Amazon S3 - Supplementary Methods€¦ · Web viewElectrophysiological recordings were performed following the methods described in Alexeev et al (3)(2)(2)(2). Full-length cDNAs for

Supplementary

Novel benzodiazepine-like ligands with various anxiolytic, antidepressant or pro-

cognitive profiles

Thomas D. Prevot1,2, Guanguan Li3, Aleksandra Vidojevic4, Keith A. Misquitta1,5, Corey Fee1,5

Anja Santrac4, Daniel E. Knutson3, Michael R. Stephen3, Revathi Kodali3, Nicolas M. Zahn3,

Leggy A. Arnold3, Petra Scholze6, Janet L. Fisher7, Bojan D. Marković8, Mounira Banasr1,2,5,

James M. Cook3, Miroslav Savic4* and Etienne Sibille1, 2,5*. 1Campbell Family Mental Health Research Institute of CAMH, Toronto, Canada2Department of Psychiatry, University of Toronto, Toronto, Canada3Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, Milwaukee,

USA4Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia5Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada6Department of Pathobiology of the Nervous System, Center for Brain Research, Medical

University of Vienna, Vienna, Austria7Department of Pharmacology, Physiology and Neuroscience, University of South Carolina

School of Medicine, Columbia South Carolina, USA8Department of Pharmaceutical Pharmacy, Faculty of Pharmacy, University of Belgrade,

Belgrade, Serbia

*Corresponding authors

Etienne Sibille, Ph.D., CAMH, 250 College street, room 134, Toronto, ON M5T 1R8, Canada

Tel: 416-535-8501, ext 36571; E-mail: [email protected]

Miroslav Savic, Ph.D. Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221

Belgrade. Tel: +3816427551447; E-mail: [email protected]

1

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

ContentsSupplementary Methods..........................................................................................................................4

Chemistry...............................................................................................................................................4

Electrophysiological recordings..........................................................................................................7

Binding studies......................................................................................................................................8

Animals.................................................................................................................................................10

Pharmacokinetic characterization.....................................................................................................11

Behavioral Assessment......................................................................................................................13

Supplementary Tables...........................................................................................................................15

Supplementary Table S1: Statistical analysis for potentiation at α1/2/3/4/5/6, β1/3,γ/δ GABAA-receptors...........................................................................................................................15

Supplementary Table S2: Preferential potentiation at α5-GABAA-R....................................16

Supplementary Table S3: Statistical comparison between α1β1γ2 and α1β3γ2 potentiation at 100nM and 1µM..........................................................................................................................17

Supplementary Table S4: Half-life and percentage of compound remaining after incubation with human or mouse liver microsomes....................................................................18

Supplementary Table S5: Electrophysiological records of the GL series for α1β3γ2 and α5β3γ2 GABAA receptor subtypes...............................................................................................19

Supplementary Table S6: Ki values for all three compounds at α1/2/3/5β3γ2 receptor....20

Supplementary Table S7: The approximated % of GABA potentiation and the values of electrophysiological potentiation obtained at α2β3γ2 and α3β3γ2 receptors (presented in Figure 1), for the estimated brain free concentrations of GL-II-73, GL-II-74 and GL-II-75, administered at the doses of 1 mg/kg and 10 mg/kg, 30 minutes after administration.........21

Supplementary Table S8: Statistical analysis for each behavioral test assessing emotionality in mice........................................................................................................................22

Supplementary Table S9: Statistical analysis for the Y-maze alternation rate after acute administration in young mice (stressed and non-stressed).......................................................23

Supplementary Table S10: Statistical analysis for the Y-maze alternation rate after acute administration in old mice..............................................................................................................24

Supplementary Table S11: Statistical analysis for the Y-maze alternation rate after sub-chronic administration in young and old mice.............................................................................25

Supplementary Figures..........................................................................................................................26

Supplementary Figure S1. General synthetic route to amides..............................................26

2

29

30

31

32

33

34

35

36

37

3839

40

4142

4344

4546

47

48495051

5253

5455

5657

5859

60

61

Supplementary Figure S2. Concentration of GL-II-73, GL-II-74 and GL-II-75 in the brain of male C57BL/6 mice after administration of a compound at doses of 1, 5 or 10mg/kg..........27

Supplementary Figure S3. Locomotor activity changes induced by DZP injection.............28

Supplementary Figure S4. Locomotor activity changes induced by αPAM injection..........29

Supplementary Figure S5. Effect of GL-II-73, GL-II-74, GL-II-75 and DZP on alternation rate in a Y-maze alternation task assessing working memory in non-stressed adult mice...30

Supplementary Figure S6. Scheme of repeated assessment of cognitive performances in the Y-Maze in function of stress exposure and α5-PAM administration...................................31

3

6263

64

65

6667

6869

70

71

Supplementary Methods

ChemistryBased on previous compounds (1) synthetized and screened which proved their potency at the

α5-subtype of the Bz/GABAAA-ergic receptors, a new series of metabolically more stable

compounds were prepared following the steps described below (2).

(R)-8-Ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid SH-053-2'F-R-CH3-acid (2)

The ethyl ester SH-053-2'F-R-CH3 1 (20.0 g, 51.6 mmol) was dissolved in DCM (200 mL) and

EtOH (500 mL), after which solid NaOH (16.6 g, 413 mmol) was added to the solution. This

reaction mixture was heated to 55 ˚C for 0.5 h and the EtOH was removed under reduced

pressure. The remaining aq solution which remained was stirred at 0 ˚C for 10 min and then aq

HCl (1M) was added dropwise to the solution until the pH was 5 (pH paper). A pale white

precipitate which formed was left in the solution for 10 min and was then collected by filtration,

and washed with cold water after which the aq layer was also allowed to stand at rt for 10 h to

yield additional acid. The combined solids were dried in a vacuum oven at 80 ˚C for 7 h to get

pure acid 2 as a white powder (18.4 g, 51.2 mmol, 99.2%): mp 196-198 °C; []D25 = +4.00 (c

0.80, CHCl3); 1H NMR (300 MHz, DMSO-d6): δ 8.42 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.81 (d, J

= 7.8 Hz, 1H), 7.65 – 7.49 (m, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 6.51 (q, J =

6.7 Hz, 1H), 4.37 (s, 1H), 1.14 (d, J = 6.8 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): δ 165.03 (s),

162.63 (s), 159.82 (d, J = 248.4 Hz), 140.49 (s), 136.40 (s), 135.52 (s), 134.78 (d, J = 1.0 Hz),

133.18 (s), 133.14 (s), 132.59 (d, J = 5.8 Hz), 131.88 (s), 129.35 (s), 128.95 (d, J = 12.6 Hz),

125.15 (d, J = 1.8 Hz), 123.97 (s), 121.05 (s), 116.37 (d, J = 20.9 Hz), 83.37 (s), 82.01 (s),

49.74 (s), 15.10 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C21H15FN3O2 360.1143; found

360.1140.

