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Discovery of Potent Benzocycloalkane Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Safety Profile for the Treatment of MRSA, VISA and LRSA Infection Baoli Li, Shuaishuai Ni, Feifei Chen, Fei Mao, Hanwen Wei, Yifu Liu, Jin Zhu, Lefu Lan, and Jian Li ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00259 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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ACS Infectious Diseases

Discovery of Potent Benzocycloalkane Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Safety Profile for the Treatment of MRSA, VISA and LRSA Infection Baoli Lia, †, Shuaishuai Nia, †, Feifei Chenb, Fei Maoa, Hanwen Weia, Yifu Liua, Jin Zhua, Lefu Lanb,*, Jian Lia,* a

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, 130 Meilong Road, Xuhui District, Shanghai, 200237, China b

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 646 Songtao Road, Pudong District, Shanghai, 201203, China †

These authors contributed equally to this work.

* To whom correspondence should be addressed: [email protected] or [email protected].

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Blocking the biosynthesis process of Staphyloxanthin has emerged as a promising anti-virulence strategy. Our previous research revealed that diapophytoene desaturase was an attractive and druggable target against infections caused by pigmented Staphylococcus aureus. Benzocycloalkane-derived compounds were effective inhibitors of diapophytoene desaturase, but limited by high hERG inhibition activity. Here, we identified a new type of benzo-hepta-containing cycloalkane derivatives as diapophytoene desaturase inhibitors. Among the fifty-eight analogues, 48 (hERG inhibition activity, IC50 = 16.1 µM) and 51 (hERG inhibition activity, IC50 > 40 µM) distinguished out for effectively inhibiting the pigment production of Staphylococcus aureus Newman and three methicillin-resistant Staphylococcus aureus strains, and enormously sensitize the four strains to hydrogen peroxide killing without bactericidal growth effect. In vivo assay, 48 and 51 displayed comparable effect with linezolid and vancomycin in livers and hearts in mice against Staphylococcus aureus Newman, and more considerable effect against Mu50 and NRS271 with normal administration. KEYWORDS:

Staphylococcus

aureus,

diapophytoene

methicillin-resistant Staphylococcus aureus, hERG safety


desaturase,

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Staphylococcus aureus (S. aureus) remains a dangerous pathogen, causing a multitude of serious and life-threatening diseases worldwide. Of growing concern, antimicrobial resistance incurred by the abuse of antibiotics,1-4 further complicates the treatment of S. aureus infections. Methicillin-resistant Staphylococcus aureus (MRSA) strains particularly represent a major problem for public health systems, as they combine resistance to methicillin and other antibiotics, resulting in high infiltration of hospital and community settings.5-9 Remarkably, in the United State, the estimated number of deaths exceeds due to MRSA infections along with HIV/AIDS.10 Antimicrobial Resistance Global Report released by World Health Organization (WHO) proved that it was a global vigilance to tackle the superbug and rekindle a drive for new, effective and safe drugs. On the basis of the previous research, staphyloxanthin (STX),11 a golden carotenoid pigment produced by S. aureus, acting as an efficient virulence factor,12-14 could enable S. aureus to evade innate immune clearance via numerous alternating single and double bonds,15 which can neutralize reactive oxygen species (ROS) from host neutrophils,16-17 and improve the lifespan of bacteria. Favorable evidence18 reveals that the essence of STX’s antioxidant properties is that unpigmented microbes are more susceptible to host killing. Therefore, blocking the STX biosynthetic pathway is an effective therapeutic strategy to compete S. aureus infections,19-24 and a new avenue to target the biosynthesis of STX in order to prevent S. aureus or even MRSA infections.25



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A previous study by our group revealed that naftifine, an FDA-approved antifungal agent, was capable of blocking the STX biosynthesis pathway of three MRSA strains by inhibiting CrtN.26 Based on the structure of naftifine, a series of new benzofuran derivatives27 and benzocycloalkane derivatives28 were synthesized, among which many compounds showed remarkable pigment inhibitory activities than naftifine. In particular, benzocycloalkane derivative 1 (Figure 1, 8[28]) displayed excellent bioactivity in vitro and in vivo, and performed obviously inhibition against STX (Pigment inhibition: IC50 = 4.1 nM). However, low water solubility, high hERG (human Ether-a-go-go Related Gene) inhibition and administration at high dosage in vivo limited further development of 1. 2-39 A

