Application of Virtual Screening to the Identification of New LpxC

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Application of Virtual Screening to the Identification of New LpxC Inhibitor Chemotypes, Oxazolidinone and Isoxazoline Patrick S. Lee, Guillaume Lapointe, Ann Marie Madera, Robert Simmons, Wenjian Xu, Aregahegn Yifru, Meiliana Tjandra, Subramanian Karur, Alice Rico, Katherine Thompson, Jade Bojkovic, Lili Xie, Kyoko Uehara, Amy Liu, Wei Shu, Cornelia Bellamacina, David McKenney, Laura Morris, Colin S. Osborne, Bret Benton, Laura McDowell, Jiping Fu, and Zachary K. Sweeney J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01287 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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Journal of Medicinal Chemistry

Application of Virtual Screening to the Identification of New LpxC Inhibitor Chemotypes, Oxazolidinone and Isoxazoline

Patrick S. Lee*, Guillaume Lapointe, Ann Marie Madera, Robert L. Simmons, Wenjian Xu, Aregahegn Yifru, Meiliana Tjandra, Subramanian Karur, Alice Rico, Katherine Thompson, Jade Bojkovic, Lili Xie, Kyoko Uehara, Amy Liu, Wei Shu, Cornelia Bellamacina, David McKenney, Laura Morris, Colin Osborne, Bret M. Benton, Laura McDowell, Jiping Fu, Zachary K. Sweeney Novartis Institutes for Biomedical Research, 5300 Chiron way, Emeryville, CA 94608 Email: [email protected] KEYWORDS: LpxC, Gram-negative, lipopolysaccharide, LPS, Pseudomonas aeruginosa, inhibitor, virtual screening, scaffold hopping

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Abstract: This report summarizes the identification and synthesis of novel LpxC inhibitors aided by computational methods that leveraged numerous crystal structures. This effort led to the identification of oxazolidinone and isoxazoline inhibitors with potent in vitro activity against P. aeruginosa and other Gram-negative bacteria. Representative compound 13f demonstrated efficacy against P. aeruginosa in a mouse neutropenic thigh infection model. The antibacterial activity against K. pneumoniae could be potentiated by Gram-positive antibiotics rifampicin (RIF) and vancomycin (VAN) in both in vitro and in vivo models.


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INTRODUCTION Infections due to multidrug-resistant (MDR) Gram-negative bacteria have become an increasing problem for healthcare systems worldwide.1,2 Illness due to these resistant species has been associated with high mortality rates, and the need for new antibacterial medicines has been widely recognized.3 New antibiotics representing novel chemical scaffolds not subject to crossresistance with established classes are needed to combat infections due to resistant Gramnegative bacteria.4 The Gram-negative outer membrane provides a significant permeability barrier that protects the bacterium from many toxic molecules including antibiotics and host innate immune factors. Lipopolysaccharide (LPS) is the major structural component of the outer leaflet of the outer membrane that provides the permeability barrier for these organisms. Lipid A constitutes the hydrophobic anchor of LPS.5 Lipid A is essential for bacterial growth and virulence, and inhibition of its synthesis is lethal to Gram-negative bacteria.6,7 In addition, LPS is a potent toxin (endotoxin) and is responsible for many of the toxic side effects associated with Gram-negative infections.8 Chemically or genetically induced loss of LPS increases the susceptibility of Gramnegative bacteria to the host immune system, as well as to antibiotics that normally have limited efficacy against these organisms.9,10 The LPS biosynthetic pathway has been characterized in multiple Gram-negative species.11 In this process, a series of enzymes located on the cytoplasmic side of the inner cellular membrane convert UDP-GlcNAc into the lipid A component of LPS. The first committed step in Lipid A biosynthesis is catalyzed by LpxC, a zinc-dependent deacetylase that represents an attractive target for new inhibitor design (Scheme 1).12,13