General procedure for amides

A mixture of the acid SH-053-2'F-R-CH3-acid 2 (2 g, 5.56 mmol), thionyl chloride (55.6 mmol)

and dry DCM (50 mL) was placed in an oven dried round bottom flask under argon. This

suspension was allowed to reflux at 60 ˚C for 2 h under argon. The absence of the starting

material was confirmed by TLC (silica gel). The organic solvent and excess thionyl chloride

were evaporated under reduced pressure, which was repeated 5 times with dry DCM (20 mL).

4

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

The yellow residue, which was obtained, was dissolved in dry DCM (50 mL) and cooled to 0 ˚C

for 10 min under argon. Then the appropriate amine was added (11.12 mmol), followed by

administration of Et3N (5.56 mmol) to the reaction mixture at 0˚C and the mixture was then

allowed to warm to rt and stirred for 2-7 h depending on the amine. After the completion of the

reaction the solvent was removed under reduced pressure. The residue was treated with ice

cold water and extracted with DCM (3 X 50 mL). The combined organic layer was washed with

brine (20 mL). The solvent was removed under reduced pressure and the residue was purified

by column chromatography on silica gel to yield the corresponding pure amides.

(R)-8-Ethynyl-6-(2-fluorophenyl)-N,N,4-trimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxam GL-II-73 (3a)

The amide 3a was prepared from 2 following the general procedure with dry dimethylamine as

the nucleophile. The crude residue was purified by column chromatography (silica gel, EtOAc

and 1% MeOH) to yield pure dimethyl amide 3a as a light yellow powder (1.5 g, 3.9 mmol,

70%): mp 189-190 °C; []D25 = +40.98 (c 0.61, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.92 (s,

1H), 7.68 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.45 – 7.35 (m,

2H), 7.21 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 9.3 Hz, 1H), 4.28 (q, J = 6.0 Hz, 1H), 3.14 (s, 1H), 3.09

(s, 3H), 2.96 (s, 3H), 1.89 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 166.10 (s), 162.69

(s), 160.19 (d, J = 250.4 Hz), 135.34 (s), 134.62 (d, J = 9.0 Hz), 133.90 (s), 133.71 (s), 132.94

(d, J = 8.4 Hz), 132.47 (s), 132.12 (d, J = 9.1 Hz), 131.29 (s), 129.18 (s), 127.57 (d, J = 11.6

Hz), 124.51 (d, J = 2.2 Hz), 122.87 (s), 121.52 (s), 116.17 (d, J = 21.3 Hz), 81.52 (s), 79.63 (s),

52.13 (s), 39.09 (s), 35.04 (s), 18.37 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for

C23H20FN4O 387.1616; found 387.1626.

(R)-N-Ethyl-8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide GL-II-74 (3b)

The amide 3b was prepared from 2 following the general procedure with dry ethylamine as the

nucleophile. The crude residue was purified by column chromatography (silica gel, 3:2 EtOAc

and hexane) to yield pure ethyl amide 3b as a white powder (1.6 g, 4.3 mmol, 78%): mp 215-

216 °C; []D25 = +44.07 (c 0.59, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.83 (s, 1H), 7.65 (dd, J =

16.8, 7.8 Hz, 2H), 7.53 (d, J = 8.3 Hz, 1H), 7.47 – 7.39 (m, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.17 (s,

1H), 7.01 (t, J = 9.3 Hz, 1H), 6.90 (q, J = 6.3 Hz, 1H), 3.61 – 3.34 (m, 2H), 3.15 (s, 1H), 1.26 (d,

J = 9.0 Hz, 3H), 1.22 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 162.81 (s), 162.51 (s),

160.13 (d, J = 250.7 Hz), 138.81 (s), 134.96 (s), 134.71 (s), 133.90 (s), 133.37 (s), 131.84 (s),

5

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

131.76 (s), 131.37 (s), 129.78 (s), 128.86 (d, J = 10.6 Hz), 124.44 (d, J = 3.4 Hz), 122.04 (s),

121.38 (s), 116.06 (d, J = 21.5 Hz), 81.55 (s), 79.52 (s), 49.89 (s), 33.70 (s), 15.02 (s), 15.01(s);

HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C23H20FN4O 387.1616; found 387.1618.

(R)-N-Cyclopropyl-8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide GL-II-75 (3c)

The amide 3c was prepared from 2 following the general procedure with dry cyclopropylamine

as the nucleophile. The crude residue was purified by a column chromatography (silica gel, 1:1

EtOAc and hexane) to yield pure cyclopropyl amide 3c as a white powder (1.8 g, 4.5 mmol,

82%): mp 231-232 °C; []D25 = +3.81 (c 0.46, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.80 (s, 1H),

7.64 (dd, J = 15.9, 7.6 Hz, 2H), 7.52 (d, J = 8.3 Hz, 1H), 7.47 – 7.37 (m, 2H), 7.30 – 7.19 (m,

2H), 7.01 (t, J = 9.3 Hz, 1H), 6.89 (q, J = 7.0 Hz, 1H), 3.15 (s, 1H), 2.93 – 2.67 (m, 1H), 1.26 (d,

J = 6.8 Hz, 3H), 0.87 – 0.76 (m, 2H), 0.65 – 0.51 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 164.02

(s), 162.82 (s), 160.13 (d, J = 249.9 Hz), 138.91 (s), 134.97 (s), 134.65 (s), 133.89 (s), 133.37

(s), 131.80 (d, J = 9.0 Hz), 131.58 (s), 131.36 (s), 129.78 (s), 128.81 (d, J = 14.6 Hz), 124.45 (d,

J = 3.4 Hz), 122.04 (s), 121.42 (s), 116.06 (d, J = 21.5 Hz), 81.53 (s), 79.55 (s), 49.88 (s), 22.10

(s), 14.99 (s), 6.54 (s), 6.50 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C24H20FN4O

399.1616; found 399.1621.