40-42 B OMe

OMe

N

OMe

N .HCl

Scaffold Hopping

N .HCl

O 1 first-generation CrtN inhibitor 1 pigment inhibition: IC50 = 0.38-5.45 nM CrtN: IC50=0.3 μM good in vivo effects (pretreatment) hERG inhibition: IC50=3.2 μM

C 43-48 second-generation CrtN inhibitor

O O

2

good in vivo effects (normal treatment) hERG inhibition: IC50 > 10 μM

O A

pigment inhibition: IC50 = 5.5±0.1 nM

OMe N 49-59 O

Figure 1. Scaffold Hopping (1-2) and three chemical modification regions (A-C) of lead compound 1.

Herein, in this text, our work focused on the design, synthesis and evaluation of CrtN inhibitors with new scaffolds, which not only demonstrated excellent activity against MRSA in vitro and in vivo at lower dosages, but also overcame the


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disadvantages of water solubility and hERG inhibition. Additionally, linezolid (LZD) and vancomycin (VAN), two widely used antimicrobial agents, were introduced as positive-controlled drugs into a murine model of S. aureus abscess formation to evaluate the effectiveness of our new compounds. First, we synthesized 2 and found its comparable activity with lead compound 1, then we designed another 46 analogues (3-48) on the premise of employing minimum structural changes. Then we chose the substituents of 3-39 of which pigment inhibition IC50 values below 10 nM and synthesized 49-59 with single oxygen atom (Figure 1). These analogues not only demonstrated excellent activity against MRSA in vitro and in vivo at lower dosages, but also overcame the disadvantages of hERG inhibition. RESULTS AND DISCUSSION Chemical modifications were performed in three cycles. First, we incorporated different substituted phenyls, thiophene, naphthalenyls, (cyclo)alkyls in region A (Figure 1), and 37 analogues (3-39) were designed (Table 1). Next, we replaced the N-methyl group in region B (Figure 1) with various steric alkyl group (including hydrogen atom) and three analogues (40-42) were designed (Table 2). Then, six analogues (43-48) were prepared to estimate if the type of linkers (allyl in region C, Figure 1) would affect pigment inhibitory activity. The details of the synthetic procedures and structural characterization are described in the Supporting Information. All of the derivatives were confirmed to have ≥ 95% purity (Table S1, Supporting Information),

and

were

identified

with

non-PAINS


on

the

web

at


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http://fafdrugs3.mti.univparis-diderot.fr/ recommended by editors from the ACS (American Chemical Society).29 To further obtain the structural scaffolds, as shown in Table 4, compound 49-59 were designed based on substituent groups of 3-39, of which the IC50 values of pigment inhibition were below 10 nM. In total, 58 novel analogues (2-59) of 1 were designed and synthesized. The chemical structures are shown in Tables 1-4. These analogues were synthesized through the routes outlined in Schemes S1-6 (Supporting Information).

In vitro pigment inhibitory activities of analogues 2-39 For the primary assay, the new analogues were assessed for pigment inhibitory activities against S. aureus Newman. The results are summarized in Tables 1-4. As seen in Table 1, substitution of phenyl could significantly affect pigment inhibition. Generally,

(cyclo)alkyl

or

heteroaromatic

groups

were

not

beneficial

(3-6).Introduction of a large naphthalene-2-yl group (8), increased the inhibition activity. Electron-withdrawing groups and electron-donating groups at the phenyl ring indistinguishably affected the activity. However, substitution positions at the phenyl ring substantially affected the inhibition activity; substitution at the para position of the phenyl ring showed improved activity (11 vs 23 vs 30 and 12 vs 25 vs 32). Remarkably, we found analog 15 displayed the most promising pigment inhibition (IC50 = 1.9±0.2 nM), inheriting the activity of 1 (IC50 = 4.1 nM). Then we fixed R1as 4-trifluoromethylphenyl for further development.


