrob Agents Chemother 2008, 52, 1510-1512. (10) Lei, Y.; Li, J. T. Affinity of penicillin-binding proteins of Escherichia coli K-12 for Furbenicillin and other beta-lactam antibiotics. Zhongguo yao li xue bao = Acta pharmacologica Sinica 1989, 10, 177-180. (11) Spratt, B. G. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur J Biochem 1977, 72, 341-352. (12) Gutmann, L.; Vincent, S.; Billot-Klein, D.; Acar, J. F.; Mrena, E.; Williamson, R. Involvement of penicillin-binding protein 2 with other penicillin-binding proteins in lysis of Escherichia coli by some beta-lactam antibiotics alone and in synergistic lytic effect of amdinocillin (mecillinam). Antimicrob Agents Chemother 1986, 30, 906-912. (13) Asli, A.; Brouillette, E.; Krause, K. M.; Nichols, W. W.; Malouin, F. Distinctive binding of Avibactam to penicillin-binding proteins of Gram-negative and Gram-positive bacteria. Antimicrob Agents Chemother 2016, 60, 752-756. 31 ACS Paragon Plus Environment


Page 32 of 35

(14) King, A. M.; King, D. T.; French, S.; Brouillette, E.; Asli, A.; Alexander, J. A.; Vuckovic, M.; Maiti, S. N.; Parr, T. R., Jr.; Brown, E. D.; Malouin, F.; Strynadka, N. C.; Wright, G. D. Structural and kinetic characterization of diazabicyclooctanes as dual inhibitors of both serine-beta-lactamases and penicillin-binding proteins. ACS Chem Biol 2016, 11, 864868. (15) Moya, B.; Barcelo, I. M.; Bhagwat, S.; Patel, M.; Bou, G.; Papp-Wallace, K. M.; Bonomo, R. A.; Oliver, A. WCK 5107 (Zidebactam) and WCK 5153 are novel inhibitors of PBP2 showing potent "beta-lactam enhancer" activity against Pseudomonas aeruginosa, including multidrug-resistant metallo-beta-lactamase-producing high-risk clones. Antimicrob Agents Chemother [online] 2017, 61, https://aac.asm.org/content/61/6/e02529-16.long (accessed Jun 15, 2018). (16) Doumith, M.; Mushtaq, S.; Livermore, D. M.; Woodford, N. New insights into the regulatory pathways associated with the activation of the stringent response in bacterial resistance to the PBP2-targeted antibiotics, Mecillinam and OP0595/RG6080. J Antimicrob Chemother 2016, 71, 2810-2814. (17) Durand-Reville, T. F.; Guler, S.; Comita-Prevoir, J.; Chen, B.; Bifulco, N.; Huynh, H.; Lahiri, S.; Shapiro, A. B.; McLeod, S. M.; Carter, N. M.; Moussa, S. H.; Velez-Vega, C.; Olivier, N. B.; McLaughlin, R.; Gao, N.; Thresher, J.; Palmer, T.; Andrews, B.; Giacobbe, R. A.; Newman, J. V.; Ehmann, D. E.; de Jonge, B.; O'Donnell, J.; Mueller, J. P.; Tommasi, R. A.; Miller, A. A. ETX2514 is a broad-spectrum beta-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii. Nat Microbiol [online] 2017, 2, https://www.nature.com/articles/nmicrobiol2017104 (accessed Jun 15, 2018). (18) Contreras-Martel, C.; Martins, A.; Ecobichon, C.; Trindade, D. M.; Mattei, P. J.; Hicham, S.; Hardouin, P.; Ghachi, M. E.; Boneca, I. G.; Dessen, A. Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex. Nat Commun [online] 2017, 8, 776, https://www.nature.com/articles/s41467-017-00783-2 (accessed Jun 15, 2018). (19) Caravano, A.; Chasset, S.; Chevreuil, F.; Faivre, F.; Lecointe, N.; Ledoussal, B.; Le Strat, F.; Richard, S.; Simon, C.; Vomscheid, S.; Brias, J.; Barbion, J. Novel Heterocyclic Compounds and their Use in Preventing or Treating Bacterial Infections. WO/2016/156348A1, 2016. Mutabilis Intellectual Property. (20) McGuire, H.; Bist, S.; Bifulco, N.; Zhao, L.; Wu, Y.; Huynh, H.; Xiong, H.; ComitaPrevoir, J.; Dussault, D.; Geng, B.; Chen, B.; Durand-Reville, T. F.; Guler, S. Heterocyclic Compounds as Beta-Lactamase Inhibitors. WO/2013/150296A1, 2013. Astrazeneca Intellectual Property. (21) Banzhaf, M.; van den Berg van Saparoea, B.; Terrak, M.; Fraipont, C.; Egan, A.; Philippe, J.; Zapun, A.; Breukink, E.; Nguyen-Disteche, M.; den Blaauwen, T.; Vollmer, W. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Mol Microbiol 2012, 85, 179-194. (22) Legaree, B. A.; Clarke, A. J. Interaction of penicillin-binding protein 2 with soluble lytic transglycosylase B1 in Pseudomonas aeruginosa. J Bacteriol 2008, 190, 6922-6926. (23) Krissinel, E.; Henrick, K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 2007, 372, 774-797. (24) Bertsche, U.; Breukink, E.; Kast, T.; Vollmer, W. In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. J Biol Chem 2005, 280, 38096-38101. (25) Chalut, C.; Remy, M. H.; Masson, J. M. Disulfide bridges are not involved in penicillin-binding protein 1b dimerization in Escherichia coli. J Bacteriol 1999, 181, 29702972.


Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Karimova, G.; Dautin, N.; Ladant, D. Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J Bacteriol 2005, 187, 2233-2243. (27) Barbion, J.; Caravano, A.; Chasset, S.; Chevreuil, F.; Lecointe, N.; Ledoussal, B.; Le Strat, F.; Richard, S.; Simon, C.; Vomscheid, S.; Brias, J.; Briet, S.; Faivre, F.; Le Fralliec, G.; Oliveira, C. Heterocyclic Compounds and their Use in Preventing or Treating Bacterial Infections. WO/2016/177862A1, 2016. Mutabilis Intellectual Property. (28) Yoshimura, F.; Nikaido, H. Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrob Agents Chemother 1985, 27, 84-92. (29) Coleman, K. Diazabicyclooctanes (DBOs): a potent new class of non-beta-lactam betalactamase inhibitors. Curr Opin Microbiol 2011, 14, 550-555. (30) Lampilas, M.; Rowlands, D. A.; Kebsi, A.; Ledoussal, B.; Pierres, C. Nitrogenous Heterocyclic Compounds, Preparation Thereof and Use Thereof as Antibacterial Medicaments. WO/2008/142285, 2008. Novexel Intellectual Property. (31) Acosta-Gutierrez, S.; Ferrara, L.; Pathania, M.; Masi, M.; Wang, J.; Bodrenko, I.; Zahn, M.; Winterhalter, M.; Stavenger, R. A.; Pages, J. M.; Naismith, J. H.; van den Berg, B.; Page, M. G. P.; Ceccarelli, M. Getting drugs into Gram-negative bacteria: rational rules for permeation through general porins. ACS Infect Dis 2018, 4, 1487-1498. (32) Oleg, T.; J., O. A. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry 2010, 31, 455-461. (33) Thulin, E.; Sundqvist, M.; Andersson, D. I. Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli. Antimicrob Agents Chemother 2015, 59, 1718-1727. (34) Lo, M. C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.; Malamas, M.; Ellestad, G. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal Biochem 2004, 332, 153-159. (35) Shapiro, A. B. Investigation of beta-lactam antibacterial drugs, beta-lactamases, and penicillin-binding proteins with fluorescence polarization and anisotropy: a review. Methods and applications in fluorescence [online] 2016, 4, https://iopscience.iop.org/article/10.1088/2050-6120/4/2/024002/meta (accessed Jun 15, 2018). (36) Shapiro, A. B.; Gao, N.; Gu, R. F.; Thresher, J. Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants. Anal Biochem 2014, 463, 15-22. (37) Kabsch, W. Xds. Acta Crystallogr D Biol Crystallogr 2010, 66, 125-132. (38) Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr 2010, 66, 133-144. (39) Vagin, A.; Lebedev, A.; Iucr. MoRDa, an automatic molecular replacement pipeline. Acta Crystallographica Section A: Foundations and Advances 2015, 71, s19. (40) Terwilliger, T. C.; Read, R. J.; Adams, P. D.; Brunger, A. T.; Afonine, P. V.; GrosseKunstleve, R. W.; Hung, L. W. Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement. Acta Crystallogr D Biol Crystallogr 2012, 68, 861-870. (41) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010, 66, 213-221.



Page 34 of 35

(42) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 2010, 66, 486-501. (43) Terwilliger, T. C.; Grosse-Kunstleve, R. W.; Afonine, P. V.; Moriarty, N. W.; Zwart, P. H.; Hung, L.-W.; Read, R. J.; Adams, P. D. Iterative model building, structure refinement and density modification with the PHENIX autoBuild wizard. Acta Crystallographica Section D 2008, 64, 61-69. (44) Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallographica Section D 2012, 68, 352-367. (45) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. Journal of Applied Crystallography 2007, 40, 658-674. (46) Lebedev, A. A.; Young, P.; Isupov, M. N.; Moroz, O. V.; Vagin, A. A.; Murshudov, G. N. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallographica Section D 2012, 68, 431-440. (47) Hough, M. A.; Wilson, K. S. From crystal to structure with CCP4. Acta Crystallogr D Struct Biol 2018, 74, 67. (48) Liebschner, D.; Afonine, P. V.; Moriarty, N. W.; Poon, B. K.; Sobolev, O. V.; Terwilliger, T. C.; Adams, P. D. Polder maps: improving OMIT maps by excluding bulk solvent. Acta Crystallogr D Struct Biol 2017, 73, 148-157. (49) Gonzalez-Dominguez, J.; Liu, Y.; Tourino, J.; Schmidt, B. MSAProbs-MPI: parallel multiple sequence aligner for distributed-memory systems. Bioinformatics 2016, 32, 38263828. (50) Waterhouse, A. M.; Procter, J. B.; Martin, D. M.; Clamp, M.; Barton, G. J. Jalview version 2, a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009, 25, 1189-1191. (51) Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 2014, 42, W320-W324. (52) Sanner, M. F. Python: a programming language for software integration and development. J Mol Graph Model 1999, 17, 57-61. (53) Webb, B.; Sali, A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 2016, 54, 5.6.1-5.6.37. (54) Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.8. In 2015. https://pymol.org/2/ (55) Janson, G.; Zhang, C.; Prado, M. G.; Paiardini, A. PyMod 2.0: improvements in protein sequence-structure analysis and homology modeling within PyMOL. Bioinformatics 2017, 33, 444-446. (56) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera, a visualization system for exploratory research and analysis. J.Comput.Chem. 2004, 25, 1605-1612. (57) Laskowski, R. A.; Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 2011, 51, 2778-2786.


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For Table of Contents Only:


Structural basis for E. coli Penicillin Binding Protein (PBP) 2 inhibition

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