(R)-(8-Ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)(pyrrolidin-1-yl)methanone GL-II-76 (3d)

The amide 3d was prepared from 2 following the general procedure with dry pyrrolidine as the

nucleophile. The crude residue was purified by column chromatography (silica gel, 6:4 EtOAc

and hexane) to yield a mixture of rotamers 3d as a white powder (3:2 ratio by 1H NMR, 1.8 g,

4.4 mmol, 80%): mp 173-174 °C; []D25 = -23.91 (c 0.70, CHCl3); 1H NMR Major rotamer (300

MHz, CDCl3): δ 7.90 (s, 1H), 7.73 – 7.58 (m, 2H), 7.51 (t, J = 9.0 Hz, 1H), 7.47 – 7.35 (m, 2H),

7.23 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 9.3 Hz, 1H), 4.31 (q, J = 6.1 Hz, 1H), 3.66 – 3.56 (m, 4H),

3.14 (s, 1H), 1.95 (d, J = 6.4 Hz, 3H), 1.93 – 1.69 (m, 4H); 1H NMR Minor rotamer (300 MHz,

CDCl3): δ 7.85 (s, 1H), 7.74 – 7.57 (m, 2H), 7.51 (t, J = 9.0 Hz, 1H), 7.47 – 7.35 (m, 2H), 7.23 (t,

J = 7.5 Hz, 1H), 7.01 (t, J = 9.3 Hz, 1H), 6.49 (q, J = 6.6 Hz, 1H), 3.82 (dd, J = 58.0, 32.3 Hz,

4H), 3.46 (d, J = 26.4 Hz, 4H), 3.14 (s, 1H), 1.28 (d, J = 6.4 Hz, 3H); HRMS (ESI/IT-TOF) m/z:

[M + H]+ Calcd for C25H22FN4O 413.1772; found 413.1772.

6

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

Electrophysiological recordingsElectrophysiological recordings were performed following the methods described in Alexeev et

al (3)(2)(2)(2). Full-length cDNAs for GABAA receptor subtypes (generously provided by Dr.

Robert Macdonald, Vanderbilt University and Dr. David Weiss, University of Texas Health

Science Center, San Antonio, TX) in mammalian expression vectors were transiently

transfected into the HEK-293T cell line (GenHunter, Nashville, TN). All subtypes were rat clones

except for α2, which was a human clone. Cells were transiently transfected using calcium

phosphate precipitation. Plasmids encoding GABAA receptor subtype cDNAs were added to the

cells in 1:1:1 ratios (α:β:γ) of 2 μg each (4)(3)(3)(3). Cells were patch-clamped at −50 mV in the

whole-cell recording configuration. GABA was diluted into the bath solution from freshly made or

frozen stocks in water. Compounds were dissolved in DMSO and diluted into the bath solution

with the highest DMSO level applied to cells of 0.01%. Solutions containing GABA or GABA +

compounds were applied to cells for 5s using a 3-barrelled solution delivery device controlled by

a computer-driven stepper motor (SF-77B, Harvard Apparatus, Holliston, MA, open tip

exchange time of <50ms). There was a continuous flow of external solution through the

chamber. Currents were recorded with an Axon 200B (Foster City, CA) patch clamp amplifier.

Whole-cell currents were analyzed using the programs Clampfit (pClamp9 suite, Axon

Instruments, Foster City, CA) and Prism (Graphpad, San Diego, CA). Concentration−response

data was fit with a four-parameter logistic equation

current=minimumcurrent+(maximumcurrent−minimumcurrent)1+(10 (logEC 50−log (modulator ) )n) where n represents the Hill

number. All fits were made to normalized data with current expressed as a percentage of the

response to GABA alone for each cell.

7

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

Binding studiesBinding studies were performed on HEK-293 cells following the methods described in (5)Human

embryonic kidney (HEK) 293 cells (AmericanType Culture Collection ATCCs CRL-1574™) were

maintained in Dulbecco's modified Eagle medium (DMEM, high glucose, GlutaMAX™

supplement, Gibco61965-059, ThermoFisher, Waltham, Massachusetts, USA) supplemented

with 10% fetal calf serum (Sigma-AldrichF7524, St. Louis, Missouri, USA), 100U/ml Penicillin-

Streptomycin (Gibco 15140-122, ThermoFisher, Waltham, Massachusetts, USA) and MEM

(Non-Essential Amino Acids Gibco 11140-035, ThermoFisher, Waltham, Massachusetts, USA)

on 10cm Cell culture dishes (Cell+, Sarstedt, Nürnbrecht, Germany) at 37 °C and 5% CO2.

HEK293 cells were transfected with cDNAs encoding rat GABAA-receptor subunits subcloned

into pCI expression vectors. The ratio of plasmids used for transfection with the calcium

phosphate precipitation method (Chen and Okayama,1987) were 3 mg α(1,2,3or5): 3 mg β3

and 15 mg γ2 per 10cm dish. Medium was changed 4–6 h after transfection. Cells were

harvested 72 days after transfection by scraping into phosphate buffered saline. After

centrifugation (10 min,12000 g, 4°C) cells were resuspended in TC50 (50 mM Tris-

CitratepH¼7.1), homogenized with an Ultra-Turraxs (IKA, Staufen, Germany) and centrifuged

(20 min,50000 g). Membranes were washed three times in TC50 as described above and frozen

at -20°C until use. Frozen membranes were thawed, re-suspended in TC50 and incubated for

90 min at 4°C in a total of 500 ml of a solution containing 50 mM Tris/citrate buffer, pH=7.1, 150

mM NaCl and 2 nM [3H]-Flunitrazepam (Perkin Elmer New England Nuclear, Waltham,

Massachusetts, USA) in the absence or presence of either 5 mM diazepam (Nycomed, Opfikon,

Switzerland) (to determine unspecific binding) or various concentrations of receptor ligands

(dissolved in DMSO, final DMSO- concentration 0.5%). Membranes were filtered through

Whatman GF/B filters and the filters were rinsed twice with 4ml of ice-cold 50 mM Tris/citrate

buffer. Filters were transferred to scintillation vials and subjected to scintillation counting after

the addition of 3 ml Rotiszint Eco plus liquid scintillation cocktail. Non-specific binding

determined in the presence of 5mM Diazepam was subtracted from total [3H]-Flunitrazepam

binding to result in specific binding. In order to determine the equilibrium binding constant KD of

3H-Flunitrazepam for the various receptor-subtypes, membranes were incubated with various

concentrations of 3H-Flunitrazepam in the absence or presence of 5 mM Diazepam. Saturation

binding experiments were analyzed using the equation Y=Bmax*X/(KD+X). Non-linear

regression analysis of the displacement curves used the equation: log(inhibitor) vs. response-

variable slope with Top=100% and Bottom=0% Y=100/(1+10^((logIC50-x)*Hill-slope)). Both

8

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

analyses were performed using GraphPad Prism (LaJolla California USA). Drug concentrations

resulting in half maximal inhibition of specific 3H-Flunitrazepam binding (IC50) were converted to

Ki values by using the Cheng-Prusoff relationship (Cheng andPrusoff, 1973) Ki=IC50/(1+(S/KD))

with S being the concentration of the radioligand (2 nM) and the KD values listed in

Supplementary Table 6.

