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Challenges in the Development of a Thiol-Based Broad-Spectrum Inhibitor for Metallo-#-Lactamases Dominik Büttner, Jan S Kramer, Franca Maria Klingler, Sandra K. Wittmann, Markus Hartmann, Christian Kurz, Daniel Kohnhäuser, Lilia Weizel, Astrid Brüggerhoff, Denia Frank, Dieter Steinhilber, Thomas A Wichelhaus, Denys Pogoryelov, and Ewgenij Proschak ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00129 • Publication Date (Web): 26 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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ACS Infectious Diseases

Challenges in the Development of a Thiol-Based Broad-Spectrum Inhibitor for Metallo-β-Lactamases

Dominik Büttner†, Jan S. Kramer†, Franca-M. Klingler†, Sandra K. Wittmann†, Markus R. Hartmann†, Christian G. Kurz†, Daniel Kohnhäuser†, Lilia Weizel†, Astrid Brüggerhoff†, Denia Frank‡, Dieter Steinhilber†, Thomas A. Wichelhaus‡, Denys Pogoryelov*§, Ewgenij Proschak*† †

Institute of Pharmaceutical Chemistry, and §Institute of Biochemistry, Goethe University

Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany; Email: [email protected] or [email protected] ‡

Institute of Medical Microbiology and Infection Control, Goethe University Hospital, Paul-

Ehrlich-Straße 40, 60596 Frankfurt, Germany

Abstract: Pathogens, expressing metallo-β-lactamases (MBLs), become resistant against most β-lactam antibiotics. Besides the dragging search for new antibiotics, development of MBL inhibitors would be an alternative weapon against resistant bacterial pathogens. Inhibition of resistance enzymes could restore the antibacterial activity of β-lactams. Various approaches to MBL inhibitors are described, among others, the promising motif of a zinc coordinating thiol moiety is very popular. Nevertheless, since the first report of a thiol-based MBL-Inhibitor (thiomandelic acid) in 2001, no steps in development of thiol based MBL inhibitors were reported that go beyond clinical isolate testing. In this study we report on the synthesis and biochemical characterization of thiol-based MBL inhibitors and highlight the challenges behind the development of thiol-based compounds, which

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exhibits good in vitro activity towards a broad spectrum of MBLs, selectivity against human off-targets, and reasonable activity against clinical isolates.

Keywords: Bacterial resistance, metallo-β-lactamases, metallo-β-lactamase inhibitor, synthesis, thiol-based, biochemical characterization

Today, it is usual to treat a bacterial infection with β-lactam antibiotics, which are the most used antibacterial drugs.1 However, the efficacy of this drug class is restricted more and more by the increasing spread of bacterial resistance worldwide.2 β-lactamases are the most common way of bacterial resistance against β-lactam antibiotics. These enzymes hydrolyze the β-lactam ring which is crucial for antibacterial activity and release the inactive hydrolyzed product.3 β-Lactamase expressing, Gram-negative bacteria among the ESKAPE4 pathogens become resistant to most classes of antibiotics. Thus, nosocomial infections caused by these pathogens become very poorly treatable.5 β-Lactamases can be categorized in two groups based on their catalytic mechanism: the serine-β-lactamases (SBLs) which cleave the β-lactam ring by a nucleophilic attack of the serine residue6 and the metallo-β-lactamases (MBLs) that contain one or two zinc ions inside the active center.7 MBLs belong to class B, which is further divided in three subclasses (B1, B2 and B3).8 According to the proposed catalytic mechanism, MBLs coordinate the β-lactam scaffold inside the active center, and hydrolyze it by a nucleophilic water molecule, which is polarized by the zinc ions. In contrast to SBLs, there is no covalent interaction between enzyme and ligand. Thus, MBLs work much faster than SBLs, and more like chemical metal-catalysts.9,10 Since we are confronted with a drying pipeline of new antibiotics, it would be useful to have an alternative defense against multi-resistant bugs. One approach is the development ACS Paragon Plus Environment

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of β-lactamase inhibitors, which have usually no intrinsic bactericidal effect, but could restore the activity of β-lactam antibiotics by inhibiting β-lactamases. Whereas numerous SBL inhibitors are already in clinical use, this does not apply to inhibitors of MBLs.11 Literature provides various approaches, and an even higher number of chemical scaffolds for the design of MBL inhibitors. These can be categorized by their mode of inhibition. Many of them contain dicarboxylate structures (1), which are coordinating the zinc ions.12 Cyclic boronates (2) represent an elegant transition state mimetic approach.13 Multivalent chelators like EDTA (3) remove the zinc ions from the active site of MBLs.14,15 Some MBLs can be inhibited by a covalent adduct to Lys211 which is generated in situ during the hydrolysis of cefaclor (4)16 (Figure 1).

