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High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes Kristin M. Bompiani, Dejan Caglic, Michelle C. Krutein, Galit Benoni, Morgan Hrones, Luke L. Lairson, Haiyan Bian, Garry R. Smith, and Tobin J Dickerson ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00033 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on July 12, 2016

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ACS Combinatorial Science

High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes Kristin M. Bompiani,¶ ‡♯ Dejan Caglič,¶ ‡♮ Michelle C. Krutein,¶ Galit Benoni,¶ Morgan Hrones,¶ Luke L. Lairson,¶ Haiyan Bian,§ Garry R. Smith,§ and Tobin J. Dickerson¶*

¶

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La

Jolla, CA, 92037 §

Fox Chase Chemical Diversity Center, 3805 Old Easton Road, Doylestown, PA, 18902

♯

Current address UCSD Moores Cancer Center, 3855 Health Sciences Drive, La Jolla, CA

92093 ♮Current address BASF Corporation, 3550 John Hopkins Ct., San Diego, CA, 92121 ‡

These authors contributed equally to this work

*Corresponding author

KEYWORDS: high-throughput screening, botulinum neurotoxin, small molecule, inhibitor, activator

ACS Paragon Plus Environment

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ABSTRACT: Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogenous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. Highthroughput screening of 97,088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent sub-micromolar inhibition (Ki 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 µM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.


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Introduction: Botulinum neurotoxins (BoNTs), which cause the disease botulism, are proteins produced by the Gram-positive bacteria Clostridium botulinum. Among the most potent toxins known, botulinum neurotoxins (BoNTs) bind to and enter host neurons, cleave intra-neuronal proteins, and impede acetylcholine neurotransmitter release [1]. Disruption of acetylcholine release by motor neurons results in flaccid muscle paralysis and, in severe cases, may result in death [2]. BoNTs have garnered considerable interest to date because of their potential use in widespread bioterrorist attacks, as reports show that several countries including Iran, Iraq, North Korea, and Syria have previously or are attempting to purify/weaponize the toxin [3, 4]. A 1995 United Nations report stated that Iraq purified 19,000 liters of toxin and weaponized approximately half of the material in delivery systems [3]. Correspondingly, researchers predict that widespread exposure of BoNTs due to a terrorist attack would cost billions of dollars in intervention and treatment, which could be significantly decreased if treatments are widely available and immediately implemented [5]. Eight immunologically distinct serotypes of BoNTs have been identified (A-H), where types A, B, and E typically cause disease in humans. Serotype A (BoNT/A) is the most studied form due to its extreme potency (LD50 in humans of approximately 1 ng/kg) [6] and long-term activity in neurons (up to 3-6 months) [7]. The mature BoNT/A toxin is a 150-kDa heterodimer comprised of a heavy and light chain linked via a disulfide bond, where both chains are required for toxicity. The heavy chain (HC) contains binding and internalization domains that facilitate toxin binding and translocation into host motor neurons, while the light chain (LC) is a zinc dependent metalloprotease that persists in neurons and hydrolyzes host proteins involved in neurotransmitter signaling [8]. HC binding to neuronal surface gangliosides and proteins triggers


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receptor-mediated endocytosis, and the translocation domain subsequently chaperones the LC from the endosome into the cytosol [9]. The translocated LC/A localizes to the inner side of the cytoplasmic membrane where it cleaves its membrane-bound substrate, SNAP-25 (synaptosomal-associated protein of 25 kDa), a member of the SNARE (soluble NSF attachment protein receptor) protein family [10]. SNAP-25 cleavage impedes acetylcholine release into the neuromuscular junction, thereby impairing muscle contraction and resulting in flaccid paralysis. Current botulism treatment is limited and includes the administration of a neutralizing equine or human anti-toxin for adult or infant cases, respectively [11, 12]. However, the antitoxins are only effective if administered within a finite time window, that is, before the toxin enters neurons and becomes unavailable to the antibodies. After cellular internalization, patients who have been exposed to large amounts of toxin can only be managed in a clinical setting and placed on a ventilator to prevent respiratory arrest [13]. Moreover, limited stockpiles of these anti-toxins are available as they are not cost effective to produce, may have batch-to batch variation, and often involve immunizing animals or collecting sera from voluntarily immunized humans. Thus, new botulism post-exposure treatments are urgently needed in the event of widespread outbreak, such as a bioterrorist attack. One current therapeutic design approach is to identify compounds that inhibit intracellular LC activity, as these can be administered postexposure to aid in long-term treatment. While peptides [14-19] and aptamers [20] have been described that inhibit LC/A activity, no compound has advanced clinically. These compounds are challenging to develop as therapeutics given that they do not readily enter cells. Thus, many researchers have focused on developing small molecule therapeutics to inhibit LC/A activity. Several small molecule HTS campaigns with recombinant LC/A (rLC/A) have been described, although the majority of inhibitory compounds initially identified possess poor (i.e.,


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micromolar) inhibitory constants (Ki) [21-24]. More recently, inhibitory compounds with submicromolar Ki values have been reported, including a betulin-derived triterpenoid with a Ki of 800 nM [25], a quinolinol inhibitor with an estimated IC50 of 40 nM in a rat synaptosomal model [26], and a “hybrid” small molecule with a Ki of 600 nM [27]. Several potent hydroxamate inhibitors have been reported, including 2,4-dichlorocinnamic hydroxamate (Ki = 300 nM) [28], a related benzothiophene hydroxamic acid (Ki = 77 nM) [29], and a recently reported adamantine hydroxamate (Ki = 27 nM) [30]. Although screening has successfully identified a number of inhibitory chemotypes, no compound has advanced to clinical testing due to a lack of efficacy in advanced cellular or in vivo models. Ideally, candidate compounds would have a high aqueous solubility, low cross-reactivity with host metalloproteases, high cellpermeability and efficacy, and low cytotoxicity. Because no BoNT small molecules have advanced to clinical trials, new small molecule LC/A inhibitors with improved efficacy ex vivo/in vivo or improved pharmacokinetic properties may provide a basis for therapeutic advancement. Herein, we describe the results of a homogenous high-throughput screening (HTS) campaign of three different drug-like small molecule libraries with the goal of identifying unrecognized BoNT inhibitor chemotypes. Screening of approximately 97,000 compounds identified numerous compounds that activate or inhibit rLC/A activity. Four inhibitory scaffolds were further studied: two previously known metalloprotease/LC scaffolds and two new scaffolds, one of which is previously unreported and among the most potent botulinum inhibitors to date.