9

219

220

221

222

223

AnimalsYoung (2-3 months) or old (21-22 months) C57BL/6 mice were were obtained from Jackson

Laboratories (US), or the Military Medical Academy (Serbia) and kept in normal housing

conditions (21±2°C, relative humidity 40-45%) with a 12hr light-dark cycle (7am ON), water and

food ad libitum. Prior to behavioral assessment, animals were handled daily for 5 min, over 5

consecutive days, to reduce acute anxiety-like responses (6). Testing took place during the light

phase, and was conducted in accordance with the Canadian or U.S. institutional animal care

committee and the Ethical Commission on Animal Experimentation of the Faculty of Pharmacy

in Belgrade (carried out in accordance with the EEC Directive 86/609).

Compound preparation and administrationFor pharmacokinetic assays and behavioral testing, compounds were diluted in a vehicle

solution containing 85% distilled H2O, 14% propylene glycol (Sigma Aldrich) and 1% Tween 80

(Sigma Aldrich) to be administered intraperitoneally (i.p.) at a volume of 10 ml/kg. Working

solutions were prepared at 1, 5 or 10 mg/kg and adjusted to body weight before injection. DZP

was used as a non-selective GABAA-R PAM with known anxiolytic and no cognitive efficacies.

DZP was administered i.p. at 1.5 mg/kg in a 10ml/kg volume, based on previous studies.

For sub-chronic administration in the drinking water for 10 consecutive days, compounds

were diluted in tap-water at 30mg/kg, stirred over night at room temperature and given to the

animals in glass bottles. Bottles were changed every other day to provide freshly prepared

solutions.

10

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

Pharmacokinetic characterization

Metabolic stability assessmentThe method used was previously described in (7). The test compounds were incubated at 10µM

with active or heat-inactivated human liver microsomes and cofactors. Aliquots were removed at

0, 10, 20, 30, 40, 50, 60 and 120 min and mixed with acetonitrile containing internal standard for

analysis. Samples were extracted and assayed using a liquid chromatography/tandem mass

spectrometry (LC-MS/MS) analytical method. On the day of the experiment, the test

compounds, prepared in DMSO, were diluted in the 100mM phosphate buffer (pH=7.4) to

achieve appropriate final concentrations. The test compounds were incubated with human or

mouse liver microsomes (0.5mg protein/mL; BD Gentest and Life Technologies) and

appropriate cofactors (2.5mM NADPH and 3.3 nM magnesium chloride) in 100mM phosphate

buffer, pH 7.4 (0.1% final DMSO), in a 37°C water bath. At selected time points, a single 100µL

aliquot was removed from each sample and mixed with 200µL of chilled acetonitrile containing

internal standard. Following brief vortexing and centrifugation, the samples were further diluted

for subsequent LC-MS/MS analysis, in triplicate. Experimental controls consisted of: (a)

incubation of all components except test compound for 0 and 60 min, (b) incubation of

Verapamil (positive control) at 10 lM for 0, 10, 20, 30, 40, 50, 60 and 120 min, and (c) incubation

of 1 and 10 µM test compound and 10 µM Verapamil with heat-inactivated microsomes (0.5 mg

protein/mL) for 0 and 60 min. Samples were analyzed by LC–MS/MS in multiple reaction

monitoring mode using positive-ion electrospray ionization. The details of the LC–MS/MS

method can be provided upon request. To determine metabolic stability, the percent remaining

at each time point was calculated:

%remaining at timeT=¿¿¿.

Pharmacokinetic profiles

Aiming to obtain the respective pharmacokinetic profiles, young mice (n = 15 per compound)

were divided into five groups of animals; each group contained three animals and corresponded

to predetermined time intervals (5, 20, 60, 180, and 720 min). The mice were treated

intraperitoneally (i.p.) at the 10 mg/kg dose and in a volume of 10 ml/kg. Also, brain

concentrations were measured 20 min after i.p. administration of the 1 mg/kg dose.

In order to parallel the design of behavioral studies, additional experiments were performed with

compounds dosed at 1, 5 and 10 mg/kg, where brain concentrations were determined 30 min

11

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

after single i.p. injection, as well as after three i.p. injections, administered 24 h, 20 h and 1 h

prior to the concentration measurement.

At the appropriate time intervals, the blood samples were collected via cardiac puncture of mice

anesthetized with ketamine solution (10% Ketamidor, Richter Pharma Ag, Wels, Austria, dosed

i.p. at 100 mg/kg) in heparinized syringes and centrifuged at 800 rcf for 10 min to obtain plasma

samples. Mice were decapitated and brains were weighed, homogenized in 1.25 ml of methanol

and centrifuged at 3400 rcf for 20 min. Compounds were extracted from plasma and

supernatants of brain tissue homogenates by solid phase extraction, using Oasis HLB

cartridges (Waters Corporation, Milford, Massachusetts). The procedure of sample preparation

and determination of concentration by ultraperformance liquid chromatography–tandem mass

spectrometry (UPLC–MS/MS) with Thermo Scientific Accela 600 UPLC system connected to a

Thermo Scientific TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo

Fisher Scientific, San Jose, California), equipped with electrospray ionization (ESI) source, has

been already described in detail. Non-compartmental pharmacokinetic analysis was performed

using PK Functions for Microsoft Excel software (by Joel Usansky, Atul Desai, and Diane Tang-

Liuwere), while graphs were constructed in commercial statistical software Sigma Plot 12

(Systat Software Inc., USA).

In vitro hydrolytic stability study in plasmaThe compounds were tested for their hydrolytic stability in vitro at 37 °C, utilizing blank mouse

plasma spiked with the respective compound and internal standard (SH-I-048A; synthesized at

the Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, USA), as

detailed in (5).

Plasma protein and brain tissue binding studies The rapid equilibrium dialysis assay used to determine free fraction of GL-II-73, GL-II-74 and

GL-II-75 in mouse plasma and brain tissue was the same as in (8). Free concentrations in the

brain were calculated by multiplying the obtained total brain concentrations with the appropriate

free fractions determined by rapid equilibrium dialysis (12.14%, 4.49% and 1.34% for GL-II-73,

GL-II-74 and GL-II-75, respectively).