Figure 1. Examples for known MBL inhibitors: A maleic acid derivate (1), a cyclic boronate (2), EDTA (3) and cefaclor(4).

Thiol containing compounds were found as an interesting scaffold for the inhibition of MBLs; as thiomandelic acid (5) was discovered as a broad-spectrum inhibitor for MBLs. Cd-NMR studies demonstrated that the sulfur atom of the compound intercalates the metal



ions within the active site and displaces the bridging water molecule that is essential for the MBL hydrolase activity.17,18 This binding mode was confirmed by crystal structures of Bacillus cereus MBL (BcII) and 519 and also of VIM-2 (Verona integrin-encoded metalloβ-lactamase) and a mercaptocarboxylate inhibitor (6).20 Skagseth et al. published investigations concerning opportunities to introduce bioisosteric groups to mercaptocarboxylic acid derived compounds. In summary, replacement of the thiol group by a hydroxyl function was not tolerated by any MBL. Substitution of the carboxylic acid by phosphonic acid or phosphonate was both accepted in principal, but only the combination of phosphonate and unprotected thiol (7) led to sub-micromolar inhibitory activity.21 A different approach to thiol-based MBL inhibitors was followed by González et al who characterized bisthiazolidines as a novel scaffold with enhanced activity on all three subclasses of MBLs.22–24 Due to their bicyclic structure and the tetrahedral oriented tertiary nitrogen, bisthiazolidines, represented by L-CS319 (8), are substrate-mimicking MBL inhibitors (Figure 2).

Figure 2. Chemical structures of thiomandelic acid (5), 2-mercapto-5-phenylpentanoic acid (6), diethyl (1-mercapto-5-phenylpentan-2-yl)phosphonate (7) and the bisthiazolidine L-CS319 (8).


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Besides the search for new chemical structures with inhibitory activity against MBLs, already approved drugs were investigated as MBL inhibitors. First of all, Captopril (9) was found as potent inhibitor for New Delhi metallo-β-lactamase (NDM-1, 7.9 µM),25 and successfully co-crystalized with NDM-1 (PDB: 4EXS). Based on this crystal structure, the binding mode of 9 and NDM-1 could be determined. Similar to the binding mode of 5, the sulfur atom of 9 intercalates between the zinc ions and additionally, the acid group interacts with Asn220.26 Recent investigations demonstrated that several approved drugs, like Tiopronin (10) and Thiorphan (11), which contain thiols, are able to inhibit MBLs in a lower micromolar range (Figure 3). Furthermore, the co-crystallization of NDM-1 and 10, a drug for the treatment of heavy metal poisoning, indicates that the described binding mode for 9 is true for other thiol containing structures.27

Figure 3. Thiol containing drugs with inhibitory activity in MBLs: Captopril (9), Tiopronin (10) and Thiorphan (11).

The general validity of the binding mode of 9 on other B1 MBLs was proved by further crystal structures, including VIM-2 (Verona integrin-encoded metallo-β-lactamase, PDB: 4C1D), and IMP-1 (Imipenem hydrolyzing metallo-β-lactamase, PDB: 4C1F). Attention must be paid to the fact that the residue, which interacts with the carboxylic group, differs, depending on the respective MBL and on the stereochemistry of the inhibitors carboxylic group.28 ACS Paragon Plus Environment