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Results and Discussion: HTS assay optimization Several in vitro and in silico high-throughput screens (HTS) with the botulinum neurotoxin light chain (BoNT LC) have been reported, where most involved screening enzymatic activity of soluble LC with a fluorescent substrate [21-24]. In our studies, we utilized a previously established FRET assay with recombinant LC/A (rLC/A) and a commercial SNAPtide® substrate to identify compounds that increase or decrease the rate of enzyme activity based on fluorescent product generation [31, 32]. Initially, we optimized and scaled down the reaction relative to previous studies [31] to facilitate cost-effective screening of a larger library. The assay was performed in 384-well low volume microtiter plates, with a total assay volume of 10 µL instead of the 100 µL total volume for 96-well format [31]. The concentration of SNAPtide substrate was decreased from 5 µM to 0.7 µM. This substrate concentration did not significantly reduce the assay signal-to-noise ratio, and pilot experiments performed under these conditions indicated that the reaction operated under steady-state conditions with a linear increase in fluorescent product formation over a period of 2 hours (data not shown). The final DMSO concentration was 2%, which had a negligible impact on the rate of substrate hydrolysis (data not shown). Collectively, the optimization and miniaturization described here led to a 70-fold decrease in the amount of commercial SNAPtide substrate used without affecting the quality of the assay readout. The Z’factor for the SNAPtide assay was 0.97 and the signal window was 171.9, suggesting the assay is well suited to screening with a high predictive value of identified hits. Additionally, we utilized automated screening equipment to significantly increase our screening capacity (Figure 1).


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Figure 1: Graphical overview of the high-throughput screening method. Primary high-throughput screening (HTS) involved single concentration screening with 97,088 compounds from the three libraries indicated. The compounds were pre-incubated with recombinant light chain, FRET substrate peptide was added, and the rate of substrate cleavage over two hours was monitored. Compounds that significantly decreased LC/A activity (greater than 3 standard deviations for ChemBridge, or greater than 90% inhibition for ChemDiv and Life) were identified as hits, where secondary screening was performed with new batches of the small molecules at a single concentration in the FRET enzyme assay. Lead compounds were identified by comparing the relative potencies (IC50s) of compounds in dose-response experiments.

Three different libraries of small molecules from separate commercial vendors, ChemBridge, ChemDiv, and Life Chemicals were chosen for screening. The libraries are commercially available diversity sets that were filtered based on the following properties: (a) the compounds have desirable drug-like properties, such as low molecular weight, favorable number of hydrogen bond acceptors and donors, and hydrophilicity according to the Lipinski’s Rule of 5 [33]; (b) the compounds were amenable to structural modifications in order to optimize activity while still retaining drug-like properties, and (c) the compounds were devoid of potentially reactive or known problematic moieties including aldehydes, hydrazines, ketones, charged compounds, disulfides, phosphates, carboxylic and sulphonic acids, phosphonates, and sulfates (Supplemental Table 1). To perform the filtering, first, a similarity (Tanimoto) coefficient of


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3σ below mean ≥90% inhibition ≥90% inhibition n/a

No. hits identified 20 36 26 82

Hit Rate (%) 0.10 0.07 0.09 0.08

Hit compounds were identified and analyzed independently for each library by clustering based on substructure similarities. From the 82 compounds analyzed, four major scaffolds were identified: an 8-hydroxyquinoline scaffold, an 8-sulfonamidoquinoline scaffold, a spiro(indolthiadiazole) scaffold, and a benzimidazole pyrazolopiperidinone scaffold (Figure 3). The 8hydroxyquinoline scaffold, which is a well-known LC/A inhibitor [35, 36], was present in all three libraries and comprised 35% of the hit pool (5 compounds in ChemBridge, 14 in ChemDiv, and 10 in Life). In addition to identifying potentially new hydroxyquinoline iterations, classification of this scaffold as hits in all three libraries indicates that the HTS was robust and sensitive. Spiro(indol-thiadiazole) compounds were present in both ChemBridge (1 compound)


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and ChemDiv libraries (2 compounds); and the 8-sulfonamidoquinoline scaffold was only present in the ChemBridge library (4 compounds). Finally, the benzimidazole pyrazolopiperidinone scaffold, which represents a previously unreported chemotype for enzyme inhibition, was present only in the ChemDiv library (5 compounds).

Figure 3: Inhibitory small molecule scaffolds identified in the HTS campaign.

Secondary screening validates the four inhibitory scaffolds identified and indicates they have a range of potencies Substructure searches with the four inhibitory scaffolds were performed within each respective commercial database to identify related compounds; these additional compounds were purchased from the vendor and included in the secondary screen. The compounds were validated in a secondary screen at the same concentration used for primary screening (12.5 µM final concentration for ChemBridge and 20 µM for ChemDiv and Life compounds) in the SNAPtide


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FRET assay [32]. To compare the relative potencies and identify lead compounds, we additionally performed dose-titrations and calculated IC50 values. The 8-hydroxyquinoline scaffold has been previously reported in the BoNT literature [37]; here we selected an additional 21 compounds from ChemBridge and ChemDiv hit lists to validate with secondary screening. All 19 compounds from ChemBridge and ChemDiv libraries that were primary hits were positive in the secondary screen (0% false positive rate). Five compounds that were not primary hits exhibited greater than 90% inhibition of LC/A (14.7% false negative rate). Dose-titrations with the 19 compounds indicated that all but one hydroxyquinoline compound had IC50 values below 10 µM, where the most potent compound (5) exhibited an IC50 of 1.6 µM (Table 2). These IC50 values are consistent with previous studies that reported low-micromolar IC50 values [26, 35, 38]. Using these compounds as a starting point, this series has been expanded and detailed structure-activity and ADME studies have been performed [37]. Table 2: Selected 8-hydroxyquinoline compounds assessed in secondary screening that showed inhibitory activity.