12

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

Behavioral Assessment Elevated plus MazeThe elevated plus maze (EPM) is designed to assess anxiety-like behaviors in rodents. The

maze is made of four white Plexiglas arms, two open arms (29x7 cm), and two enclosed arms

(29x7x17 cm) that formed a cross shape with the two open arms opposite each other. The maze

is 90 cm above the floor and illuminated at 100 lux. Young male and female (50%) mice

(Jackson Laboratories) were injected i.p. with vehicle, 5 or 10mg/kg dose of test compound or

1.5mg/kg of DZP, 30 minutes prior to testing. They were placed individually on the central

platform, facing an open arm, and allowed to explore the apparatus for 10 min. Behavior was

recorded with a digital camera and analyzed using ANY-maze Video Tracking System software

(Stoelting Co, Wood Dale, IL, USA). The time in the open arm was used to calculate the

percentage of time in the open arm presented in the results section using the following formula:

PercentageTime∈openarm=Time∈the openarmTotalTime

x 100.

Forced Swim Test Young male mice were tested in the forced swim test (FST), assessing for antidepressant

efficacy. Mice (Jackson Laboratories) were sub-chronically injected via i.p. with vehicle or 1, 5 or

10 mg/kg of test compounds 24 h, 20 h and 1 h before testing, as per standard methods in the

field for testing potential antidepressant compounds. Following standard protocol for DZP,

animals were injected only once at the dose of 1.5 mg/kg, 1 h prior testing. The protocol used

was initially described by Porsolt (9), where mice were placed in an inescapable transparent

tank filled with water (25 cm, 26±1oC). Animals were recorded for a period of 6min and a manual

count of the immobile time in the tank was measured. Immobility is defined as the minimum

amount of movement to stay afloat, between the second and the sixth minute of testing.

Compounds that reduced immobility in the FST are considered to have potential antidepressant

actions.

Spontaneous alternation in the Y-MazeYoung male and female (50%) mice (Jackson Laboratories, Bar Harbor, USA) and old males

(22 months-old) were single-housed. Young animals were subjected to a chronic stress protocol

(CS) to induce a working memory deficit. They were placed in a 50 ml Falcon® tube, twice a

day, for 1 hr during their diurnal cycle. CS was not applied on testing days. The apparatus was a

black plastic Y-maze with 3 arms, 26cm long, 8cm wide with sidewalls 13cm high and all

13

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

separated by 120°; each arm having a sliding door. The protocol used was a modified version of

the one described in Vandesquille et al (10). Briefly, mice were first habituated to the apparatus

and to distal cues during 2 consecutive days over a 10 min free exploration session. The

following day, animals performed a training session consisting of seven successive trials where

they have to alternate between the 2 goal arms with an inter trial interval (ITI) of 30 sec. The

same general procedure used in the training session was implemented 24h later, except that

the ITI was lengthened to 90s or 60s, for young or old animals respectively. Animals were

acutely injected i.p. with vehicle, 1, 5 or 10 mg/kg dose of the test compounds or 1.5mg/kg DZP,

30 min before the beginning of the test. To dissociate memory deficits from an eventual

progressive loss of motivation, an 8th trial was added to the series which was separated from the

7th trial by a shorter ITI (5s). All animals failing to alternate at the 8 th trial were excluded from the

analysis. The alternation rate was calculated and was expressed in percentage:

Alternationrate= AlternationMaximumalternation possible

x100. The percentage of alternation during the

entire task was considered as an index of working memory performance (50% of alternation

corresponding to a random alternation rate). This test can be repeated weekly, with no

requirement for habituating the animals again. This allows us to reduce the number of animals

and test multiple doses or compounds in the same animals, with a week of wash-out between

experiments. We confirmed that the compounds were washed out of the organism of the mouse

by conducting a pilot experiment that validated our design (for more details, see

Supplementary Figure 6).

Spontaneous locomotor activityBased on previous studies from our team, we analyzed the influence of each compound on

spontaneous locomotor activity (SLA) of young mice (n=27). The apparatus was a white and

opaque Plexiglas chamber (40×25×35 cm) under dim red light (20 lux). A digital camera

mounted above the apparatus recorded the animal activity, which was tracked and analyzed

using ANY-maze Video Tracking System software (Stoelting Co, Wood Dale, IL, USA).

Compounds or vehicle was applied i.p. and a single mouse was immediately placed in the

center of the chamber. The activity was tracked for a total of 60min. Chambers were cleaned

with 70% ethanol after every trial.

14

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

Supplementary Tables

Supplementary Table S1: Statistical analysis for potentiation at α1/2/3/4/5/6, β1/3,γ/δ GABAA-

receptors

N t-test p N t-test p N t-test p N t-test p N t-test p N t-test p N t-test p N t-test pα1ß3γ2 5 2.9 0.04 5 8.05 0.0013 5 3.37 0.02 5 3.17 0.033 6 6.08 0.0017 5 6.9 0.0023 5 15.9 0.0001 5 6.68 0.0026α2ß3γ2 5 2.1 0.1 5 4.31 0.012 6 3.8 0.011 5 7.61 0.0016 6 4.1 0.009 5 3.4 0.026 5 34.04 0.0001 5 11.67 0.0003α3ß3γ2 6 7.6 0.0006 6 8.03 0.0005 5 10.2 0.0005 5 6.22 0.003 6 5.91 0.002 5 6.05 0.0038 6 5.33 0.0031 6 4.34 0.0074α4ß3γ2 5 0.35 0.73 5 2.12 0.1 5 -0.38 0.7 5 -0.44 0.68 5 0.99 0.37 5 2.01 0.11 6 1.41 0.2169 6 1.29 0.25α5ß3γ2 5 5.1 0.006 5 9.07 0.0008 5 2.9 0.04 5 7.07 0.0021 5 3.29 0.03 5 4.67 0.0095 6 4.04 0.0099 6 5.02 0.004α6ß3γ2 5 1.8 0.14 5 0.73 0.5 5 1.8 0.14 5 1.59 0.18 5 2.21 0.09 6 0.46 0.67 4 2.04 0.13 4 0.13 0.9

α1ß1γ2 4 2.35 0.077 4 4.67 0.0095 4 2.7 0.054 4 5.09 0.007 5 10.99 0.0001 5 9.97 0.0002 4 2.09 0.1046 4 8.7 0.0009

α1ß3δ 4 0.151 0.887 3 -0.47 0.669 4 2.27 0.0852 4 -0.77 0.4866

GL-II-74 GL-II-75 GL-II-76GL-II-731µM 100 nM 1µM100 nM 1µM 100 nM 1µM 100 nM

175.380 40 5.962 <.0001116.500 4 2.945 .0422162.420 4 8.052 .0013117.320 4 3.373 .0280189.680 4 3.173 .0338203.183 5 6.087 .0017346.600 4 6.926 .0023117.280 4 15.945 <.0001144.500 4 6.683 .0026

Moy. DDL t pa1, Totala1, Gl-II-73 (100nM)a1, Gl-II-73 (1uM)a1, Gl-II-74 (100nM)a1, Gl-II-74 (1uM)a1, Gl-II-75 (100nM)a1, Gl-II-75 (1uM)a1, Gl-II-76 (100nM)a1, Gl-II-76 (1uM)

Test-t univarié Eclaté par : GLMoy. théorique = 100

171.336 41 5.562 <.0001115.020 4 2.102 .1034134.100 4 4.315 .0125119.333 5 3.874 .0117199.580 4 7.612 .0016194.217 5 4.106 .0093324.820 4 3.425 .0267126.620 4 34.044 <.0001162.820 4 11.670 .0003


Test-t univarié Eclaté par : GLMoy. théorique = 100

169.693 44 6.785 <.0001123.017 5 7.625 .0006136.567 5 8.036 .0005126.640 4 10.296 .0005199.220 4 6.221 .0034183.617 5 5.917 .0020321.740 4 6.055 .0038124.267 5 5.333 .0031165.567 5 4.347 .0074


Test-t univarié Eclaté par : GLMoy. théorique = 100t-test comparison to 100%.