The structure of 9 seemed to be a promising chemical landmark for the further development of MBL inhibitors. Li et al. synthesized a library of simplified Captopril analogues, where they substituted the pyrrolidine ring by various amide-coupled residues, and tested them on their inhibitory potency against NDM-1. Li et al. could demonstrate that the carboxylic acid, located at the pyrrolidine ring of 9, is highly important for activity. Removal of the carboxylic acid goes along with a dramatically decreased potency, right up to complete loss of activity.29 Lienard et al. synthesized thiol derivatives of diverse amino acids. Compound 12 displayed good Ki values on distinct members of each MBL-subclass,30 what was supplemented by inhibitory activities within a bacterial imipenem-induced growth inhibition assay for the structurally related compound 13.31 Latest research yielded in compound 14 which combined tryptophan and a (S)-3-mercapto-2-methylpropanyl moiety of Captopril and showed good IC50 values on IMP-1 and several VIM mutants32 (Figure 4).

Figure 4. Chemical structures of 12, 13 and 14.

Taken together, there are strong reasons in favor of development of thiol-based MBL inhibitors and a large number of potent compounds was described in literature. However, the aim to develop a clinically useful thiol-based MBL inhibitor seems to be far away. Although most of the presented inhibitors showed good in vitro activity, they often lacked in broad-spectrum activity, or were tested on only a minor number of MBLs. Furthermore, ACS Paragon Plus Environment

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promising inhibitors failed or were not evaluated against clinically relevant isolates. Finally, most of the inhibitors were more or less structurally related to approved drugs and therefore addressing human enzymes as well. Nevertheless, almost none of the showed compounds were investigated in the context of the relevant human targets. The aim of this study is to perform a broad investigation of a series of thiol-based MBL inhibitors, the latter mentioned factors, and to highlight opportunities how to handle them. Therefore, we designed a series of thiol containing inhibitors, based on the chemical structure of 9. We modified both, the pyrrolidine ring, including the position of the acidic group, and the methyl group. Interactions, between inhibitors and MBLs (NDM-1, VIM1/2 and IMP-7) were investigated by several biochemical and biophysical methods. Furthermore, we examined the logD7.4 values and activity in clinical isolates as well as off-target activity of selected compounds in order to investigate the challenges and opportunities in development of a thiol-based MBL inhibitor.

RESULTS AND DISCUSSION The synthetic procedure to substitute the methyl group by different aromatic residues is outlined in Scheme 1. Frost et al. described an elegant method to prepare acrylic esters, starting from an aldehyde.33 Therefore, benzaldehyde derivatives 15b and 15c or 2thenaldehyde (15d) reacted with Meldrum´s acid (16) in an acid-catalyzed, Knoevenagellike reaction at 75 °C for 2 h. The condensation product (17b-d) was reduced towards 18b-d using in situ generated NaHB(OAc)3 in ice bath cooled DCM. The corresponding acrylic ester derivatives 19b-d was obtained by the reaction of 18b-d with Eschenmoser´s salt (20) in methanol at 70 °C for 18 h. Ester hydrolysis of 19b-d was performed using aqueous NaOH and heating to 100 °C for 1 h under microwave irradiation. The resulting acrylic acid (21b-d) was activated through ACS Paragon Plus Environment


by thionyl chloride in methylene chloride at 40 °C for 3 h. The following amide coupling reaction with 22 and DIPEA was carried out in 1,2-dichloro ethane at 90 °C for 1 h under microwave irradiation. The obtained acrylic amides (23b-d) reacted with thioacetic acid in a Michael addition at room temperature for three days to yield 24b-d. Synthesis of 25a was accomplished as shown in Scheme 2. Final ester hydrolysis were carried out with TFA in methylene chloride at 40 °C overnight and subsequently treated with aqueous ammonia for 3 h at room temperature to obtain 25a-d.

Scheme 1: Synthesis route for derivatives 25a-d.a

a

Reaction conditions: (a) 15b-d, AcOH, iPrOH, 75 °C, 2 h; (b) NaBH4, AcOH, DCM, 0 °C, 1 h, r.t., 1 h; (c) 20, MeOH, 70 °C, 18 h; (d) NaOH, H2O, MW, 100 °C, 1 h; (e) SOCl2, 40 °C, 3 h and 22, DIPEA, 1,2-DCM, MW, 90 °C, 1 h; (f) AcSH, r.t., 3 d; (g): TFA, DCM, 40 °C, o.n. and NH3 (aq), r.t., 3 h.