Compound ID

Vendor ID

R1

R2

1

CB5175086

H

H

2

CB6948044

R3

H

R4

IC50 (µM)

H

9.3

H

6.7


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3

CB7012006

CH3

H

2.7

4

CB7633607

CH3

H

1.7

5

CB7637950

CH3

H

1.6

6

7706-0972

H

Cl

4.0

7

8397-0126

H

H

5.6

8

8397-0127

H

H

1.9

9

8397-0140

H

H

7.1

10

8397-0166

H

H

2.9

11

8397-0180

H

H

4.9

12

8397-0181

H

H

4.2


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13

8397-0271

H

H

4.2

14

8397-0490

H

H

5.5

15

8397-0558

H

H

5.1

16

8397-0559

H

H

4.4

17

8397-0572

H

H

5.2

18

8397-0573

H

H

2.3

19

8398-1033

H

H

10.9

Next, we determined the potency of 27 spiro(indol-thiadiazole) compounds (1 hit and 26 additional structures) available from the ChemBridge small molecule repository. Structurally, these compounds possess a distinct similarity to a previously reported compound that potently inhibits the matrix metalloprotease ADAMTS-5 [39]. The single compound that was a primary hit exhibited inhibitory activity towards LC/A in the secondary screen (0% false positive rate). All spiro(indol-thiadiazole) compounds in the initial ChemBridge library were identified as hits (0% false negative rate). Dose-titrations identified 9 compounds with IC50 values below 5 µM,


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and 3 compounds (25, 31 and 33) had submicromolar IC50 values (Table 3). The remaining 15 compounds had no impact on LC/A enzyme activity when tested at 100 µM (Supplemental Table 3). The most potent compound in the spiro inhibitor series, 33, displayed an IC50 value of 0.64 µM. Some SAR trends could be identified based on this series. Substituted (compounds 27 and 29) as well as unsubstituted (compounds 25, 26, 28, 30, 31, 32 and 33) benzyl rings at the R1 position were well tolerated. Halogenation at the R4 position was tolerated (compounds 28 and 30), and as was methylation (compound 26) or ethylation (compounds 25 and 27). N-methylacetamide substitution at the R1 position and/or N-methyl-acetamide at the R3 position were not tolerated, as these compounds were completely inactive (compounds 134-149; Supplemental Table 3). Interestingly, addition of a 5-chloro group on the existing 2-chloro-benzyl substitution at the R2 position of compound 31 rendered compound 132, inactive. Our data agree with the previously published data for this scaffold [39] and suggest that these compounds may be broad metalloprotease inhibitors with modest inhibitory constants. SAR analysis of the spiro(indolthiadiazole) LC/A inhibitor series described here implicates a relative plasticity of the LC/A, which agrees with previous observations with other inhibitory chemotypes [37, 40]. Table 3: Spiro(indol-thiadiazole) compounds assessed in secondary screening that showed inhibitory activity.

Compound ID

Vendor ID

25 26

R1

IC50 (µM)

R2

R3

R4

CB5668345

H

H

-CH2CH3

0.71

CB5668437

H

H

CH3

1.62


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27

CB5672440

H

H

-CH2CH3

1.33

28

CB5677976

CH3

H

Br

2.90

29

CB5679472

CH3

H

H

2.82

30

CB5681294

CH3

H

H

3.44

31

CB5825244

H

H

0.95

32

CB5830995

H

Br

2.25

33

CB5833514

H

H

0.64

H

We expanded upon the four initial 8-sulfonamidoquinoline hit compounds and screened an additional 47 compounds from the ChemBridge repository. This core is a well-known zinc chelator [41], and related compounds inhibit metalloproteases [42]. IC50 values were determined for all 51 compounds (Table 4 and Supplemental Table 4). Three compounds identified as primary hits were positive in the secondary screen, whereas one hit from the primary screen showed only moderate inhibition of LC/A in the secondary screen and was a false positive (7.7% false positive rate). There were no 8-sulfonamidoquinoline compounds in the initial ChemBridge library that were unidentified as LC/A inhibitors (0% false negative rate). A majority of the additional compounds (84%) were inactive and had IC50 values greater than 25 µM. Closer analysis and correlation with the primary screen data indicated that 12 of the inactive compounds


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were present in the initial library and showed no inhibitory activity in the primary or secondary screens, validating our screening protocol. The sulfonamidoquinoline compounds tested consisted of compounds with quinoline and naphthalene backbones (Table 4 and Supplemental Table 4). The two most potent compounds, 35 and 37, exhibited low micromolar inhibitory activities with IC50 values of 3.5 and 3.2 µM, respectively. Interestingly, only these two hits and one sulfonamidoquinoline compound with moderate inhibitory activity (36; IC50 = 6.7 µM) have a methoxy substitution at the R4 position of the quinoline backbone, apparently increasing inhibitory activity. Substitution of the R4 thio-triazole group on the naphthalene backbone was tolerated, where these compounds have IC50 values between 9.7 and 25.5 µM (compounds 3841). Finally, a moderately active sulfonamidoquinoline compound, 34, with 2,4,6-trimethylbenzyl substitution at the R1 position and the quinoline backbone, had an IC50 value of 18.8 µM. Overall, the sulfonamidoquinoline compounds exhibited only moderate LC/A inhibition with IC50 values in the micromolar range, similar to what has been previously observed for zincdependent matrix metalloproteases [42]. This scaffold does not appear as potent as other compounds, such as the hydroxamates, which have sub- to low-micromolar IC50 values [29]. While these compounds have higher IC50 values, they may be easier to optimize for solubility or stability in future DMPK studies. These factors may prove to play a large a role in clinical success. Table 4: 8-sulfonamidoquinoline compounds assessed in secondary screening that showed inhibitory activity.