15

363

364

365

366

367

Supplementary Table S2: Preferential potentiation at α5-GABAA-R

Subunit N

ANOVA values

p-value NANOVA values



p-value

α1 5 0.6 5 0.0015 5 0.21 5 0.0007α2 5 0.04 5 0.0017 5 0.38 5 0.059α3 6 0.44 6 0.0068 6 0.84 6 0.007α4 5 0.0002 5 <0.0001 5 0.0029 5 <0.0001α5 5 * 5 * 5 * 5 *α6 5 0.0007 5 0.0002 5 0.006 5 <0.0001α1 5 <0.0001 5 <0.0001 5 0.12 5 0.008α2 5 <0.0001 5 0.0002 5 0.19 5 0.04α3 6 <0.0001 6 0.0002 6 0.25 6 0.001α4 5 <0.0001 5 <0.0001 5 0.02 5 <0.0001α5 5 * 5 * 5 * 5 *α6 5 <0.0001 5 <0.0001 5 0.01 5 <0.0001

F(5,20)=8.2;

p=0.0002

F(5,15)=7.7;

p=0.0009

F(5,15)=11.8;

p<0.0001

F(5,15)=9.6;

p=0.0003

GL-II-73 GL-II-74 GL-II-75 GL-II-76

100n

M

1µM

F(5,20)=6.1;

p=0.0013

F(5,20)=44.3;

p<0.0001

F(5,20)=6.1;

p=0.0013

F(5,20)=16.9;

p=0.002

* refers to the group used as reference for post-hoc statistical analysis.

16

368369

370

371

Supplementary Table S3: Statistical comparison between α1β1γ2 and α1β3γ2 potentiation at

100nM and 1µM

N ANOVA p-value N ANOVA p-value N ANOVA p-value N ANOVA p-valueß3 5 * 5 * 6 * 5 *ß1 5 0.59 5 n/a 6 n/a 5 n/aß3 5 * 5 * 5 * 5 *ß1 5 0.033 5 n/a 5 n/a 5 n/a

100nM

1µM

GL-II-73 GL-II-74 GL-II-75 GL-II-76

F(1,16)=5.7 ; p=0.02

F(1,16)=0.2 ; p=0.6

F(1.19)=0.14 ; p=0.7

F(1,16)=0.19 ; p=0.66


17

372

373

374

375

376

Supplementary Table S4: Half-life and percentage of compound remaining after incubation

with human or mouse liver microsomes

Compound Name

Half-life (min) (HLM)

% left after 2 hr. (HLM)

Half-life (min)(MLM)

% left after 2 hr. (MLM)

GL-II-73 280 ± 31 73.34 ± 0.6 472.7 ± 56 81.6 ± 0.27

GL-II-74 403 ± 40 82 ± 0.52 72 ± 6.7 32.1 ± 0.54

GL-II-75 163 ± 12 61.9 ± 0.9 106 ± 8 50 ± 0.55

GL-II-76 154 ± 5 75 ± 0.1 23 ± 1 18.6 ± 0.3

18

377

378

379

380

Supplementary Table S5: Electrophysiological records of the GL series for α1β3γ2 and

α5β3γ2 GABAA receptor subtypes

α1β3γ2 α5β3γ2

Compound#

cellsAverage EC50

(±SEM)

Average max potentiation

(±SEM)

# cell

s

Average EC50

(±SEM)

Average max potentiation

(±SEM)

GL-II-73 3 892.3 ± 287.0 nM 219.9 ± 42.7% 3 890.8 ± 287.0 nM 382.8 ± 36.2%

GL-II-74 4 1136.2 ± 170.7 nM 367.3 ± 17.3% 3 206.9 ± 58.5 nM 371.2 ± 26.6%

GL-II-75 3 215.3 ± 46.2 nM 409.5 ± 16.8% 3 190.8 ± 58.8 nM 326.4 ± 19.3%

Diazepam 3 48.5 ± 6.3 nM 335.7 ± 13.0% 4 78.2 ± 8.5 nM 266.0 ± 10.8%

19

381

382

383

384

Supplementary Table S6: Ki values for all three compounds at α1/2/3/5β3γ2 receptor

132 232 332 532

GL-II-73 55 ± 4µM 30 ± 5 µM 63 ± 7µM 5 ± 2 µM***

GL-II-74 1060 ± 100 nM 809 ± 130nM 1250 ± 51nM 83 ± 12nM***

GL-II-75 542 ± 88 nM 789 ± 130 nM 480 ± 62 nM 79 ± 2 nM**

;DZP 22.4 ± 5.4 nM 13.4 ±1.1 nM 20.4 ± 2.8 nM 12.1 ± 1.2 nM

Significance towards 132 ; *:p<0.05,**:p<0.01,***:p<0.001

20

385

386

387

388

Supplementary Table S7: The approximated % of GABA potentiation and the values of

electrophysiological potentiation obtained at α2β3γ2 and α3β3γ2 receptors (presented in Figure 1), for the estimated brain free concentrations of GL-II-73, GL-II-74 and GL-II-75, administered

at the doses of 1 mg/kg and 10 mg/kg, 30 minutes after administration.