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Subsequently, we varied the groups on nitrogen atom, as seen with analogues 40-42, to assess whether the inhibition activity was enhanced or not. Evidently, once the N-methyl group was removed (40) or converted into ethyl and isopropyl groups (41-42), pigment inhibition lessened (Table 2). Analyzing the data in Table 3, we found that allyl linker moiety was pivotal to regulating pigment inhibitory activity. When the allyl linker was replaced with vinylogue (44, IC50 = 1.5±0.1 nM) or propargyl (46, IC50 = 5.4 ±0.1 nM) or trienes(48, IC50 = 2.3±0.0), the pigment inhibitory activities remained. Elimination of double bonds (47) led to the loss of pigment inhibitory activities (IC50 > 1000 nM). Moreover, introduction of branched methyl (43) or additional methylene (45) resulted in the decrease of pigment inhibitory activities (IC50 > 1000 nM). As showed in Table 4, we chose the substituent groups of 3-39 with values of IC50 (for pigment inhibition in S. aureus Newman) less than 10 nM and synthesized 49-59, and remarkably, they all showed similar and excellent activity (IC50 = 2.1-10 nM, Table 4). Table 1. Chemical structures of 3-39 and their pigment inhibitory activities against S. aureus Newman.

S. aureus Compd.

R1

S. aureus Compd.

Newman


R1 Newman


IC50 (nM)a

Page 8 of 35

IC50 (nM)a

3

>1000

22

4.0±0.6

4

52.7±7.0

23

4.5±0.5

5

91.8±0.3

24

>1000

6

21.8±1.1

25

29.6±0.4

7

>1000

26

100.3±7.5

8

3.5±0.0

27

68.1±3.7

9

5.5±0.1

28

>1000

10

2.0±0.2

29

32.6±1.3

11

4.5±0.8

30

25.6±0.4

12

2.0±0.4

31

29.1±3.1

13

3.4±0.8

32

>1000


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a

14

10.8±1.9

33

85.6±4.9

15

1.9±0.2

34

73.7±3.3

16

16.5±1.2

35

6.4±0.6

17

34.6±6.4

36

3.4±0.0

18

9.3±0.4

37

3.3±0.2

19

3.1±0.4

38

10.8±1.1

20

11.5±6.5

39

3.6±0.2

21

4.2±0.0

The IC50 values were tested with two independent experiments and reported as the

average ± S.D. Table 2. Chemical structures of 40-42 and their pigment inhibitory activities against S. aureus Newman.



Page 10 of 35

S. aureus Newman Compd.

R

3

IC50 (nM)a

a

40

H

> 1000

41

ethyl

107.9 ± 37.0

42

isopropyl

> 1000


average ± S.D. Table 3. Chemical structures of 43-48 and their pigment inhibitory activities against S. aureus Newman.

S. aureus Newman Compd.

Linker (X) IC50 (nM)a

a

43

> 1000

44

1.5 ± 0.1

45

> 1000

46

5.4 ± 0.1

47

> 1000

48

2.3 ± 0.03


average ± S.D.


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Table 4. Chemical structures of 49-59 and their pigment inhibitory activities against S. aureus Newman.

S. aureus Compd.

R1

Newman

S. aureus Compd.

IC50 (nM)a

a

R1

Newman IC50 (nM)a

49

6.0±0.1

55

4.2±0.1

50

2.3±0.1

56

2.1±0.1

51

2.7±0.1

57

9.2±0.2

52

3.2±0.3

58

3.0±0.1

53

2.5±0.3

59

10.0±0.1

54

2.4±0.0


average ± S.D.