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Compound ID

Vendor ID

X

34

CB5469620

35

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R2

R3

R4

IC50 (µM)

N

CH3

H

H

18.8

CB6626965

N

H

H

-O-CH3

3.5

36

CB6633275

N

H

H

-O-CH3

6.7

37

CB6991782

N

H

H

-O-CH3

3.2

38

CB7788159

C

H

OH

15.3

39

CB7796127

C

H

OH

9.7

40

CB7803009

C

H

OH

25.5

41

CB7816522

C

H

OH

12.3

R1

Substructure searches with the benzimidazole pyrazolopiperidinone scaffold identified 80 additional commercial ChemDiv compounds, 54 of which were present in the initial library but inhibited less than 90% of enzyme activity at the screening concentration of 20 µM and were not identified as hits. All 85 compounds were evaluated in parallel with a secondary screen, where 21 compounds were validated (inhibited >80% of LC/A activity at 20 µM, data not shown). All five compounds that were present in the initial screen hit list (≥90% inhibition in the primary screen, compounds 69, 70, 94, 97 and 98) were positive in the secondary screen and had a


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statistically similar percentage of inhibition (90 ± 12%; 0% false positive rate). Four compounds that were present in the initial library but displayed poor (0-75%) inhibition in the primary screen inhibited greater than 90% of enzyme activity in the secondary screen (7.4% false negative rate; compounds 66, 67, 84 and 100; data not shown). In addition to the 85 commercial compounds, 67 additional benzimidazole pyrazolopiperidinone compounds or scaffold substructures were synthesized and evaluated in dose-titrations to further evaluate SAR and identify active moieties within the scaffold (Tables 5 and 6, Supplemental Tables 5 and 6). Similar to other previously reported LC/A inhibitory scaffolds, the SAR landscape for the benzimidazole compounds appears relatively flat, and a variety of substitutions were well tolerated. Compounds containing a phenyl R1 substituent were extremely potent and demonstrated submicromolar IC50 values (Table 5). Substitution with fluorine (43-45), chlorine (57-59), and bromine (54-56) at the 2, 3, or 4 position on the phenyl was well tolerated compared to phenyl alone (42; IC50 = 0.14 µM). Di-substitutions with the same halogen (46-51, 60-63) or different halogens (64-68), as well as tri-fluoro methyl substituents (51-53) were also well tolerated at each position of the phenyl ring. Bromo-ether (69, 70), ester (72), nitrile (73), nitro (74-76), and O-trifluoromethyl (77) substituents were tolerated, although carboxylation at the 4-position significantly decreased potency (71; IC50 = 23.5 µM) (Table 5; panel of representative IC50 curves Supplemental Figure 1). Collectively, these data indicate that the phenyl R1 substituent may play a steric, but not an electrostatic role in LC/A inhibition, as the presence and position of electronegative substitutions on the phenyl did not significantly increase or decrease potency.


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Table 5: Commercial benzimidazole pyrazolopiperidinone compounds that showed inhibitory activity toward BoNT LC/A.

Compound ID

ChemDiv or FoxChase ID

42

R1

R2

IC50 (µM)

J094-0647

CH3

0.14

43

J094-0172

CH3

0.36

44

J094-0173

CH3

0.27

45

J094-0168

CH3

0.32

46

J094-0170

CH3

0.18

47

J094-0169

CH3

0.15

48

J094-0171

CH3

0.10

49

J094-0655

CH3

0.14


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50

J094-0184

CH3

0.18

51

J094-0182

CH3

0.15

52

J094-0197

CH3

0.20

53

J094-0183

CH3

0.12

54

J094-0177

CH3

0.09

55

J094-0176

CH3

0.37

56

J094-0178

CH3

0.16

57

J094-0179

CH3

0.12

58

J094-0180

CH3

0.12

59

J094-0181

CH3

0.28

60

J094-0648

CH3

0.06


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61

J094-0199

CH3

0.19

62

J094-0649

CH3

0.33

63

J094-0198

CH3

0.48

64

J094-0167

CH3

0.27

65

J094-0174

CH3

0.19

66

J094-0175

CH3

0.51

67

J094-0195

CH3

0.26

68

J094-0196

CH3

0.11

69

J094-0185

CH3

0.88

70

J094-0186

CH3

0.28


22

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71

J094-0187

CH3

23.50

72

J094-0188

CH3

0.30

73

J094-0190

CH3

0.78

74

J094-0194

CH3

0.14

75

J094-0192

CH3

0.43

76

J094-0193

CH3

0.74

77

FC2538

CH3

0.43

78

J094-0652

CH3

8.89

79

FC2549

CH3

2.23


23


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Page 24 of 51

80

FC2556

CH3

0.75

81

FC2557

CH3

0.24

82

J094-0626

CH3

0.88

83

J094-0633

CH3

0.43

84

J094-0625

CH3

0.70

85

J094-0628

CH3

0.66

86

J094-0634

CH3

0.10

87

J094-0632

CH3

1.13

88

FC3027

CH3

0.11

89

J094-0630

CH3

0.30


24

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90

J094-0631

CH3

0.53

91

FC3025

CH3

0.17

92

FC3026

CH3

0.11

93

J094-0627

CH3

0.25

94

J094-0640

CH3

0.48

95

FC2537

CH3

0.42

96

FC3024

CH3

0.11

97

J094-0642

CH3

6.44

98

J094-0643

CH3

4.52

99

J094-0644

CH3

8.06


25


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Page 26 of 51

100

J094-0645

CH3

3.43

101

FC2592

CH3

>50

102

J094-0897

CH3

0.45

103

FC2539

CH3

14.60

104

FC2548

CH3

0.85

105

FC-3138

CH3

0.33

106

FC2838

0.13

107

FC2839

0.25

108

FC2840

0.44

109

FC2841

0.31


26

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Pyridyl substitution at the R1 position significantly decreased potency compared to phenyl substitution (compound 78 IC50 = 8.89 µM vs. compound 42 IC50 = 0.14 µM). However, conjugation of a pyrrolidine ring to the pyridine increased potency, particularly when added to the 4-position (80; IC50 = 0.75 µM). Similarly, conjugation of a piperidine to the 4-position further increased potency, resulting a compound with similar affinity to the R1-phenyl compounds (81; IC50 = 0.24 µM). Compounds with a thiophene at the R1 position were generally potent inhibitors with IC50s comparable to the phenyl compounds (82, IC50 = 0.88, 83 IC50 = 0.43). Placement of the sulfur within the ring had a minor (less than 2-fold) impact on potency, and methylation and ethylation of the thiophene also had a minor impact on potency (84, 85); in contrast, dimethylation appreciably increased potency (86; IC50 = 0.10 µM). Addition of a t-butyl to the thiophene significantly decreased activity (87; IC50 = 1.13 µM), while a nitro- (93), bromo(90-92), chloro- (88), or dichloro- (89) substituent increased thiophene compound potency and resulted in compounds with similar potencies to the phenyl compounds. Conjugation of additional aromatic rings to the thiophene R1 substituent was also well tolerated (94-96). Pyrazole substitution at R1 severely decreased inhibitory activity, and these compounds demonstrated micromolar IC50s (97-100). R2 substitution with a t-butyl was well tolerated, as compounds with this moiety (106-109) displayed sub-micromolar IC50 values similar to the methylated analogs (Table 5). Interestingly, a majority of the cognate benzimidazole compounds that contain an Nmethyl imidazole have severely decreased inhibitory, suggesting that imidazole methylation may result in an activity cliff. The R2 methylated analogs of compounds 64 (IC50 = 0.27 µM), 45 (IC50 = 0.32 µM), and 47 (IC50 = 0.15 µM), all have a greater than 300-fold decrease in activity (compounds 202, 190 and 192, IC50 > 100 µM; Supplemental Table 5). R2 methylation resulting