DoseCompound

Estimated brain free concentrations (ng/g)

Estimatedbrain free concentrations (nmol/kg)

Approximated % of GABA potentiation

α1β3γ2

α2β3γ2

α3β3γ2

α5β3γ2

1

mg/k

g

GL-II-73 5.67 14.67 100.96<120

%

<120

%103.93

GL-II-74 4.07 10.53 100.71<120

%

<120

%118.68

GL-II-75 2.29 5.76 100.34<120

%

<120

%99.97

10

mg/k

g

GL-II-73 117.29 303.53 131.68 124.25 129.08 179.62

GL-II-74 94.93 245.67 146.18 150.02 154.85 256.67

GL-II-75 18.85 47.32 162.90 153.24 139.49 136.76

21

389

390

391

392

393

394

Supplementary Table S8: Statistical analysis for each behavioral test assessing emotionality in

mice

AssayDose

(mg/kg) NANOVA values

p-valueDose


p-valueDose


p-valueDose


p-value

0 (i.p.) 13 - 0 (i.p.) 14 - 0 (i.p.) 13 - 0 (i.p.) 11 -5 (i.p.) 13 0.24 5 (i.p.) 13 0.32 5 (i.p.) 14 - 1.5 (i.p.) 10 0.02

10 (i.p.) 14 0.009 10 (i.p.) 14 0.005 10 (i.p.) 13 -0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 12 -1 (i.p.) 8 0.53 1 (i.p.) 8 0.08 1 (i.p.) 8 0.96 1.5 (i.p.) 12 0.0045 (i.p.) 6 0.58 5 (i.p.) 8 0.02 5 (i.p.) 8 0.031

10 (i.p.) 8 0.0003 10 (i.p.) 8 0.0004 10 (i.p.) 9 0.0031

F(2,37)=2.8;

p=0.07

F(3,29)=5.4 ;

p=0.004

F(2,37)=3.7 ;

p=0.03

F(3,26)=7.2 ;

p=0.001

Forced Swim test

Elevated Plus Maze

GL-II-73 GL-II-74 GL-II-75 DZP

F(1,22)=9.9 ; p=0.004

F(1,19)=6.6;p=0.02

F(2,37)=4.45 ;

p=0.02

F(3,28)=5.5 ;

p=0.0042


22

395

396

397

398

399

400

Supplementary Table S9: Statistical analysis for the Y-maze alternation rate after acute

administration in young mice (stressed and non-stressed)

Assay Dose (mg/kg) NANOVA values

p-value Dose (mg/kg) NANOVA values


p-valueDose


p-value

0 (i.p.) - NS 10 0,0002 0 (i.p.) - NS 8 0,0023 0 (i.p.) - NS 8 0,0007 0 (i.p.) - NS 6 0,030 (i.p.) -CS 10 - 0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 6 -1 (i.p.)- CS 5 0,99 1 (i.p.) 4 0,98 1 (i.p.) 6 0,01 1.5 (i.p.) 6 0,945 (i.p.)-CS 10 0,74 5 (i.p.) 10 0,97 5 (i.p.) 4 0,04510 (i.p.)-CS 12 0,01 10 (i.p.) 4 0,98 10 (i.p.) 9 0,0370 (i.p.) - NS 10 - 0 (i.p.) - NS 8 - 0 (i.p.) - NS 8 - 0 (i.p.) - NS 6 -5 (i.p.) - NS 5 n/a 5 (i.p.) - NS 9 0,49 1.5(i.p) 6 0,00510 (i.p.) - NS 10 n/a 10 (i.p.) - NS 5 0,002 10 (i.p.) - NS 7 n/a

F(1,10)=12.41 ;

p=0.0055

F(2,15)=5.2 ; p=0.01

GL-II-75 DZP

F(1,13)=1.126 ;

p=0.31

GL-II-73 GL-II-74

F(4,29)=9.5 ; p<0.0001

F(4,30)=7 ;p=0.0004

F(5,46)=9.3 ;

p<0.0001

Acut

e

Y-Maze Spontaneous Alternation (Young)

Y-Maze Spontaneous Alternation (Young non exposed to CS)

F(2,22)=2.41 ;

p=0.11

F(2,19)=8.87 ;

p=0.002


23

401

402

403

404

405

Supplementary Table S10: Statistical analysis for the Y-maze alternation rate after acute

administration in old mice



p-value

0 (i.p.) - Young 5 0,0005 0 (i.p.) - Young 5 0,0020 (i.p.) - Old 5 - 0 (i.p.) - Old 5 -

5 (i.p.) 6 0,03 5 (i.p.) 4 0,013

F(2,11)=12.3 ;

p=0.0015

F(2,13)=14.7 ;

p=0.0005

Acut

e

GL-II-73 GL-II-75

Y-Maze Spontaneous Alternation (Ageing)


24

406

407

408

409

410

Supplementary Table S11: Statistical analysis for the Y-maze alternation rate after sub-chronic

administration in young and old mice



p-value

0 (p.o.) - NS 6 0,001 0 (p.o.) - NS 6 0,00510 (p.o.) -CS 5 - 0 (p.o.) 5 -30(p.o.)-CS 6 0,005 30(p.o.) 6 0,33

0 (p.o.) - Young 5 0,0001 0 (p.o.) - Young 6 0,00120 (p.o.) - Old 6 - 0 (p.o.) - Old 8 -

30(p.o.) 4 0,0001 30(p.o.) 5 0,46

Chro

nic

Y-Maze Spontaneous Alternation (Young)

F(2,14)=28.1 ;

p=0.0001

F(2,14)=8.1 ; p=0.0045

Y-Maze Spontaneous Alternation (Ageing)

F(2,12)=73.83 ;

p=0.0001

F(2,16)=10.8 ;

p=0.001

GL-II-73 GL-II-75


25

411

412

413

414

415

Supplementary Figures

Supplementary Figure S1. General synthetic route to amides

Summarized in these schemes are the principal steps for the synthesis of the GL-II

series compounds from the parent compound SH-053-2'F-R-CH3 (1). A detailed

synthesis of SH-053-2'F-R-CH3 (1) is summarized in the US Patent 7,618,958.

26

416

417

418

419

420

421

422

423

Supplementary Figure S2. Concentration of GL-II-73, GL-II-74 and GL-II-75 in plasma and

brain of male C57BL/6 mice after administration of a compound at doses of 1, 5 or 10 mg/kg, 30

min after single i.p. injection (A), as well as after three i.p. injections, administered 24 h, 20 h

and 1 h (B) prior to concentration measurement.

In order to parallel the design of behavioral studies (elevated plus maze, Y maze and forced

swim test), plasma and brain concentrations were determined 30 min after single i.p. injection

27

424

425

426

427

428

429

430

(A), as well as after three i.p. injections, administered 24 h, 20 h and 1 h (B) prior to

concentration measurement with compounds dosed at 1, 5 and 10 mg/kg. In the appropriate

time intervals, the samples were collected from mice anesthetized with ketamine solution (10%

Ketamidor, dosed i.p. at 100 mg/kg). Compounds were extracted from plasma and supernatants

of brain tissue homogenates by solid phase extraction. The procedure of sample preparation

and determination of concentration by ultraperformance liquid chromatography–tandem mass

spectrometry (UPLC–MS/MS) has been already described in Obradovitch et al.