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The synthesis routes for the 2-benzyl-3-mercaptopropionic acid derivatives are shown in Scheme 2. Amino acids (26a,b,d-f) were suspended in tert-butyl acetate and treated with perchloric acid to obtain the corresponding tert-butyl esters (27a,b,d-f). The carboxylic acid

group

of

the

commercial

available

starting

compound

3-(acetylthio)-2-

benzylpropanoic acid (28) was activated by thionyl chloride in dichloromethane at 40 °C. The corresponding acid chloride (29) was directly subjected to amide coupling with the protected amines (27a,b,d-f) in ACN (or ACN/DMF) using potassium carbonate (or DIPEA) as base at room temperature overnight. The resulting amides (30a,b,d-f) were heated with TFA in methylene chloride overnight and subsequently treated with aqueous ammonia for 3 h at room temperature to obtain the final 1-(2-benzyl-3mercaptopropanoyl)amino acid derivatives (31a,b,d-f). For unprotected amino acids (32c,g-i), an alternate amide coupling reaction (33c,g-i) was performed in a mixture of ACN and DMF (or water/DMF) using DIPEA (or NaHCO3) as base at room temperature overnight. The final cleavage of the thioester was carried out in aqueous ammonia (31c,gi).



Scheme 2: Synthesis routes for introduction of different amino acids, starting from 28.a

a

Compounds 31a,b,d-f and 25a were synthesized via route A, and 31c,g-i via route B. Reaction conditions (h1/h2) TBAC, HClO4, 0 °C, r.t., o.n.; (i) SOCl2, DMF, DCM, 40 °C, 3 h; (j1) K2CO3, ACN, r.t., o.n.; (j2) DIPEA, ACN/DMF, r.t., o.n.; (k1) TFA, DCM, 40 °C, o.n. and NH3 (aq), r.t., 3 h; (k2) HCl (37 %) reflux, 3 h; (k3) NH3 (aq), r.t., 3 h; (l1) DIPEA, ACN/DMF, r.t., o.n. (l2) NaHCO3, H2O/ACN, r.t., o.n.

The preparation of 4-N-substituted piperazine-2-carboxylic acid building blocks is shown in Scheme 3. 1-N-boc-piperazine-2-carboxylic acid (34) served as a starting material for both derivatives 35a and 35b. Benzoic acid was pre-stirred with EDC and HOBt in DCM for 1 h and subsequently reacted with 34 at room temperature for 72 h. The crude intermediate was treated with trifluoroacetic acid in DCM at 40 °C for 3 h to yield 35a. Phenyl isocyanate and 34 were mixed in dioxane and kept at 110 °C for 30 minutes under microwave irradiation to obtain the tertiary urea 36. The benzyl ester 37 was introduced by the reaction of 36 with benzyl bromide and K2CO3 in ACN at room temperature for


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8 h. The tert-butyloxycarbonyl protecting group was hydrolyzed with TFA in DCM at 40 °C for 3 h to receive the free amine 35b. The respective amide coupling reactions (36a,b) were performed as described above (Scheme 2). To obtain 37b, as distinct from the remaining derivatives, final ester cleavage was carried out with aqueous LiOH at room temperature overnight. (For further synthetic details see Supporting Information)

Scheme 3: Synthesis of 35a and 35b.a O

O

NH Boc

n

N

N Boc

O

N

o

N H

N Boc

N

N H

OH O

34

OH

O

36

37

O m

O

N HN O

N HN

OH

35a

OBn

O

N H

p

OBn

35b

a

Reaction conditions: (m) benzoic acid, EDC, HOBt, DCM, r.t., 1 h and 34, r.t., 72 h; (n) PhCNO, dioxan, MW, 110 °C, 0.5 h; (o) K2CO,3, BnBr, ACN, r.t., 8 h; (p) TFA, DCM, 40 °C, 3 h. 35a was further reacted according route B and 35b according route A (Scheme 2).

