OX1 (EC50 = 8.29 nM); OX2 (EC50 = 187 nM)

Two peptides OXA (15–33) with EC50 of 64 nM and OXA (17–33) with EC50 = 8.29 nM displayed the highest potency among all analogues. In contrast to previous reports,19 orexin A (15–33) showed modest selectivity for the OX1 receptor (∼15-fold) in our hands. OXA (17–33) had slightly higher selectivity for OX1 (∼23-fold), which represents the shortest analogue with OX1 selectivity to date. Although the reason for this discrepancy is unclear, it has been proposed that orexin agonist discrimination is dependent on the expression system.

OXA (17–33) was chosen for further SAR studies because of its good potency and OX1 preference. An alanine scan was first performed to determine the contributions of the side chains to the overall activity (Table 2). Substitution of the first six N-terminal amino acids (17–22) partially retained the agonist activity at the OX1 receptor, except for [Ala20] (EC50 > 3 μM), whose analogue with the acetyl cap had no OX1 activity at up to 10 μM. [Ala18] substitution showed the least effect on activity (EC50 79 nM). Alanine substitution at the 24–28 positions resulted in a more significant drop in activity, whereas changes in the last 5 amino acids (C-terminal) led to a complete loss in activity, confirming that modifications on the C-terminal region are not tolerated. A similar trend is present at the OX2 receptor. The preference for the OX1 receptor was generally maintained with substitution at 17–22 positions but was abolished with substitution at the 24–26 positions. Acylation on the N-terminus displayed minimal effects on agonist activity in this series of peptides (see Supporting Information).

The effect of side-chain chirality on the activity was examined by a d-amino acid scan on OXA (17–33) (Table 3). Similarly, changes on the C-terminal side of the peptide (28–33) led to a complete loss of activity. Changes in the remaining positions (17–27) revealed that [d-Glu18], [d-Leu19], and [d-Leu20] analogues retained some of the activity, whereas all other analogues had noticeable decreases in agonist potency. Interestingly, substitution to d-amino acids at positions 19–27 seemed to reverse the binding preference to favor OX2. For example, [d-Leu20] orexin (17–33) analogue had no change in activity at OX2 and a 64-fold drop in activity at OX1; [d-His26] OXA (17–33) had 9-fold reduction in activity at OX2 and almost 700-fold drop in activity at OX1. These results suggest a more pronounced impact of chain orientation on orexin receptor binding in this region. [Tyr17] and [Glu18] retained slight selectivity for OX1, although agonist activity of both analogues significantly decreased.

The effect of more dramatic changes on secondary structure of peptides was investigated using proline scans of OXA (17–33). Given that modification in the C-terminus resulted in total loss of activity in previous scans, only the first half from the N-terminus (17–22) was investigated. The analogues resulting from proline substitution at 17–19 position retained some of the original activity, with [Pro18] OXA (17–33) being the least affected by change (Table 4). A more significant drop in activity was observed for analogues at 20–22 positions. Selectivity of OX1 was slightly maintained or abolished in this series [1].

Calcium Mobilization Assays for OX1 and OX2 [1]
Two individual stable cell lines were created by over-expressing human OX1 and OX2 receptors in CHO-RD-HGA16 cells, a CHO cell line stably over-expressing the promiscuous Gq-protein Gα16. The day before the assay, cells were plated into 96-well black-walled assay plates at 25,000 cells/well in Ham’s F12 supplemented with 10% fetal bovine serum, 100 units of penicillin and streptomycin, and 100 μg/mL normocinTM. After incubating at 37°C, 5% CO2 overnight, the growth medium was removed and the cells were gently washed with 100 μL of pre-warmed (37°C) assay buffer (1X HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4). The cells were incubated for 45 minutes at 37°C, 5% CO2 in 200 μL of a calcium-sensitive fluorescent dye (½ the manufacturer’s recommended concentration diluted in assay buffer without probenecid, calcium 5 assay kit). During the incubation period, 8-point concentration curves of the test compounds were prepared at 10X final concentration in assay buffer/0.25% BSA/1% solvent and aliquoted into 96-well polypropylene plates. After 45 minutes, 25 μL of pretreatment (assay buffer/2.5% BSA/10% solvent) was added to the wells and incubated at 37°C for 15 min. Calciummediated changes in fluorescence were monitored in a FlexStation II plate reader. Fluorescence intensity was measured every 1.52 seconds for 19 seconds to establish baseline fluorescence followed by FlexStation II addition of 25 μL of test compound and further readings for a total of 60 seconds (excitation at 485 nm, detection at 525 nm). Peak kinetic reduction relative fluorescent units (RFU) were plotted against compound concentration. Data were fit to a three-parameter logistic curve to generate EC50 values