28

431

432

433

434

435

436

437

Supplementary Figure S3. Locomotor activity changes induced by DZP injection

The effect of the referent non-selective PAM DZP dosed i.p. at 10 mg/kg was assessed on

distance traveled in 5-min bins during recording in the locomotor activity test in male mice.

Animals were injected and immediately placed in an opaque Plexiglas chamber under dim red

light (20 lux) for 1hr. Chambers were cleaned with 70% ethanol after every trial. Distance

travelled per 5-min bin was quantified a posteriori using ANYmaze Video Tracking System. A

two-way repeated measures ANOVA showed a significant effect of factor Treatment (Treatment:

F(1,132)=17.376, p=0.001; Time: F(11,132)=1.822, p=0.056; Interaction: F(11,253)=0.809,

p=0.631). Post-hoc comparisons demonstrated that DZP induced a hypolocomotor response in

time intervals 10-35 min and 40-55 min suggesting a sedative effect in the spontaneous

locomotor activity test in mice, in accordance with the approximated substantial potentiation of

α1-containing GABAA receptors.

29

438

439

440

441

442

443

444

445

446

447

448

449

450

Supplementary Figure S4. Locomotor activity changes induced by αPAM injection

The effect of GL-II-73, GL-II-74 and GL-II-75 all dosed i.p. at 10 mg/kg were assessed on

distance traveled in 5-min bins during recording in the locomotor activity test in male mice.

Animals were injected with vehicle (VEH) or the test compounds and immediately placed in an

opaque Plexiglas chamber under dim red light (20 lux) for 1 hr. Chambers were cleaned with

70% ethanol after every trial. Distance travelled per 5-min bin was quantified a posteriori using

ANYmaze Video Tracking System. A two-way repeated measures ANOVA revealed a

significant effect of all factors (Treatment: F(3,253)=6.52, P=0.002; Time: F(11,253)=14.59,

P<0.001; Interaction: F(33,253)=3.25, P<0.001). Post-hoc comparisons of 5-min bins

demonstrated that GL-II-74 induced a hyperlocomotor response in time intervals 0-5 min and

50-55 min, while GL-II-75 elicited a similar stimulant-like effect in the first 5 min, but a consistent

hypolocomotion in the whole period 15-40 min.

30

451

452

453

454

455

456

457

458

459

460

461

462

463

464

Supplementary Figure S5. Effect of GL-II-73, GL-II-74, GL-II-75 and DZP on alternation rate in

a Y-maze alternation task assessing working memory in non-stressed adult mice

The effect of each compound and the non-selective PAM DZP was assessed on alternation

performance in the Y-maze to evaluate potential cognitive deficit induced by the compound

under baseline conditions. Animals received 5 or 10mg/kg of the compounds, or 1.5mg/kg of

DZP, 30 min prior to testing. GL-II-74 was the only compound to show decreased alternation

rate after 10 mg/kg administration, similarly comparable to DZP. **p<0.001 compared to “No

Stress-Vehicle” group.

31

465

466

467

468

469

470

471

472

473

No drug -No stress

No Drug -Stress

Drug - Stress

No Drug -Stress

0102030405060708090

100

ControlStress

Alte

rnati

on R

ate

(%)

** ***

Supplementary Figure S6. Scheme of repeated assessment of cognitive performances in the

Y-Maze in function of stress exposure and α5-PAM administration

The same animals (N=10 per condition: Control or Stress) were evaluated weekly in the

Y-Maze task, assessing working memory abilities. The first week, after habituation to

the maze, the animals were tested without receiving the compound and without being

exposed to chronic stress yet. After that first assessment, animals belonging to the

Stress group were exposed to 1 week of chronic stress (CS), and re-evaluated. Results

showed that 1 week of CS induced alternation deficit in that task. The following week,

while the stress group was still exposed to CS, animals were injected with GL-II-73, 10

mg/kg. Results showed that the compound administration restored the alternation rate

that was altered the week before due to CS exposure. However, if we keep exposing

the animal to CS, without injecting them, the alternation rate drops down again,

suggesting that the single drug administration does not have long term effect, and that

the compound is washed out from the organism.

32

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

References1. Cook JM, Zhou H, S. H, Sarma PVVS, Zhang C (2009): Stereospecific anxiolytic and anticonvulsant agents with reduced muscle-relaxant, sedative hypnotic and ataxic effects. PCT WO2006/004945A1.2. Li G, Stephen MR, Kodali R, Zahn NM, Poe MM, Tiruveedhula VVNPB, et al. (2018): Synthesis of chiral GABAA receptor subtype selective ligands as potential agents to treat schizophrenia as well as depression. ARKIVOC.158-183.3. Alexeev M, Grosenbaugh DK, Mott DD, Fisher JL (2012): The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABA(A) receptors. Neuropharmacology. 62:2507-2514.4. Fisher JL, Zhang J, Macdonald RL (1997): The role of alpha1 and alpha6 subtype amino-terminal domains in allosteric regulation of gamma-aminobutyric acida receptors. Molecular pharmacology. 52:714-724.5. Stamenic TT, Poe MM, Rehman S, Santrac A, Divovic B, Scholze P, et al. (2016): Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the alpha5 subunit. European journal of pharmacology. 791:433-443.6. Ghosal S, Nunley A, Mahbod P, Lewis AG, Smith EP, Tong J, et al. (2015): Mouse handling limits the impact of stress on metabolic endpoints. Physiology & behavior. 150:31-37.7. Namjoshi OA, Wang ZJ, Rallapalli SK, Johnson EM, Jr., Johnson YT, Ng H, et al. (2013): Search for alpha3beta(2)/(3)gamma2 subtype selective ligands that are stable on human liver microsomes. Bioorganic & medicinal chemistry. 21:93-101.8. Obradovic A, Joksimovic S, Poe MM, Ramerstorfer J, Varagic Z, Namjoshi O, et al. (2014): Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: the approximated activation of receptor subtypes may explain behavioral effects. Brain research. 1554:36-48.9. Porsolt RD (1979): Animal model of depression. Biomedicine / [publiee pour l'AAICIG]. 30:139-140.10. Vandesquille M, Krazem A, Louis C, Lestage P, Beracochea D (2011): S 18986 reverses spatial working memory impairments in aged mice: comparison with memantine. Psychopharmacology. 215:709-720.

33

490491492493494495496497498499500501502503504505506507508509510511512513514515516517518

519

Amazon S3 - Supplementary Methods€¦ · Web viewElectrophysiological recordings were performed following the methods described in Alexeev et al (3)(2)(2)(2). Full-length cDNAs for

Documents