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in an activity cliff holds true for 25 of 27 of the methylated compounds, except for compounds 197 (IC50 = 0.78 µM) and 211 (IC50 = 0.75 µM). We hypothesize that the amine hydrogen may play a critical role in LC/A binding and inhibition (e.g., potentially coordinate the active site zinc), or the methyl group may be too bulky for incorporation into the binding site. However, given the small size of this addition and the relatively known plasticity of the enzyme, such a dramatic drop in activity due to steric interference seems unlikely [37, 40]. Substructure analysis of the benzimidazole pyrazolopiperidinone scaffold was performed by synthesizing a series of compounds with various scaffold fragments from one of the most potent compounds (47). Strikingly, analogs of compound 47 that lack the benzimidazole and have a hydrogen or methyl as R3 (110, 115) were largely inactive, save compound 125. In this case, installation of a trifluoro-methyl group at R1 restored inhibitory activity (125; IC50 = 0.75 µM), indicating that the benzimidazole moiety is not absolutely required for potent inhibition. Benzimidazole pyrazolopiperidinone substructures bearing a hydrogen (121), cyclopropyl (122), isopropyl (123), or t-butyl (124) R3 group were inactive (Table 6). Table 6: Substructure analysis of the benzimidazole pyrazolopiperidinone scaffold.

Compound ID 110

R1

R2

R3

IC50 (µM)

CH3

H

>100


28

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111

H

>100

112

H

>100

113

H

>100

114

H

>100

CH3

>100

116

CH3

>100

117

CH3

>100

115

CH3

>100

118


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>100

119

CH3

>100

CH3

>100

122

CH3

>50

123

CH3

>50

124

CH3

>50

125

CH3

0.75

120

121

H

Compounds where the benzimidazole was replaced with other aromatic substitutions were also assessed to determine if activity could be rescued. Initially we mined the original HTS library and filtered the screening data for compounds containing solely the pyrazolopiperidinone


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core. Analogs containing a benzathiazole, benzoxazole, pyrimidine, and triazole-pyridazine in place of the benzimidazole were present in the primary screen; all of these compounds had significantly decreased activity compared to their benzimidazole counterparts and were not identified as hits (Supplemental Table 6). Additionally, we synthesized analogs that possessed a pyridine (251-269) or pyrimidine (270-273), which had no activity, and a ketophenyl group (284, 285), which had decreased activity (284; IC50 = 10 µM) (Supplemental Table 6). Compounds 274 and 276, which have a methoxybenzothiazole substitution, did possess some weak inhibitory activity with micromolar IC50s. Collectively, these data indicate that the benzimidazole core is specifically required for high affinity LC/A inhibition, and that while conjunction with the pyrazolopiperidinone may not be required, the presence of this group may increase potency. Strikingly, the most potent benzimidazole compound (60) has an IC50 of 60 nM, which places it among the most potent botulinum inhibitors reported. While several groups have previously described LC/A inhibitors that contain a benzimidazole, presumably as a zinc chelator, the combined benzimidazole pyrazolopiperidinone scaffold has not yet been reported in the context of any small molecule enzyme inhibitor. Eichorn et al. performed in silico screening of the NCI library and identified a benzimidazole dendrimer (NSC348884) that had protective activity in BoNT-intoxicated mice, although its effect was modest [43]. Cardinale et al. screened libraries from ChemBridge, ChemDiv, and Microsource with a similar FRET-based enzyme assay and identified several benzimidazole acrylonitrile and vinylbenzimidazole compounds that are hypothesized to be covalent inhibitors. The most potent compound displayed an IC50 of approximately 10 µM. While this compound was not active in cells, a derived compound, MBX1533, prevented SNAP-25 cleavage in chick neuronal cells; however, both compounds


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were cytotoxic to human cells [21]. Compared to the previously reported compounds, the benzimidazole scaffold described here is significantly more potent. Detailed kinetic and cytotoxic analyses of lead benzimidazole compounds Lead compounds 45, 47, and 90 were further evaluated in an alternative enzyme assay with SNAP-25 derived substrate peptide (SNAP141-206) [44]. Unlike the fluorophore SNAPtide substrate, which only engages the LC/A active site and utilizes a fluorescent substrate, this unmodified 66mer peptide represents the critical residues of endogenous SNAP-25 that interact with the LC/A (active site and exosites). Substrate cleavage under steady state conditions is monitored via semi-quantitative liquid chromatography/mass spectrometry (LC/MS). Dose titration with 45 indicated dose-dependent inhibition. Initially, a non-competitive curve fit was used to estimate a Ki value of 368 nM (Figure 4a), which is similar to the IC50 value from the SNAPtide assay (IC50 = 0.32 µM). Similarly, 45 and 47 titrations demonstrated dose-dependent inhibition with Ki = 213 and 211 nM, respectively (Figure 4b,c). These data support that these novel compounds are among the most potent botulinum LC inhibitors to date.