SAR of 1-derived benzo-hepta-containing cycloalkane analogues SAR analysis of a set of 1-derived compounds provided important insights of the essential structural requirements for effective pigment inhibition. Analysis of the data shown in Tables 1-4 reveals some noteworthy observations of the SAR for compounds 2-59: (1) in region A (Figure 1), in the studied set of the R1 group (Table 1), the potency is increased in the order phenyl > naphthalenyl > thiophene > (cyclo)alkyl. The para position is the best substituted position at the phenyl ring (12 vs 25 vs 32, Table 1), and while the electronic effect of the substituent is limited, the replacement of phenyl with various types of groups could remarkably affect the pigment inhibitory activities; (2) in region B (Figure 1), the fitting bulky N-substituents are critical for high potency (40-42, Table 2), too small (40) or too big (41-42) are not beneficial, leading to the loss of activity; (3) in region C (Figure 1), among different linkages, the potency increase in the order vinylogue > trienes > propargyl > 2-methyl allyl = propenyl = propyl; (4) when we reduced oxygen atoms by one in benzocycloalkane (Figure 1), there was no obvious difference. Taken together, the SARs are unambiguous. Encouragingly, ten analogues (10, 12, 15, 44, 48, 50, 51, 53, 54 and 56) with IC50 (for pigment inhibition in S. aureus Newman) less than 3 nM could be kept for further development. Water solubility and hERG inhibition activity of the potential analogues The ten potential analogues (10, 12, 15, 44, 48, 50, 51, 53, 54 and 56) were tested for water solubility. Adding the oxygen atom (one or two) to the alkane ring was favorable, leading to the increase of water solubility. Especially, the water solubility


Page 12 of 35

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of analog 12 (13 mg/mL) was 3 folds better than that of lead compound 1 (Table 5). Then we investigated hERG inhibition activity (IC50) of the ten analogues. As shown in Table 5, it could be clearly found that the values of IC50 increase with the length of the linker (15 vs 44 vs 48). Remarkably, the values of IC50 of 51 (one oxygen atom in the alkane ring) was more than 40.0 µM, which could be much safer than the drug needed. Considering our top priority was to decrease hERG inhibition and keep the structural diversity, 48 and 51 were chosen as candidate compounds for further investigation in vitro and in vivo. Table 5. Water solubility and hERG inhibition (IC50) of 1 and its ten representative analogues. Solubility

hERG inhibition

Compd.

Solubility

hERG inhibition

(mg/mL)

activity IC50 (µM)

Compd. (mg/mL)

activity IC50 (µM)

1

4.2

3.2

50

3.3

6.4

10

8.0

3.7

51

2.8

>40.0

12

13.0

1.9

53

2.6

7.9

15

9.6

2.0

54

0.2

9.4

44

0.5

15.5

56

3.5

24.5

48

4.2

16.1

In vitro CrtN enzymatic inhibitory activity and MRSA pigment inhibitory activity of 48 and 51



Page 14 of 35

We evaluated 48 and 51 for their ability to inhibit the enzymatic activity of CrtN in vitro. Using our previous protocol,19 both compounds were found to significantly inhibit the enzymatic activity of CrtN at nanomolar concentrations (Table 6). USA400 MW2

and

USA300

LAC30-31,

two

clones

liable

for

the

epidemic

of

community-acquired MRSA (CA-MRSA) infectious diseases in the United States, Mu50, a hospital-acquired MRSA (HA-MRSA) strain with VAN-intermediate resistance (VISA/MRSA), along with NRS271, a LZD resistance (LRSA/MRSA)32-33, the four MRSA strains were taken to investigate inhibition activity of 48 and 51. The results of pigment inhibition were shown in Table 6. We were delighted to find that the color of all strains (USA400 MW2, USA300 LAC, Mu50 and NRS271) faded by the inhibition of the two compounds to a large extent. The pigment inhibitory activities against MRSA were comparable with that against S. aureus Newman. Moreover, 48 and 51 did not affect the growth of S. aureus strains at 0.2 mM and differed from antibiotics (Figure 2). Table 6. Enzyme (CrtN IC50), and IC50 values of 48 and 51 in the pigment formation of S. aureus USA400 MW2, USA300 LAC, and Mu50. CrtN

USA400 MW2 USA300 LAC

Mu50

NRS271

Compd.

a

IC50 (µM)a

IC50 (nM)a

IC50 (nM)a

IC50 (nM)a IC50 (nM)a

48

0.39±0.02

7.6±0.2

4.4±0.2

1.0±0.1

8.1±0.3

51

0.43±0.05

3.4±0.2

5.5±0.2

0.7±0.1

10.3±0.6

The IC50 values were tested with two independent experiments and and reported as

the average ± S.D.