The MBL inhibitory potential of the synthesized compounds was investigated in a kinetic fluorescence-based activity assay using Fluorocillin™ as substrate. The IC50 values of D/L-Captopril (9) and compounds 31a-d are listed in Table 1. Captopril showed similar activities towards all tested MBLs with slight preference towards IMP-7. In comparison, the introduction of the unsubstituted phenyl ring (25a) led to comparable values on NDM-1 and IMP-7, but resulted in a slight loss of activity on VIM-1. Additional substituents on the phenyl ring led to further decrease in activity. While the 2-



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methoxyphenol derivative (25c) was not well tolerated by all three MBLs, the brominated compound 25b showed only slightly decreased IC50 values. On the contrary, introduction of the thiophene ring (25d) led to a gap in activity on NDM-1 and IMP-7, but in comparison to the derivatives containing a phenyl ring, an increased activity on VIM-1.

Table 1. In vitro activity of compounds 9 and 25a-da

IC50 [µM]

R1

NDM-1

VIM-1

IMP-7

H

6.4 ± 2.50

6.8 ± 1.20

2.9 ± 0.90

25a

3.2 ± 0.29

16.3 ± 2.63

4.6 ± 0.17

25b

6.2 ± 0.25

16.1 ± 4.2

7.7 ± 1.65

25c

25.2 ± 4.66

23.1 ± 2.37

30.4 ± 5.43

25d

17.0 ± 1.09

6.8 ± 0.74

8.7 ± 1.73

9

a

Results are mean ± SD of three different experiments.

The IC50 values of derivatives 31a-i, containing different amino acids, are shown in Table 2. All substitution patterns were well tolerated on NDM-1 and showed a trend towards improved activity in comparison with Captopril. Compound 31d was characterized by its sub-micromolar activity, especially in comparison with its enantiomer 31e. The S-


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configured alanine (31a), the thiazolidine ring (31h) and likewise the four membered ring (31c) were not well tolerated by VIM-1. The sarcosine substructure (31b) and all piperidine derivatives (31d-g, i) showed slightly increased activities. On IMP-7, the piperidine motive displayed the best inhibitory activities, independent from position and absolute configuration of the carboxylate. Compound 31i, containing a sulfone group, which is bioisosteric to carboxylic acids, showed reasonable activity on NDM-1 and VIM1, but not on IMP-7.

Table 2. In vitro activity of compounds 31a-ia

IC50 [µM]

R2

NDM-1

VIM-1

IMP-7

31a

1.7 ± 0.15

12.1 ± 1.48

6.1 ± 0.90

31b

2.6 ± 0.71

4.9 ± 0.70

4.7 ± 0.79

31c

2.2 ± 0.48

n.d.b

7.4 ± 1.06

0.7 ± 0.05

4.6 ± 0.85

0.9 ± 0.06

4.5 ± 0.95

4.9 ± 0.62

1.1 ± 0.06

31d

N

O

31e

OH



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31f

1.6 ± 0.25

1.4 ± 0.16

2.2 ± 0.03

31g

1.8 ± 0.51

2.6 ± 0.32

1.6 ± 0.09

31h

6.7 ± 0.57

16.2 ± 2.81

2.9 ± 0.27

31i

2.4 ± 0.51

2.9 ± 0.27

7.9 ± 0.48

a

Results are mean ± SD of three different experiments; bnot determinable.

Both piperazine derivatives 37a and 37b together with their IC50 values are presented in Table 3. While the activities of 37a, what carries a benzoyl group in position 4, were comparable with that of 31d and 31e on NDM-1 and VIM-1, the tertiary urea moiety of 37b was not tolerated on NDM-1. However, both compounds provided a further improved activity on IMP-7 and were well tolerated by VIM-1.

Table 3. In vitro activity of compounds 37a and 37ba

IC50 [µM]

R3

NDM-1

VIM-1


IMP-7

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37a

2.5 ± 0.33

4.5 ± 0.69

0.3 ± 0.01

37b

47.7 ± 9.48

7.0 ± 0.78

0.3 ± 0.25

a

Results are mean ± SD of three different experiments.