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Figure 4: LC/MS data from the LC/A enzyme assay with 66mer substrate in the presence of the three lead benzimidazole compounds. Dose-response Ki titrations with lead benzimidazole pyrazolopiperidinone small molecules. Dose titrations with A) compound 45; B) compound 47; and C) compound 90. The data represent the mean ± SEM of two independent experiments. The data were fit with a non-competitive model of enzyme inhibition to calculate inhibitory constants with GraphPad Prism software. Representative Lineweaver-Burk linear replots with various concentrations of D) compound 45; E) compound 47; and F) compound 90. The data represent the linear replot of the average velocities shown in panels A, B, and C. Note that compounds 45 and 47 show significant deviation from linearity at high substrate and high compound concentrations, indicating more than one active enzyme species is present.

Closer analysis of the non-competitive fit for the LC/MS data indicated the model did not fit well at high substrate and high inhibitor concentrations. Lineweaver-Burk linear replots of the LC/MS kinetic data for 45 and 47 indicated noticeable curvature and deviation from linearity at high substrate concentrations and high inhibitor concentrations (Figure 4d,e). In contrast, the data for 90 appear more linear (Figure 4f). The deviation from linearity with the fluorophenyl benzimidazoles at high substrate concentrations implies that there is a mixture of enzyme species present. Two enzyme forms that have similar Km values, but different Vmax values may be mistaken for a single enzyme species, as the Vmax observed is the sum of the individual enzyme species[45]. Such non-linear data is often initially mistaken for non-competitive enzyme inhibition unless carefully assessed at high substrate concentrations, as we have done here. The data indicate that multiple rLC/A species are present in the LC/MS assay, and we propose that all three compounds are true competitive inhibitors. Compounds 45 and 47 appear to preferentially


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inhibit one form of the enzyme species, while 90 may either inhibit both (or all) enzyme forms present, or may bias the equilibrium between the species and stabilize one conformation. While several papers have discussed the plasticity of rLC/A, no reports have described the presence of multiple enzyme forms. Typically screens are performed with a truncated form of the rLC/A because of its increased solubility and stability [46]. However, it’s possible that truncated rLC/A may exist in multiple forms in this screening assay. Whether any of these forms is representative of native toxin on cell membranes remains unknown and should be assessed for future screening campaigns. Lead small molecules show specificity toward the LC/A over other zinc metalloproteases A majority of BoNT LC/A small molecule scaffolds reported in the literature are competitive inhibitors that presumably coordinate the catalytic zinc, although a few noncompetitive inhibitors also have been reported [47, 48]. Correspondingly, screening with the SNAPtide substrate, which is a small peptide that only engages the LC/A active site, may bias hit identification toward classical competitive inhibitors. Previous studies have shown that quinolinols chelate divalent cations [36], and consequentially this scaffold has been previously identified through HTS against BoNT/A LC [35]. Similarly, spiro(indol-thiadiazole) and 8sulfonamidoquinoline scaffolds have been reported to inhibit zinc metalloproteases. Crystal structures of 8-sulfonamidoquinoline compounds indicate that the amide nitrogens coordinate zinc [41]. While the benzimidazole pyrazolopiperidinone scaffold has not been previously described, reports have shown that compounds bearing a benzimidazole moiety can chelate zinc. A crystal structure of the well-known serine protease inhibitor BABIM (bis(5-amidino-2benzimidazolyl)methane) in complex with trypsin shows that the zinc is coordinated by the two


34

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imidazole nitrogens [49]. Therefore, we hypothesize that all four scaffolds inhibit LC/A activity through zinc chelation. To test potential off-target effects, we characterized the inhibitory activity of lead compounds 4 (IC50 = 1.7 µM; 242-fold molar excess compound to LC/A), 33 (0.64 µM; 91x), 37 (IC50 = 3.2 µM; 457x), 47 (0.15 µM; 21x), and 90 (0.53 µM; 75x) toward other zinc metalloproteases. Kinetic assays with recombinant matrix metalloproteinases (MMP) 1 and 12 show that the compounds have 2,000 molar excess) the compounds inhibited 9-33% activity (Supplemental Figure 2a, b). In contrast, the MMP inhibitor marimastat completely inhibited activity of both MMPs at 100 nM (not shown). Similarly, these five lead compounds showed no inhibition of thermolysin activity at 1 µM (14-fold molar excess) and only 10-30% inhibition at 30 µM (400-fold molar excess) (Supplemental Figure 2c), while a positive control compound (phosphoramidon) inhibited >90% of thermolysin activity at 5 µM (data not shown). Collectively, these data suggest that the lead compounds, particularly the benzimidazole compounds 47 and 90, may have increased specificity toward the LC/A. Additional kinetic studies or crystallography data are needed to determine the mechanism of enzyme inhibition and define the inhibitor binding site. Previous reports have described small molecules bearing a benzimidazole moiety that showed protection in cellular assays, yet were cytotoxic to human cells [43]. Dose titrations with 47 and 90 from 0-25 µM in an MTS viability assay yielded LD50 values of 4.4 ± 0.2 µM and 6.4 ± 0.2 µM, respectively (Supplemental Figure 3). While these compounds appear cytotoxic at micromolar concentrations, there may be an optimal dose that engenders LC/A inhibition without imparting cellular toxicity, as only nanomolar concentrations are required for robust


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LC/A inhibition in vitro. Additional DMPK and cellular studies are needed to determine compound solubility and cell permeability. Preliminary SAR analysis of all four of the scaffolds suggests a relatively flat landscape, where a majority of substitutions are well tolerated. This phenomenon has been previously reported with several LC/A inhibitory scaffolds [37, 40], and it has been hypothesized that the LC/A adopts a flexible conformation in solution that can accommodate a variety of spatial combinations and chemical substituents. Correspondingly, several recent studies have suggested that LC/A can exist in multiple conformations in solution, which agrees with our kinetic data suggesting two enzyme forms are present [50-54]. It is currently unknown if any of the rLC/A forms present in solution are representative of the LC/A active on cell membranes in vivo. Consequentially, rLC/A enzyme plasticity may contribute to activity discrepancies between in vitro and ex vivo assays. Assays utilizing the full-length LC are challenging, given that the recombinant protein is difficult to purify and tends to precipitate rapidly. Future cell-based HTS assays may provide better information about compounds that can inhibit LC/A within cells. Although such systems are currently being developed [55], none has yet been applied to botulinum HTS. Typically, LC/A inhibitors identified through homogeneous HTS are subsequently tested in cell-based assays, and if functional, progress to in vivo studies with mice. However, given that most compounds identified to date have significant liabilities such as poor inhibitory constants [23, 24], poor specificity [56], instability [57], poor solubility [25], and/or cytotoxicity [21, 22, 58] in vitro and ex vivo, only a few compounds have been tested in vivo [22, 26, 43, 59]. No botulinum therapeutic has advanced to phase I clinical trials due to lack of efficacy in animal models. In this context, new scaffolds such as those reported here are critical to the long-term


36

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development of a viable botulism therapeutic that could treat exposure to this potent toxin. Early integration of drug metabolism and pharmacokinetic (DMPK) studies during the discovery process may further select for better lead compounds and hasten progression into advanced models.