Scheme 1. Lipid A biosynthesis pathway from UDP-GlcNAc for E. coli

Antibacterial LpxC inhibitors were originally discovered in whole-cell chemical screens conducted at Merck.14 Lead compound 1 (Chart 1) inhibited galactose uptake into E. coli, and optimized compounds in this series exhibited moderate minimum inhibitory concentrations (MICs) against both Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) isolates. Replacement of the oxazoline contained in 1 with a threonine motif eventually led to the discovery of CHIR-090 (2), a well characterized LpxC inhibitor with activity against a broad spectrum of Gram-negative bacteria.15,16 Since then, LpxC inhibitors with diverse scaffolds have been reported and selective examples are summarized in Chart 1.17–19 ACHN-975 (3), a compound developed by Achaogen, was the first LpxC inhibitor to enter clinical development and exhibited enzymatic and microbiological activity against E. coli, K. pneumoniae, and P. aeruginosa.20 We also have reported a novel hydroxamic acid LpxC inhibitor with antipseudomonal activity in vitro and in vivo (4).21 Pfizer disclosed several series of LpxC inhibitors that contain a sulfone moiety adjacent to the hydroxamic acid (i.e. 5).22–24 Actelion reported a series of indazole-containing compounds with good overall microbial activity (i.e. 6).28 Those compounds all share those features: 1) a hydroxamate head group 2) a central linker and 3) a lipophilic tail, which mimics the Zn binding natural substrate. Central heterocycle rings instead of the benzamide group as illustrated in 2, 3, and 4, proved to be critical for optimal physicochemical properties, including solubility and low protein binding. In particular, pyridone


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analogs exemplified by compound 5 have potent inhibitory activity against P. aeruginosa, while maintaining favorable physicochemical properties. With the variability observed in the central linkers represented in Chart 1, we embarked on an effort to explore alternatives in this particular region using computational methods.

Chart 1. Structure of selected LpxC inhibitors

Computational scaffold-hopping is one of many methods for lead identification.25,26 This approach employs multiple methodologies using either 2D chemical structural features, 3D conformation of the molecule, and/or the biological fingerprint of the compound.27 Scaffolds 5 ACS Paragon Plus Environment


proposed by these programs can be further filtered by virtual library enumeration, calculation of physicochemical properties, pharmacophore queries, and docking. In this contribution, we report the identification of novel LpxC inhibitors through computational scaffold hopping methods. This effort led to the preparation of oxazolidinone and isoxazoline inhibitors with excellent MIC against P. aeruginosa and other Gram-negative bacteria.

CHEMISTRY The synthesis of the representative oxazolidinone hydroxamic acid 13f is illustrated in Scheme 2. Formation of the benzyl carbamate of aniline 7 followed by nucleophilic addition to (R)-glycidyl butyrate gave the corresponding hydroxyl oxazolidinone 8. Conversion to iodide 9 and subsequent displacement with ethyl 2-(methylsulfonyl)propanoate yielded 11 as a mixture of diastereomers, which could be separated by silica gel column chromatography. Palladiumcatalyzed coupling between bromide 11 and cyclopropylpropiolic acid afforded ester 12. The final product 13f was obtained via a three step sequence involving saponification of ester 12, coupling of the acid intermediate with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, and acid deprotection.

Scheme 2. Synthesis of Oxazolidinone Hydroxamic Acids 13f


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Reagents and condition: (a) (i) Cbz-Cl, NaHCO3, acetone, water, 5 ˚C to rt, 3h, 90%; (ii) nBuLi, THF, −75 ˚C, 1h; (R)-glycidyl butyrate, −75 ˚C to rt, overnight, 66%; (b) I2, PPh3, Imidazole, rt, 8h, 53%; (c) NaH, DMF, 0 ˚C to rt, 2h, 15%; (d) 3-cyclopropylpropiolic acid, PdCl2(PPh3)2, dppb, DBU, DMSO, 90 ˚C, 4h, 68%; (e) LiOH, THF/MeOH/water, rt, 3h, 81%; (f) NH2OTHP, EDC·HCl, HOBT, TEA, DCM, rt, 18h, 80%; (g) aqueous HCl, EtOH, rt, 4h, 10%.

Hydroxamic acid 13j was obtained following the route depicted in scheme 3. 4(methoxyphenyl)methanamine 14 was converted to iodide 15, followed by nucleophilic displacement with ethyl 2-(methylsulfonyl)propanoate 10 under basic conditions to afford the (PMB)-protected oxazolidinone 16 as a separable mixture of diastereoisomers. Deprotection with ceric ammonium nitrate (CAN) revealed the free oxazolidinone 17. Copper-mediated coupling between dibromopyridine and oxazolidinone 17 afforded bromide 18, which was converted to ester 19 by Suzuki coupling with phenyl boronic acid. The final product 13j was obtained via a three step sequence involving saponification of ester 19, coupling of the acid intermediate with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, and acid deprotection.