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Figure 2. The growth curve and effects of 48 and 51 on the bacterial growth of S. aureus Newman (A and E), USA400 MW2 (B and F), USA300 LAC (C and G), and Mu50 (D and H). Data are presented as means ± SEM, n = 2 independent experiments.

Effects of 48 and 51 on sensitizing S. aureus to immune clearance We explored whether the inhibition of pigment of all strains translated to susceptibility of the four colonies to innate immune clearance by hydrogen peroxide killing and human whole blood killing. As shown in Figure 3, after incubation with either 48 or 51 (1 µM), the resulting white S. aureus Newman were more susceptible to be killed by 1.5% H2O2, compared with the untreated S. aureus (mock) (survival, 1.2% vs 26.7%), declining survival percentage by a factor of ~30. The survival percentage of USA400 WM2, USA300 LAC, and Mu50 reduced by a factor of ~10 (2.7% vs 26.3%), ~15 (2.3% vs 29.7%), and ~10 (2.0% vs 23.3%), respectively. In parallel, the survival of the known antioxidant nacetylcysteine (NAC)-treated S. aureus cells was higher than the mock treatment as expected (57.3% vs 26.7%),



declining survival percentage by a factor of ~2. Similarly, while the survival rates of the three NAC-treated MRSA strains were inclined to worsen (56.0% vs 26.3% in USA400 MW2, 52.7% vs 29.7% in USA300 LAC, 53.3% vs 23.3% in Mu50). All results exhibited that the addition of H2O2 (with strong oxidation) exerted an impact on the MRSA strains’ survival, and the pigment definitely acted as the protective antioxidant. We then repeated the same experiment for 51, and the analysis was identical to that of 48 (Figure 3E-H).

Figure 3. Effects of 48 and 51 on the susceptibility to hydrogen peroxide killing. S. aureus Newman (A and E), USA400 MW2 (B and F), USA300 LAC (C and G), and Mu50 (D and H), **p < 0.01***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).

Next, fresh human blood was introduced for further simulating authentic immune clearance. Dealt with the same concentration of 48 in hydrogen peroxide


Page 16 of 35

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killing, the nonpigmented S. aureus were more susceptible to be killed in freshly isolated human whole blood, causing Newman strain survival percentage to decline by a factor of ~20 (2.1% vs 37.5%). The survival percentage of USA400 WM2, USA300 LAC, and Mu50 fell down by a factor of ~8 (2% vs 18.7%), ~80 (0.8% vs 66.7%), and ~20 (1.2% vs 26.7%), respectively. We then repeated the same experiment for 51, and the analysis was identical to that of 48. The above results suggested that 48 and 51 indeed made S. aureus more susceptible to immune clearance in human blood.

Figure 4. Effect of 48 and 51 on the susceptibility to human whole blood killing. S. aureus Newman (A and E), USA400 MW2 (B and F), USA300 LAC (C and G), and



Page 18 of 35

Mu50 (D and H), *p< 0.05, **p< 0.01, ***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).

In vitro metabolic stability of 48 and 51 in mouse liver microsome Mouse liver microsome assay was utilized to preliminarily evaluate the effect of the stability of compounds in liver. As shown in Table 7, 48 and 51 showed acceptable stability (t1/2 = 3.28 mins of 48 and t1/2 = 7.45 mins of 51). Table 7. Mouse microsome stability of 48 and 51. Compd.

k

t1/2 (min)

48

0.211

3.28

51

0.093

7.45

In vitro anti-fungal activity of 48 and 51. Because leading compound 1 originated from the anti-fungal agent naftifine, we would like to investigate whether our compound reserved the capacity of antifungal. As shown in Table 8, three fungus strains and three first-line drugs were chosen to proceed with the in vitro assay. Compared with the three positive control groups, 48 and 51 exhibited weak activities against all three dermatophytes, which verified our compounds losing their antifungal activities. Table 8. Antifungal activities of 48 and 51. Antifungal activities, MIC80 (µg /mL)

Compd.