[1]. Truncated Orexin Peptides: Structure-Activity Relationship Studies. ACS Med Chem Lett. 2013;4(12):1224-1227.

Orexin receptors are involved in many processes including energy homeostasis, wake/sleep cycle, metabolism and reward. Development of potent and selective ligands is an essential step for defining the mechanism(s) underlying such critical processes. The goal of this study was to further investigate the structure-activity relationships of these peptides and to identify truncated form of the orexin peptides active at OX1. Truncation studies have led to OXA (17-33) as the shortest active peptide known to date with a 23-fold selectivity for OX1 over OX2. Alanine, D-amino acid and proline scans have highlighted the particular importance of Tyr17, Leu20, Asn25 and His26 for agonist properties of OXA(17-33). The conformation of the C-terminus might also be a defining factor in agonist activity and selectivity of the orexin peptides for the OX1 receptor. [1]

C79H125N23O22

1748.97871756554

1747.936952

343268-91-7

OXA(17-33) TFA

77846288

Tyr-Glu-Leu-Leu-His-Gly-Ala-Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Leu-NH2; H-Tyr-Glu-Leu-Leu-His-Gly-Ala-Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Leu-NH2; L-tyrosyl-L-alpha-glutamyl-L-leucyl-L-leucyl-L-histidyl-glycyl-L-alanyl-glycyl-L-asparagyl-L-histidyl-L-alanyl-L-alanyl-glycyl-L-isoleucyl-L-leucyl-L-threonyl-L-leucinamide

YELLHGAGNHAAGILTL-NH2; YELLHGAGNHAAGILTL

White to off-white solid powder

-4.2

24

25

55

124

3640

16

O=C([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])C(C([H])([H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N=C([H])N1[H])N([H])C([C@]([H])(C([H])([H])C(N([H])[H])=O)N([H])C(C([H])([H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C(C([H])([H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N=C([H])N1[H])N([H])C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C(=O)O[H])N([H])C([C@]([H])(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H])N([H])[H])=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)[C@@]([H])(C([H])([H])[H])O[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H]

JPRBZUXULYLVPJ-HYNYKCRDSA-N

InChI=1S/C79H125N23O22/c1-15-41(10)64(78(123)100-55(25-40(8)9)77(122)102-65(45(14)103)79(124)95-52(66(82)111)22-37(2)3)101-62(108)34-86-68(113)43(12)91-69(114)44(13)92-73(118)57(28-48-31-84-36-89-48)99-76(121)58(29-59(81)105)93-61(107)33-85-67(112)42(11)90-60(106)32-87-71(116)56(27-47-30-83-35-88-47)98-75(120)54(24-39(6)7)97-74(119)53(23-38(4)5)96-72(117)51(20-21-63(109)110)94-70(115)50(80)26-46-16-18-49(104)19-17-46/h16-19,30-31,35-45,50-58,64-65,103-104H,15,20-29,32-34,80H2,1-14H3,(H2,81,105)(H2,82,111)(H,83,88)(H,84,89)(H,85,112)(H,86,113)(H,87,116)(H,90,106)(H,91,114)(H,92,118)(H,93,107)(H,94,115)(H,95,124)(H,96,117)(H,97,119)(H,98,120)(H,99,121)(H,100,123)(H,101,108)(H,102,122)(H,109,110)/t41-,42-,43-,44-,45+,50-,51-,52-,53-,54-,55-,56-,57-,58-,64-,65-/m0/s1

(4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[[2-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S,3S)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-2-oxoethyl]amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-5-oxopentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)