Scheme 3. Synthesis of Oxazolidinone Hydroxamic Acid 13j



Reagents and condition: (a) (i) Cbz-Cl, NaOH, acetone, water, 0 ˚C to rt, 3h, 93%; (ii) nBuLi, THF, -70 ˚C, 1h; (R)-glycidyl butyrate, −70 ˚C to rt, 5h, 79%; (b) I2, PPh3, Imidazole, rt, 7h, 70%; (c) NaH, DMF, 0 ˚C to rt, 2h, 31%; (d) CAN, CH3CN:H2O (9:1), rt, 24h, 84%; (e) 2,5dibromopyridine, CuI, trans-cyclohexane-1,2-diamine, Cs2CO3, 1,4-dioxane, 125 ˚C, 4h, 66%; (f) Phenylboronic acid, PdCl2(dppf), Cs2CO3, 1,4-dioxane, 90 ˚C, 3h, 66%; (g) LiOH, THF/MeOH/water, rt, 30 minutes; (h) NH2OTHP, EDC.HCl, HOBT, NMM, rt, 24h, 70%; (i) HCl, EtOH, rt, 1h, 30%.

The synthesis of isoxazoline 25e (Scheme 4) began with the generation of 4-bromo-Nhydroxybenzimidoyl chloride 21, which underwent cycloaddition with enantiomerically pure alkene 22 to provide isoxazoline 23 as a separable mixture of diastereoisomers (1:1). Palladiumcatalyzed coupling of bromide 23 with ethynyl cyclopropane yielded ester intermediate 24, which was converted into hydroxamic acid 25e by the standard three step procedure.

Scheme 4. Synthesis of Isoxazoline Hydroxamic Acid 25e


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Reagents and condition: (a) NCS, DMF, 50 ˚C, 2h, 85%; (b) NaH, 0 ˚C, 1h; Allyl bromide, rt, 5h, 30%; (c) TEA, Diethyl ether, 0 ˚C to rt, 3h, 35%; (d) ethynyl cyclopropane, PdCl2(PPh3)2, CuI, PPh3, Et2NH, DMF, 100 ˚C, 4h, 83%; (e) LiOH, THF/MeOH/Water, rt, 2h, 90%; (f) NH2OTHP, EDC.HCl, HOBt, TEA, rt, 24h, 87%; (g) HCl, EtOH, rt, 2h, 94%.

Results and Discussion In the initial scaffold selection for computational queries, we selected a modified version of compound 6 due to the size of the central ring. We also simplified the tail region to serve as an anchor vector, as illustrated below in Figure 1a (internal crystal structure). We utilized Cresset’s SPARK module28 using the bioactive conformation of this simplified indazole 26 (Figure 1) to search for bioisosteres. The input filters were that “linker 1 atom” with any C, “atom 2” to be any atom and that the proposed molecules have at least 1 ring. The output was further refined to exclude compounds with reactive functionalities.



O

S

O 1

O NH HO

O 2

N N 26

(a)

(b) Figure 1. (a) LpxC tool molecule 26. The red portion is defined as the replacement portion. (b) Illustration of field for reference molecule

Our search was performed on “common” and “very common” databases as provided by Cresset which includes fragments that have been seen 100-500 times or more than 500 times, respectively, from a database which includes compounds from ZINC drug-like set,29 ChEMBL,30 and theoretical rings (VEHICLe).31 The total score was calculated using 0.5 of the score from shape similarity. Following the scoring, a manual selection of the top 100 clusters were made based on synthetic feasibility, introduction of hydrophilic groups in the area of the indazole moiety and calculated physicochemical properties. The field comparison of the query and representative hit molecules are illustrated in Figure 2.


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(a)

(b) Figure 2. (a) Field illustration with oxazolidinone scaffold (smooth spheres) and reference compound (icosahedrons). (b) Field illustration with isoxazoline scaffold (smooth spheres) and reference compound (icosahedrons).

The oxazolidinone scaffold 13 and the isoxazoline scaffold 25 were recognized among several proposals that effectively linked the methylene atom adjacent to atom 1 to the hydrophobic element 2 via a structurally well-defined hydrophilic linker. The field illustration of isoxazoline and oxazolidinone scaffolds overlaid with the input structure 26 is shown in Figure 2. The initial analogs selected for synthesis replaced the suggested methoxy group with a more hydrophobic bromine atom. This led to the preparation of 13a and 25a. As both analogs had 64

>64

1

13b

0.17

0.5

4

32

1.6

13c

0.12

2

32

>64

1.5

CHIR-090 (2)

-



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13d

0.1

1

4

32

1.4

13e

0.063

0.5

2

16

1.4

13f

0.065

0.5

0.125

1

1.9

13g

0.026

1

0.25

4

2.3

13h

0.2

1

0.5

1

1.6

13i

0.12

2

0.25

1

1.2

13j

0.12

2

2

32

1.8

a) Organisms used in this study: PA (P. aeruginosa ATCC 27853), EC (E. coli ATCC 25922), KP (K. pneumoniae ATCC 43816) b) Measured at pH 7.4.