Trichophyton rubrum

Microsporum gypseum

Tinea barbae

Ketoconazole

0.5

2

0.0625


Page 19 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


Voriconazole

0.03125

0.25

0.03125

Fluconazole

1

8

2

48

32

64

>64

51

16

32

32

Cytotoxicity of human embryonic kidney cell (HEK-293T) and hepatocellular carcinoma cell (HepG-2) 48 and 51 were tested for cytotoxicity evaluation in HEK-293T and HepG-2. As shown in Table 9, in comparison with amphotericin B, 48 and 51 displayed far less cytotoxicity (48: CC50 = 13.7 µM for HEK-293T, CC50 = 23.1 µM for HepG-2; 51: CC50 = 32.9 µM for HEK-293T, CC50 = 39.5 µM for HepG-2; amphotericin B: CC50 = 8.1 µM for HEK-293T, CC50 = 7.8 µM for HepG-2). Table 9. Cytotoxicities of 48 and 51 on human embryonic kidney cell (HEK-293T) and hepatocellular carcinoma cell (HepG-2). Compd.

HEK-293T, CC50 (µM)

HepG-2, CC50 (µM)

Amphotericin B

8.1

7.8

48

13.7

23.1

51

32.9

39.5

In vivo effects of 48 and 51 on attenuating the virulence of S. aureus Newman Next, we extended our assessment on the contribution of STX to abscess formation in a systematic S. aureus Newman infection model. 9 administrations (every 12 h)



totally for the pretreatment groups (including VAN- and LZD-treatment groups) and 7 administrations (every 12 h) totally for the normal groups were given. Before infection with 2.3 × 107 colony-forming units (CFU) S. aureus Newman bacteria via retro-orbital injection, 2 administrations were given to mice (24 h and 12 h before, day 0), which were the pretreatment. Then, 6 h after infection (day 1), the third administration for pretreatment groups and the first for normal groups were given. After 6 administrations (day 2-4) and a 12 h interval, the mice were sacrificed (day 5), and we measured bacterial survival in host organs. As shown in Figure 5A, in general, each case showed a significant reduction (*P < 0.1 and more than 85 % descend in surviving bacteria) compared to non-treatment group (6.6 log10 CFU) in livers. In addition, 48 with the low-dosage case (5 mg/kg/dose) and 51 with the high-dosage case (20 mg/kg/dose) in normal treatment exhibited the same protective effect as the two positive cases with the high-dosage (20 mg/kg/dose) in pretreatment (5.0 log10 CFU by 48 vs 4.9 log10 CFU by 51 vs 5.6 log10 CFU by VAN vs 5.5 log10 CFU by LZD). The analysis of Figure 5B was just like that of Figure 5A, but the most potential group of 48 and 51 was not comparable to VAN.


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Figure 5. Effect of 48 and 51 treatment on S. aureus Newman bacteria survival in the livers (A) and hearts (B) of mice (n = 10) challenged with 2.3×107 CFU S. aureus Newman bacteria. P = pretreatment, drugs or compounds were intraperitoneally administered 24 h before infection compared with the normal treatment (drugs or compounds were intraperitoneally administered 6 h after infection). Statistical significance determined by Mann-Whitney Test (two-tailed): *p < 0.05, **p < 0.01, ***p < 0.001. Each symbol represents the value for an individual mouse. Horizontal bars indicate observation means and dashed lines mark limits of detection.

In vivo effects of derivatives 48 and 51 on attenuating the virulence of Mu50. To determine whether the effect of derivatives 48 and 51 could also be reflected in multi-drug resistant MRSA strains in vivo, a VAN-intermediate S.aureus (Table S3, Supporting Information), Mu50 infection model was established. The data in Figure 6A revealed that derivative-treatment groups significantly decreased the Mu50 staphylococcal loads in livers. All of them corresponded to over 95.0 % decrease in surviving bacteria (significance **P < 0.01). The best group of 48 (20 mg/kg/dose, in normal treatment) exceeded VAN (significance ***P < 0.001), and was comparable to LZD (4.6 log10 CFU by 48 vs 5.3 log10 CFU by VAN vs 4.5 log10 CFU by LZD), while 51-treatment groups were not comparable to those of 48. As for Figure 6B, all the derivative-treatment groups still significantly decreased Mu50 staphylococcal loads in hearts, and the most potential ones of 48 (20 mg/kg/dose, in normal treatment) and 51 (20 mg/kg/dose, in pretreatment) could be comparable to VAN, but not to



LZD (4.7 log10 CFU by 48 vs 4.9 log10 CFU by 51 vs 4.5 log10 CFU by VAN vs 3.2 log10 CFU by LZD).