In summary, derivatives containing the piperidine substructure provided the best overall activity on all three tested MBLs. On NDM-1, the absolute configuration of the carboxylic acid seemed to be of special importance regarding the inhibitory potency, as 31d provided a six-time better activity compared to 31e. On VIM-1 and IMP-7, this effect was not found. Molecular docking analysis of 31d in complex with NDM-1 reveals the possible reason for this behavior – an ionic interaction of the carboxylate moiety towards Lys211 which is unique for NDM-1 (see Supporting Information, Figure S2). The introduction of additional substituents to the piperazine ring improved the inhibition of IMP-7. According to the activities of the r and s configured 31d and 31e on NDM-1, it could be worthwhile to prepare the piperazine ring with an absolute orientation of the acid group. Likewise, a shift of the acid group towards positon 3 could be a promising approach for further studies. Compounds 9, 25a, 31d and 37b were further investigated by differential scanning fluorimetry (DSF) (Table 4). Neither of the synthesized compounds was able to stabilize VIM-1 in a significant manner, except 9, which showed a weak stabilization. On IMP-7, the shifts of melting points correlated well with the corresponding IC50 values. As expected, the highest stabilization was found for the most active compound 37b. In contrast, the thermal shifts on NDM-1 did not correlate with the determined IC50 values. The enzyme was stabilized most by 37b, which exhibited the weakest potency in the ACS Paragon Plus Environment


activity assay; in contrast to this 31d, which had 68-fold better inhibition values on NDM1 compared to 37b, thermal shift was determined as half as strong.

Table 4. Thermal shifts (in ∆ °C)a NDM-1 b

VIM-1 b

IMP-7 b

9

1.9 ± 0.6

1.5 ± 0.5

2.5 ± 0.2

25a

0.9 ± 0.5

0.2 ± 0.4

1.8 ± 0.2

31d

1.3 ± 0.2

0.7 ± 0.6

2.7 ± 0.3

37b

2.6 ± 0.3

0.7 ± 0.6

6.0 ± 0.2

a

Results are mean ± SD of three different b

experiments. Melting points without inhibitor [in °C]: 56.3 ± 0.1 (NDM-1), 66.1 ± 0.6 (VIM-1) and 72.5 ± 0.3 (IMP-7). Results are mean ± SD of three different experiments.

The outcomes from DSF experiments, concerning the stabilization of NDM-1 by 31d and 37b, required a more closely look at this enzyme-ligand interactions by isothermal titration calorimetry (ITC) experiments (Figure 5 and Supporting Information, Figure S1). 9, 31d and 37b exhibit comparable Kd values in low micromolar range (Table 5). However, while 9 displays a fully enthalpically driven binding, the binding event between NDM-1 and 31d was mainly entropically driven.34 This could have been an explanation for the lower thermal shift of NDM-1 caused by 31d. On the other hand, binding of 37b was found as distinctly more enthalpically driven, which led to an increased thermal shift. Although 37b provided a poorer IC50 value towards NDM-1 in the activity assay, the 4benzoyl piperazine substructure seemed to offer additional interaction points towards the ACS Paragon Plus Environment

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enzyme, what could than again lead, with the right substitution pattern, to an increased activity.

Figure 5. Titration curves of ITC experiments. A: NDM-1: 130 µM, 31d: 700 µM, 10.98 µL/titration step; B: NDM-1: 100 µM, 37b: 1000 µM, 10.98 µL/titration step.



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Table 5. Dissociation constant, binding enthalpies and entropies of 9, 31d and 37b at 25°C Kd [µM]

∆G [kJ/mol]

∆H [kJ/mol]

-T∆S [kJ/mol]

n

9

17.51

-27.15

-40.16

13.01

1.21

31d

7.84

-29.14

-4.86

-24.28

0.85

37b

13.16

-27.78

-14.79

-12.99

0.89

The direct binding of 31d to MBLs was confirmed by co-crystallization and X-ray structure determination of VIM-2 from Pseudomonas aeruginosa in complex with 31d. To avoid bias in model interpretation and ligand identification, the structure of VIM-2 at 1.5 Å resolution was obtained ab initio, by single wavelength anomalous dispersion (SAD) using zinc atoms as anomalous scatterers. The obtained crystals in the space group