Structure-activity relationships (SAR) at R1 for the isoxazoline series were similar to oxazolidinone scaffold (Table 2). Although the binding of isoxazoline analogs to P. aeruginosa enzyme is weaker compared to oxazolidones based on the Kd values, the MICs against ATCC strains are comparable. Compounds 25b, 25c and 25d with shorter tails were less effective at


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inhibiting the growth of K. pneumoniae isolate (25b, K. pneumoniae MIC = 16 µg/mL). As with the oxazolidinone scaffold (i.e. 13f, Table 1), cyclopropyl alkyne 25e strongly inhibited the growth in all three species (P. aeruginosa MIC = 1 µg/mL, K. pneumoniae MIC = 0.5 µg/mL and E. coli MIC = 0.125 µg/mL).

Table 2. Isoxazoline SAR

MIC (µg/mL)a

PA

logDb

R Kd (nM)

PA

EC

KP

0.48

2

0.125

0.5

25a

2.6

4

32

>64

1.2

25b

0.2

1

4

16

1.7

25c

0.3

1

2

16

2.0

25d

0.44

0.5

1

8

1.4

25e

0.17

1

0.125

0.5

2.2

CHIR-090 (2)

-

a) Organisms used in this study: PA (P. aeruginosa ATCC 27853), EC (E. coli ATCC 25922), KP (K. pneumoniae ATCC 43816) b) Measured at pH 7.4. 15 ACS Paragon Plus Environment


In order to gain insight into the binding mode of these inhibitors, the co-crystal structure of 13f complexed with the P. aeruginosa LpxC enzyme was obtained and refined to 1.8 angstrom resolution (Figure 3). The sulfone and methyl group are oriented as previously described for Pfizer inhibitor 5.22–24 The hydroxamic acid functionality is bound to the active site zinc atom and forms a network of interactions with H78, H237, T190, E77, and D241 that line this polar region. The sulfone oxygen atoms are located close to well-defined water molecules, and the methyl group attached to the sulfone functionality is engaged in hydrophobic interactions with F191. As suggested by the conformation observed in scaffold hopping virtual screen, the oxazolidinone group links the sulfone/hydroxamic acid portion of the inhibitor with the hydrophobic phenyl group while maintaining a low-energy conformation. Although the oxygen atom of the oxazolidinone group is near to the alpha-methyl group, the carbonyl oxygen of the oxazolidinone forms a favorable polar interaction in the largely hydrophobic binding pocket with the C2 CH group of His19. The phenyl group and the cyclopropyl alkyne functionality interact with many of the hydrophobic residues that line the lipophilic tunnel, and the cyclopropyl group extends to the solvent exposed region of the binding pocket.


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Figure 3. Cocrystal oxazolidinone 13f with P. aeruginosa LpxC enzyme (pdb code: 6mae).

The physicochemical properties and pharmacokinetics of compound 13f were collected and summarized in Table 3. With a measured logD of 1.9, 13f has plasma protein binding (PPB) less than 90% across different species. The solubility of 13f at pH 7.4 is 126 µg/mL and is significantly higher at pH 9.0. In mouse, rat and dog, 13f shows medium to high plasma clearance relative to hepatic blood flow following i.v. administration. The variation in half-life across species could be attributed to species specific clearance-mechanisms. In all three species, the Vss is low, which coupled with medium clearance resulted in short half-lives.