Experimental Methods: Compound libraries The 20,000-member ChemBridge small molecule library (ChemBridge, Inc., San Diego, CA), a representative subset of a much larger library from ChemBridge, was designed based on the following criteria: (a) the compounds have desirable drug-like properties, such as low molecular weight, a favorable number of hydrogen bond acceptors and donors, and hydrophilicity; (b) the compounds are amenable for structural modifications in order to optimize activity, while still retaining drug-like properties, and (c) the compounds are devoid of potentially reactive or known problematic moieties including aldehydes, hydrazines, ketones, charged compounds, disulfides, phosphates, carboxylic and sulphonic acids, phosphonates, and sulfates. The stock compounds were dissolved in DMSO to a final concentration of 2.5 mM and transferred into 63, 384-well plates. All compounds were subsequently screened at a final concentration of 12.5 µM. Selected individual compounds were ordered from ChemBridge for secondary screening and potency determination. The ChemDiv library (ChemDiv, Inc., San Diego, CA) used in this study is derived from a larger commercial diversity set and contained 48,928 compounds stored in 139, 384-well


37


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Page 38 of 51

plates; the stock compounds were dissolved in DMSO to a final concentration of 2 mM and screened at a final concentration of 20 µM. Library compounds were initially filtered based on the criteria previously described. Selected individual compound hits or related compounds were ordered from ChemDiv for secondary screening and additional studies. The Life library (Life Chemicals, Inc., Ontario, Canada) is a subset of a commercial diversity set and contains 28,160 compounds in 80, 384-well plates; stock compounds were dissolved in DMSO to a final concentration of 2 mM and screened at a final concentration of 20 µM. Library compounds were initially filtered based on the previously described guidelines.

High-throughput screening Library compounds were evaluated for their impact on recombinant BoNT/A LC enzymatic activity towards SNAPtide® FITC/DABCYL fluorogenic substrate (List Biological Laboratories, Campbell, CA). The assay was performed in 40 mM Hepes, pH 7.4, with 0.01% Tween-20 at room temperature (22 °C). Recombinant BoNT/A LC (expressed in Escherichia coli and purified as described elsewhere[46]) was diluted in Hepes assay buffer to 8.75 nM and 8 µL of the enzyme solution was dispensed into a low-volume 384-well black assay plate (Greiner) using a MultiFlo microplate dispenser (BioTek, Winooski, VT). ChemBridge small molecule compound libraries (2.5 mM stock concentration; 50 nL), as well as ChemDiv and Life small molecule compound libraries (2.0 mM stock concentration; 100 nL) were transferred from 384well source plates using a 50- or 100-nl 384-slot pin tool (V&P Scientific, San Diego, CA) and a Biomek FXP liquid handler (Beckman Coulter, Brea, CA). Compounds were preincubated with the enzyme for at least 15 min at room temperature. Finally, 2 µL of 3.5 µM SNAPtide solution


38

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in Hepes buffer with 10% DMSO was dispensed using a MultiFlo microplate dispenser (BioTek). The final DMSO concentration in the screening assay was 2%. Stacks of 20 assay plates were simultaneously transferred to a stacked EnVision multilabel plate reader (PerkinElmer, Waltham, MA); rapid fluorescence reads with λex = 490 nm, λem = 523 nm were utilized to sequentially read all 20 plates within time points. Fluorescence was read every 30 minutes for 3 hours, and DMSO negative controls showed linear fluorescent product generation over the entire 3 hours. Slopes were calculated for each compound and compared to the slopes of the uninhibited enzyme (DMSO negative control). Normalized rates were calculated based on the average DMSO negative control rate for each respective individual library of compounds.

Statistical analyses Assay performance measures of the low-volume 384-well SNAPtide assay were determined as follows. Z’-factor for this assay was determined by comparing relative velocities of BoNT LC/A cleavage of SNAPtide in the presence of the positive (2,4-dichloro-cynnamyl hydroxamate[28]) and negative (DMSO) controls in low-volume 384-well plates. It was calculated as Z’ = 1 – 3(st + sb)/(mt - mb), where st and sb are standard deviations of the negative and positive controls, respectively, and mt and mb mean velocities of negative and positive controls, respectively[60]. Signal window was determined using the same data set as for determining the Z’-factor and was subsequently calculated as SW = ((mt – mb) – 3(st + sb)) / st [61].

Determination of compound potencies (IC50 values)


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Serial dilutions of compounds were prepared in DMSO, and 1 µL of each compound dilution was preincubated with 10 µl of 70 nM BoNT LC/A and 79 µl of 40 mM Hepes assay buffer in a 96-well black plate (Greiner, Monroe, NC) for 5 min at room temperature. Subsequently, 10 µL of 7 µM SNAPtide® was added to initiate the reaction. The final assay concentrations were 7 nM BoNT LC/A and 0.7 µM SNAPtide® in 2% DMSO. The well fluorescence was recorded every 5 minutes for a total of 1 hr and 45 min at room temperature on a Synergy MX plate reader (BioTek; λex = 490 nm, λem = 523 nm). Enzyme velocities used to determine the IC50 values were calculated from the linear portion of the fluorescence versus time data. IC50 values were determined with a quadratic four-parameter fit with the BioTek Gen5 software.

66mer LC/MS kinetics Recombinant BoNT/A LC activity toward SNAP141-206 (66mer) substrate was assessed at ambient temperature with 2% final DMSO in 40 mM Hepes, pH 7.4 and analyzed via LC/MS as previously

described[44].