C2 contained two VIM-2 molecules in the asymmetric unit (Supporting Information). In the initial mFo-DFc electon density map, obtained after SAD phasing and automatic model building, the pronounced bulky positive electon densities were identified in the vicinity of dizinc-containing active sites of both VIM-2 molecules of the asymmetric unit. The properties of the obtained 2mFo-DFc (Figure 6a) maps allowed unambiguous placement of 31d molecule in the unique orientation in the active sites of both VIM-2 molecules. The correctness of the ligand placement in the final model was further corroborated by the polder omit map (Figure 6a). 35 In its binding pose the thiol moiety of

31d intercalates directly between zinc atoms of the VIM-2 active. We found that ACS Paragon Plus Environment

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orientation of 31d in the active site is very similar to that one of the structurally related inhibitor L-captopril in the relevant structure of VIM-2 (PDB ID:4C1E

28

). As shown in

Figure 6b, similarly to 31d, thiol moiety of L-captopril is coordinated between the both zinc cations inside the active center of VIM-2 while replacing

the polarized water

molecule required for catalysis. Furthermore, 31d and L-captopril displays similarities of coordination network with the protein backbone. Thus, the carbonyl oxygens of both amide groups formed a hydrogen bonds with Asn210, the nonpolar parts of the heterocycles fit well into the lipophilic subpocket of the active site, and the acid groups directed towards the polar surface of the active site (Figure 6b). The additional phenyl ring of 31b pointed in the direction of the solvent, apparently, due to the observed weak electron density around this ring (Figure 6a), orients itself in a flexible manner, without any steady interactions towards the enzyme.

Figure 6. A: Ligand interactions in the X-ray structure of VIM-2 active site in complex with 31d. The positive 2mFo-DFc difference map (blue mesh contoured at 1σ) and mFo-DFc polder omit map (green mesh) with solvent exclusion radius of 3Å and contoured at 3σ around the 31d ligand were calculated from the final model (PDB: 5O7N). 31d molecule. Ligand and neighboring protein environment are shown as stick models. The dashed lines



indicate the interactions of 31d with both Zinc ions (light blue spheres) and the key residues of the active site. B: Superposition of the bound ligands 31d (cyan) and D-Captopril (orange, PDB: 4C1E) within VIM-2 active site.

For 25a, 31d, and 37b the inhibitory potential on VIM-2 was evaluated (table 6). Studies concerning activity of Captopril on VIM-2 were published elsewhere.28 In brief, activity depended on the exact stereochemistry, whereby only one of the four possible stereoisomers offered a nanomolar IC50. The other three showed activities in a low micromolar range, comparable to VIM-1. In the case of 25a, IC50 values were found only as slightly lower than those on VIM-1. For 31d and 37b the difference in the obtained results was more obvious, as activity of 31d was increased by factor 15 and for 37b even by factor 35. All tested compounds provided a significantly higher stabilization of VIM-2 (Table 6). These variabilities between the single subtypes could be an additional difficulty in the development of an “pan” MBL inhibitor.

Table 6. IC50 values and thermal shifts on VIM-2 a IC50 [µM]b

∆ °C b

9

0.07 / 4.4c

6.3 ± 0.2d

25a

13.7 ± 8.30

2.4 ± 0.1

31d

0.3 ± 0.09

6.3 ± 0.2

37b

0.2 ± 0.12

10.1 ± 0.2

a

Melting point without inhibitor [in °C]: 61.9 ± 0.1.

b

Results are mean ± SD of three different

experiments. cD-/L-Captopril, values from the literature28; drac-Captopril


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Next, influence on cell viability of a human cell line was evaluated for 9, 25a, 31d, and 37b. In a LDH release assay, compounds 9, 25a and 31d showed only slight in vitro toxicity to HeLa cells at a concentration of 100 µM (Figure 7a). Cytotoxicity of 37b was found as marginally higher. In a WST-1 assay, compounds 9, 25a and 37b had only little influence on cell proliferation at 100 µM. A more intensified anti-proliferative effect was found for 31d (Figure 7b).

Figure 7. Cytotoxicity data of 9, 25a, 31d and 37b. A: LDH release assay; B: WST-1 assay.