Figure 6. Effect of 48 and 51 treatment on Mu50 bacteria survival in the livers (A) and hearts (B) of mice (n = 10) challenged with 1.1×109 CFU Mu50 bacteria. P = pretreatment, drugs or compounds were intraperitoneally administered 24 h before infection compared with the normal treatment (drugs or compounds were intraperitoneally administered 6 h after infection). Statistical significance determined by Mann-Whitney Test (two-tailed): *p< 0.05, **p< 0.01, ***p< 0.001. Each symbol represents the value for an individual mouse. Horizontal bars indicate observation means and dashed lines mark limits of detection.

In vivo effects of derivatives 48 and 51 on attenuating the virulence of NRS271. To assess the spectrum of activities of 48 and 51, we evaluated their antibacterial activity against NRS271 strain in vivo (LZD-resistant S.aureus, LRSA, Table S3, Supporting Information). As shown in Figure 7A, although the value of the LZD group was superior to non-treatment group (significance *P < 0.1 reduction), it still displayed weakly inhibitory activity compared with other groups in the experiment. In


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contrast to the LZD-treatment group suffered from NRS271, the derivative-treatment groups always exhibited a significant effect in comparison with non-treatment group in livers (all analog-treatment groups significance ***P < 0.001, over 99% decrease in surviving bacteria), especially 48 with 5 mg/kg/dose (in pretreatment) and 51 with 20 mg/kg/dose (in pretreatment) exceeded LZD with significance ***P < 0.001. However, the best two groups showed no significant difference compared to VAN (4.9 log10 CFU by 48 vs 5.0 log10 CFU by 51 vs 3.7 log10 CFU by VAN vs 6.1 log10 CFU by LZD). As shown in Figure 7B, similar to the effect in livers, the strains were weakened continuously in the LZD-treatment group in hearts, and 48-treatment group with 5 mg/kg/dose in normal treatment was still comparable to LZD (4.7 log10 CFU by 48 vs 4.6 log10 CFU by LZD). However, notably, the whole groups of 51 not only exceeded that of LZD, but caught up on that of VAN (2.6 log10 CFU by 51 vs 2.7 log10 CFU by VAN), which gave us much confidence for further development.

Figure 7. Effect of 48 and 51 treatment on NRS271 bacteria survival in the livers (A) and hearts (B) of mice (n = 10) challenged with 2.3×108 CFU NRS271 bacteria. P = pretreatment, drugs or compounds were intraperitoneally administered 24 h before infection compared with the normal treatment (drugs or compounds were



intraperitoneally administered 6 h after infection). Statistical significance determined by Mann-Whitney Test (two-tailed): *p< 0.05, **p< 0.01, ***p< 0.001. Each symbol represents the value for an individual mouse. Horizontal bars indicate observation means and dashed lines mark limits of detection.

In general, 48 and 51 were capable of decreasing the bacterial survival in the abscess formation model of S. aureus Newman, Mu50 (VISA) and NRS271 (LRSA) strains. What’s more, some of 48 or 51 treatment groups were more efficacious than the two positive groups. Additionally, the experiment demonstrated that there was no significant difference between the pretreatment regiments with the normal treatment. To further evaluate the efficacy of 48 and 51 at affecting the outcome of S. aureus sepsis, we challenged animals with 4 × 108 CFU Newman bacteria. The untreated mice nearly died out (93.3% animal) within 4 days, while 48-treated resulted in 60% animal survival and 51-treated in 66.7%. By the eighth day, the rest of the 48-treated and 51-treated mice were still alive (Figure 8). This investigation clearly proved that both 48-treatment and 51-treatment weakened the virulence of S. aureus Newman in vivo.