Table 3. Physicochemical properties and PK profiles of 13f. measured logD

1.9

PPB (%) mouse/rat/dog/human

87/89/86/86

solubility (mg/mL)

0.126

pH 7.4



mouse i.v. (60 mg/kg)

rat i.v. (5 mg/kg)

dog i.v. (10 mg/kg)

pH 9

>20

Cl (mL/min/kg)

49

Vss (L/kg)

1.6

t1/2 (h)

5.5

Cl (mL/min/kg)

24

Vss (L/kg)

1.0

t1/2 (h)

0.9

Cl (mL/min/kg)

26

Vss (L/kg)

0.8

t1/2 (h)

0.7

Next, the in vivo efficacy of 13f was evaluated in the mouse neutropenic thigh infection model. Mice were rendered neutropenic by administration of cyclophosphamide and infected with P. aeruginosa cells. Two hours post infection, treatment was started with 13f administered every 6 hours at a range of doses. The bacterial burden in thighs was evaluated at 24 hours post infection with the results shown in Figure 4. Bacterial stasis was achieved at dose of 105 mg/kg/day. Doses of 160 mg/kg/day and higher reduced bacterial levels to the limit of detection.


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Figure 4. Efficacy of 13f in the mouse neutropenic thigh infection model. Mice were infected with P. aeruginosa and treated every 6 hours with 13f at doses ranging from 0-640 mg/kg/day. 0 hour control is bacterial levels in thighs at the start of therapy, LOD = limit of detection.

One of the proposed attributes of LpxC inhibition is that depletion of LPS should render cells more vulnerable to innate immune factors as well as enhance permeability to antibacterial agents.10 A recent publication exemplified this hypothesis by showing that LpxC inhibitors, used either alone or in combination with vancomycin, were efficacious in vivo against Acinetobacter baumannii (A. baumannii) that were insensitive to growth inhibition in vitro.34,35 In order to assess the potential of our LpxC inhibitors in a similar manner, we measured the MIC of selected oxazolidinone and isoxazoline inhibitors in combination with the Gram-positive antibiotics rifampicin (RIF) and vancomycin (VAN). RIF had modest activity against the K. pneumoniae strain used in our studies (MIC, 32 µg/mL), therefore a sub-lethal concentration of 2 µg/mL (1/16th MIC) was chosen for these studies. Likewise, VAN had no detectable activity against the 19 ACS Paragon Plus Environment


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K. pneumoniae strain used in our studies (MIC, >128 µg/mL), so a sub-lethal concentration of 4 µg/ml (≤1/64th MIC) was selected. As shown in Table 4, the observed MIC of several LpxC inhibitors against K. pneumoniae was significantly reduced when assessed in combination with sub-lethal concentrations of RIF or VAN. The range of potentiation range from 1 for 13h with VAN (MIC = 1 µg/mL, MIC + VAN = 1 µg/mL) to 64 fold for 13e when combined with RIF (MIC = 16 µg/mL, MIC + RIF = 0.25 µg/mL). These data suggest that for certain compounds, the concentration of LpxC inhibitor required to increase outer membrane permeability and thus enhanced susceptibility to RIF and VAN, is lower than the concentration required to completely inhibit growth via the LpxC inhibitor alone.

Table 4. MIC of antibiotic combinations MIC (µg/mL) alone and in combination with RIF Compound

Potentiation factora

and VAN against K. pneumoniae Alone

+RIF

+VAN

+RIF

+VAN

13b

32

1

8

32

4

13e

16

0.25

4

64

4

13f

1

0.06

0.25

16

4

13h

1

0.5

1

2

1

25d

8

0.25

2

32

4

25e

0.5

0.03

0.125

16

4

RIF and VAN were included in the MIC assay at fixed concentrations of 2 and 4 µg/mL, respectively a

Fold reduction in MIC in the presence of RIF or VAN


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To investigate if these combinations could improve in vivo activity, the efficacy of 13f in combination with VAN was evaluated in a mouse lung infection model. Briefly, immunocompetent mice were infected intranasally with a K. pneumoniae strain. Eighteen hours post-infection, treatment was initiated with mice dosed every 6 hours with vehicle, VAN (200 mg/kg/dose), 13f (20 mg/kg/dose) or VAN and 13f combined. Animals were euthanized 48 h after the first treatment, the lungs were harvested, and the bacterial load in the lung determined. As can be seen in Figure 5, VAN alone was not efficacious, while 13f alone gave a decrease of approximately 4.5-log10. An additional 2.5-log10 drop was observed when the combination of 13f and VAN was tested. This experiment corroborates similar published results,34 and suggests that for clinically important bacterial species the efficacy of LpxC inhibitor might be improved by coadministration with other antibiotics.



Figure 5. Efficacy of 13f and VAN in a mouse lung infection model. Mice were infected with K. pneumoniae and treated with VAN and 13f alone and in conbination. Results are mean ± standard deviation, n=5 mice per group). ** p

Application of Virtual Screening to the Identification of New LpxC

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