The angiotensin-converting enzyme (ACE) is an important player within the regulation of blood pressure, and its inhibition leads to hypotension. In the case of an acute sepsis, which goes along with an uncontrolled hypotension, a further blood pressure lowering induced by application of an ACE inhibiting drug could have fatal consequences. Since all synthesized compounds were derived from the molecular structure of Captopril, an ACS Paragon Plus Environment


approved ACE inhibitor, 9, 25a, 31d and 37a were analyzed concerning their inhibitory activity on ACE (Figure 8a). As expected, Captopril and 25a showed full inhibition, even at 10 µM. For compound 31d, which differed in ring size of the heterocycle, a slightly decreased inhibition was measured (10 µM: 85.8 ± 2.1 %, 30 µM: 95.0 ± 4.1). For 37a, which contains the enlarged piperazine substructure, no inhibition could be detected at concentrations up to 30 µM. Thus, the piperazine derivatives might serve best as a scaffold for further investigations. Orning et al. had demonstrated, that Captopril is also an inhibitor for LTA4H,36 which is part of the biosynthesis of pro-inflammatory mediator LTB4. In these studies a percentage inhibition of 73.9 ± 4.5 % (10 µM) and 86.3 ± 3.1 % (30 µM) was found for 9 (Figure 8b). Only minor inhibitory potentials were determined for the further evolved derivatives 25a (10 µM: 12.4 ± 7.2 %, 30 µM: 17.6 ± 8.5), 31d (10 µM: 0.3 ± 2.8 %, 30 µM: 8.8 ± 8.0) and 37a (10 µM: 12.1 ± 9.3 %, 30 µM: 17.0 ± 9.4).


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Figure 8. Off-target activity of 9, 25a, 31d and 37a. A: percentage inhibition of ACE; B: percentage inhibition of LTA4H.

Compounds 25a and 31d were evaluated for their potential to inhibit bacterial MBLs inside bacteria and their ability to restore the antibacterial activity of imipenem within an antimicrobial susceptibility testing. Results are presented in table 7. In summary, according to its IC50 values, 25a was less active in most E. coli strains, compared with 9, which showed the best activity values. The increased activity of 31d in comparison to 25a was reflected in the MIC experiments, nevertheless, without reaching the activity of 9. In the case of clinical isolates, neither of the compounds, except 9 on an NDM-1 carrying K. pneumoniae T2301 strain, was able to restore activity of imipenem in a significant manner.

Table 7. MBL inhibitor (9, 25a, 31d) induced reduction of imipenem MIC in resistant clinical isolates. Imipenem MIC in mg/Lb (fold change5 in MIC)

Isolatesa

+ 9,c,d

+ 25a c,d

+ 31d c,d

E. coli TOP10 T2359 (NDM-1)

128

4 (32)

32 (4)

16 (8)

E. coli TOP10 T2360 (IMP-7)

2

0.5 (4)

1 (2)

1 (2)

E. coli TOP10 T2361 (VIM-1)

1

0.5 (2)

0.5 (2)

1 (1)

E. coli pET24a T2377 (NDM-1)

32

1 (32)

2 (16)

2 (16)

E. coli pET24a T2378 (IMP-7)

4

0.5 (8)

1 (4)

0.5 (8)

E. coli pET24a T2379 (VIM-1)

64

2 (32)

32 (2)

2 (32)

K. pneumoniae T2301 (NDM-1)

16

4 (4)

16 (1)

8 (2)

P. aeruginosa T2226 (IMP-7)

64

32 (2)

32 (2)

32 (2)



K. pneumoniae T2216 (VIM-1) a

8

4 (2)

Page 24 of 44

8 (1)

4 (2)

Transformants with respective MBL in TOPO-vector, pET24a-vector and clinical isolates; b 9, 25a and

31d in combination with imipenem was tested at a constant concentration of 128 mg/L; c 9, 25a and 31d did not exhibit any intrinsic antibacterial activity at the given concentration; d Fold change was calculated by MIC imipenem / MIC imipenem + inhibitor. Significant fold change (≥ 4) is indicated in bold.

The results of MIC testing were surprising at the first glance but could be explained by the capability of compounds to penetrate the outer membrane of Gram-negative pathogens. Brown et al. could demonstrate that antibacterial agents should have low logD7.4 values to enable the permeation through the outer membrane of Gram-negative pathogens. In the case of E. coli, a logD7.4 of

Challenges in the Development of a Thiol-Based ... - ACS Publications

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