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Figure 8. Effects of 48 and 51 on S. aureus survival in protecting mice (n=15) from lethal S. aureus infection challenged with 4×108 CFU Newman bacteria.

CONCLUSIONS In this study, 58 compounds were designed, synthesized, and evaluated the activity against wild-type S. aureus Newman and several MRSA strains. According to the results of the pigment inhibitory activities against S. aureus Newman, unambiguous SARs were obtained. Ten derivatives (10, 12, 15, 44, 48, 50, 51, 53, 54 and 56, with IC50 values of pigment inhibition less than 3nM) were selected and subjected to further investigation. Finally, derivatives 48 and 51 were carefully selected from respective series, since they exhibited favorable characteristics in vitro including hERG inhibition (IC50 = 16.1 µM and IC50 > 40 µM, respectively), solubility (4.2 mg/mL and 2.8mg/mL, respectively), enzymatic activities and pigment inhibitory activities of four other MRSA strains. The experiments of bacterial growth assays and immune clearance proved that 48 and 51 could sensitize S. aureus strains to be killed by H2O2 and human blood without the interference of the bacterial growth.



Derivatives 48 and 51 also displayed potent antibacterial spectrum in vivo against pigmented S. aureus Newman, Mu50 (VISA/MRSA) and NRS271 (LRSA/MRSA). With four different regiments in vivo, 48 and 51 were proven efficacious in S. aureus Newman and multidrug-resistant MRSA strains (Mu50 and NRS271) model. As for S. aureus Newman, 48-treatment with low-dosage in normal treatment group (5 mg/kg/dose, 35 mg/kg in total dose) and 51-treatment with high-dosage in normal treatment group (20 mg/kg/dose, 140 mg/kg in total dose) showed comparable effect with the two positive groups (LZD and VAN) in livers and hearts. As for the two strains of MRSA, 48-treatment and 51-treatment could exceed the positive drugs, which were resisted by the MRSA strains. The favorable in vitro and in vivo activities of 48 and 51, combined with their superior hERG safety profile and solubility, make them promising drug candidates. More evaluation of these two drug candidates are currently under investigation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxx HPLC analysis data of derivatives 2-59, experimental procedures and characterizations of compounds, pigment inhibition assay, the hERG cardiac toxicity assay, CrtN enzyme inhibition assay, MIC values of compounds against MRSA strains, bacterial growth assays of S. aureus Newman and MRSA strains,


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hydrogen peroxide killing and human whole blood killing assay, metabolic stability of mouse liver microsome, anti-fungal assay, cytotoxicity of HEK-293T and HepG-2, S. aureus systemic infection assay and NMR spectra of representative compounds

AUTHOR INFORMATION Corresponding Authors *

For

J.L.:

phone,

86-21-64252584;

Fax,

86-21-64252584;

Email,

[email protected]; * For L.L.: Phone, +86-21-50803109; E-mail, [email protected]. ORCID Feifei Chen: 0000-0002-7251-1956 Fei Mao: 0000-0001-5871-6878 Jin Zhu: 0000-0003-3934-0312 Lefu Lan: 0000-0002-5551-5496 Jian Li: 0000-0002-7521-8798 Author Contributions †

B.L. and S.N. contributed equally to this work.

Notes The authors declare no competing financial interests. ACKNOWLEDGEMENT Financial support for this research provided by the National Key R&D Program of China (Grant 2017YFB0202600), the National Natural Science Foundation of China



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(Grant 21672064), the “Shu Guang” project supported by the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (Grant 14SG28), and the Fundamental Research Funds for the Central Universities is gratefully acknowledged. ABBREVIATIONS S. aureus, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; STX, staphyloxanthin; CrtN, diapophytoene desaturase; Compd., compound; SAR, structure-activity relationship; IC50, half maximal inhibitory concentration; MIC, minimum inhibitory concentration; HPLC, high-performance liquid chromatography; MS, mass chromatography; CFU, colony-forming unit; PBS, phosphate-buffered saline; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol; EtOAc,

ethyl

acetate;

MeOH,

methanol;

THF,

tetrahydrofuran;

CH2Cl2,

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