SNAP141-206

66mer

peptide

substrate

(H2N

–

ARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRA TKMLGSG-COOH) and

13

C standard internal control peptide (H2N-R-(13C)A-TKM-(13C)L-

(13C)G-S-(13C)G-COOH) were custom peptides purchased from Bachem (Bubendorf, Switzerland). Briefly, LC/A (20 µL; 2 nM final) was added to assay buffer (40 mM HEPES, pH 7.4; 20 µL) and appropriate concentrations of diluted small molecule (5 µL in 10% DMSO and assay buffer). The mixture was incubated at ambient temperature for 10 min, and the assay was initiated with the addition of various concentrations of 66mer (0-30 µM; 5 µL in 10% DMSO and assay buffer). After 30 minutes, 40 µL aliquots were removed and quenched with 5 µL of


40

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15% TFA (1.5% final), and 13C-labeled internal control was added to a final concentration of 200 nM (5 µL) to yield 50 µL total sample. Ten microliters were injected onto a Zorbax 300SB-C8 column and analyzed with an Agilent 1100 LC/MS system. The product and internal control peak areas were integrated with ChemStation software (Agilent, Santa Clara, CA) and the concentration of product formed was calculated by normalizing the product peak area to the internal control peak area. Binding inhibitory constants (KI) were calculated using models of non-competitive enzyme inhibition with the Prism GraphPad software (GraphPad, La Jolla, CA).

Cytotoxicity and cell viability Murine neuroblastoma cells (Neuro-2as, ATCC #CCL-131) were seeded in 96-well tissue culture plates (Corning Life Sciences, Corning, NY) at a density of 10,000 cells per well (100 µL) in quadruplicate. Cells were grown in DMEM media (Life Technologies, Carlsbad, CA) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA) for 24 hours at 37°C with 5% CO2. The media was removed and replaced with various concentrations of smallmolecules diluted in a serum-free neuronal media comprised of a 1:1 mixture of DMEM and F:12 media (Life Technologies) supplemented with 1x N-2 supplement (Life Technologies) and 1x B-27 supplement without Vitamin A (Life Technologies). The small molecules were diluted in 100 µL of serum-free neuronal media at a final DMSO concentration of 1%. Cells were incubated with compound or vehicle DMSO control for an additional 24 hours at 37°C with 5% CO2. Cell viability was quantified with a CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Seattle, WA) according to the manufacturer’s instructions. The cell viability


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data for the experimental absorbance at A495 nm was normalized to the average absorbance for the DMSO negative control to calculate the percent viability.

Matrix metalloproteinase and thermolysin enzyme assays Recombinant MMP-1 and MMP-12 were purchased from Anaspec (Fremont, CA). Enzymatic activity was quantified with an MMP activity kit (Anaspec) in a 96-well plate according to the manufacturer’s instructions. Briefly, rMMP (50 µL) was incubated with small molecule compound (1 µL) at room temperature for 10 minutes. MMP substrate (50 µL) was added and the fluorescence (λex = 490 nm, λem = 523 nm) was measured with a Synergy MX plate reader (BioTek) every 5 minutes for 60 minutes total. The final assay volume was 101 µL per well, which contained 20 ng rMMP, 0-30 µM small molecule compounds or DMSO vehicle control (1% DMSO final), and 1X substrate. Marimastat (100 nM; Sigma Aldrich, St. Louis, MO) was used as a positive control. Fluorescent values were corrected for background fluorescence and the enzyme velocity was calculated with BioTek Gen5 software. Velocities were normalized to the DMSO vehicle control. Recombinant thermolysin was purchased from Sigma Aldrich, and enzyme activity was quantified with a protease activity assay kit from Abcam (ab112153; Cambridge, MA) in a 96-well plate according to the manufacturer’s instructions. Briefly, thermolysin (10 µL) was incubated with small molecule compound (1 µL) and 80 µL 1X assay buffer at room temperature for 10 minutes. Fluorescent substrate (10 µL) was added and the fluorescence (λex = 540 nm, λem = 590 nm) was measured with a Synergy MX plate reader (BioTek) every 5 minutes for 45 minutes total. The final assay volume was 101 µL per well, which contained 70 nM thermolysin, 0-30 µM small molecule compounds or DMSO vehicle


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control (1% DMSO final), and 1X substrate. Phosphoramidon (5 µM; Sigma Aldrich, St. Louis, MO) was used as a positive control. Fluorescent values were corrected for background fluorescence and the enzyme velocity was calculated with BioTek Gen5 software. Velocities were normalized to the DMSO vehicle control.

ASSOCIATED CONTENT

Supporting Information. Supplemental Figure 1 shows representative dose-response curves for the 3 lead benzimidazole compounds 45, 47, and 90 in the SNAPtide FRET-based LC/A enzyme assay. Supplemental Figure 2 shows off target zinc metalloprotease activity for compounds 4, 33, 37, 47, and 90 with MMP-1, MMP-12 and thermolysin. Supplemental Figure 3 shows the murine neuroblastoma cytotoxicity data for compounds 45 and 90. Supplemental Table 1 lists the potentially reactive moieties and problematic structures that were filtered for during the initial library generation. Supplemental Table 2 contains the structures of inactive spiro(indolthiadiazole) compounds, Supplemental Table 3 contains the structures of inactive 8sulfonamidoquinoline compounds, and Supplemental Tables 4, 5 and 6 include the structures of inactive benzimidazole pyrazolopiperidinone compounds. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *Tobin J. Dickerson - phone: +1 (858) 784-2522; e-mail: [email protected] Author Contributions K. Bompiani and D. Caglic conceived and performed experiments, analyzed data, and co-wrote the manuscript; M. Krutein, M. Hrones, B. Haiyan, and G. Benoni performed experiments and analyzed data; L. Lairson, G. Smith, and T. Dickerson conceived experiments, co-analyzed data, and co-wrote the manuscript. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by a grant from the National Institutes of Health (AI082190 to T.J.D.) and the California Institute of Regenerative Medicine (TB1-01186 and CL1-00502). Conflict of Interest The authors have no conflicts of interest to declare.

ACKNOWLEDGMENTS The authors would like to thank Sujata Godbole for technical assistance.

ABBREVIATIONS BoNT: botulinum neurotoxin; LC: light chain; HC: heavy chain; HTS: high-throughput screen; FRET: Förster resonance energy transfer, SAR: structure activity relationship


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