Chemokine; Antibiotic; antibacterial; Synthetic cationic peptide; Innate defense modulator/peptide

Regulation of the immune system by immunomodulatory agents, such as the synthetic innate defense regulator (IDR) peptides, has been proposed as a potential strategy to strengthen host immune responses against infection. IDR peptides confer protection in vivo against a range of bacterial infections and have been developed as components of single-dose vaccine adjuvants due to their ability to modulate innate immunity, correlating with an increased recruitment of monocytes to sites of infection or immunization. However, the mechanisms by which IDR peptides augment monocyte recruitment remain poorly defined. Anti-infective peptide IDR-1002 was demonstrated here to lack direct monocyte chemoattractive activity yet enhance, by up to 5-fold, the ability of human monocytes to migrate on fibronectin towards chemokines. This effect correlated with an increased adhesion of monocytes and THP-1 cells to fibronectin by IDR-1002 and other IDR peptides and the adhesion of THP-1 cells to fibronectin occurred in a β(1)-integrin-dependent manner, corresponding with an increased activation of β(1)-integrins and the phosphoinositide 3-kinase (PI3K)-Akt pathway. PI3K- and Akt-specific inhibitors abrogated IDR-1002-induced adhesion and activation of β(1)-integrins, whereas p38 and MEK1 inhibitors did not affect, or moderately inhibited, adhesion, respectively. Furthermore, IDR-1002 enhancement of monocyte migration towards chemokines and activation of β(1)-integrins was abrogated in the presence of PI3K- and Akt-specific inhibitors. In summary, IDR-1002 enhanced monocyte migration on fibronectin through promotion of β(1)-integrin-mediated interactions regulated by the PI3K-Akt pathway, revealing a mechanism by which IDR-1002 promotes monocyte recruitment [1].

Fibronectin Migration Assay [1]
All migration assays were performed using a 48-well microchemotaxis chamber. Freshly enriched human peripheral blood monocytes were adjusted to RPMI 1640 with 1% FBS and incubated at 37°C for 1 h. For inhibitor studies, monocytes were pretreated with chemical inhibitors, or a DMSO vehicle control, at the indicated concentrations for 1 h. Similarly in β1-integrin blocking experiments, monocytes were pretreated with 20 µg/ml of a HUTS-4-blocking antibody or an isotype-matched mouse IgG1 control antibody for 1 h; 5 × 104 monocytes were added to the upper wells of the chamber, and indicated concentrations of chemokines in RPMI 1640 with 1% FBS were added to the lower wells. Lower wells containing RPMI 1640 and 1% FBS were used as negative controls. In IDR-1002-chemokine synergy studies, treatment with IDR-1002 was done by adding peptide to both upper and lower wells to avoid a concentration gradient. The upper and lower wells were separated by a polycarbonate membrane with 5-µm-diameter pores. Membranes were precoated with 50 µg/ml of fibronectin at 4°C overnight, washed with PBS and air-dried prior to use. After 1 h of incubation, non-migrated monocytes were removed by PBS washing and scraping with a rubber blade and adherent cells on the underside of the membrane were stained with the Diff-Quick staining kit.

Control experiments assessing the effects of IDR-1002 on monocyte migration independent of chemokines were similarly done. To investigate any direct chemotactic properties of IDR-1002, different concentrations of IDR-1002 were added to the lower wells of the chemotaxis chamber and separated from the monocyte-containing chamber with a non-coated polycarbonate membrane. Likewise, migration experiments investigating the chemotactic or chemokinetic properties of IDR-1002 in a fibronectin system were done by the addition of the indicated amount of peptide to both the upper and lower wells in chemokinesis experiments, and lower wells alone in chemotaxis experiments. The wells were then separated with a fibronectin-coated polycarbonate membrane and processed using the method stated above.

Migration for each treatment was measured by averaging the number of migrated cells per HPF over 5 fields with each treatment condition done in duplicate. Fold change over control values were calculated by dividing the average cell count per HPF of each treatment by the average cell count per HPF of basal migration in media alone.

[1]. A Novel Citrullinated Modification of Histone 3 and Its Regulatory Mechanisms Related to IPO-38 Antibody-Labeled Protein. Front Oncol. 2019 Apr 18;9:304.

Although the current study focused on the ability of IDR-1002 to utilize the PI3K-Akt pathway in modulating β1-integrin affinity via ‘inside-out’ activation, there are other mechanisms through which this pathway can regulate integrin function. The PI3K-Akt pathway is known to promote the recycling of internalized integrins to the cell surface, although the lack of effect by IDR-1002 on total surface β1-integrins levels makes this an unlikely explanation for IDR-1002-enhanced adhesion. However, we cannot exclude the role of PI3K-Akt-mediated increases in integrin avidity via its promotion of integrin mobility and clustering. Taking into consideration the multiple effects of IDR peptides and natural peptides on cellular signaling, as well as the complexity of interactions within signaling networks, IDR-1002 may affect other signaling networks through cross-talk. For example, in various cell models, PI3K has been shown to activate various small GTPases and phospholipase C, which are among the best-characterized mediators of integrin activation. Similarly, other signaling pathways used by IDR-1002 are known to impact the PI3K-Akt pathway, including the G-protein-coupled receptor-induced networks. Despite these complexities, we propose that the PI3K-Akt pathway is central to IDR-1002 modulation of β1-integrin function and subsequent enhancement of monocyte migration and adhesion to fibronectin.

Monocyte adhesion and migration on fibronectin within tissues is only one process coordinating overall leukocyte infiltration. This study provides insights into other mechanisms by which IDR-1002 potentially promotes monocyte mobilization to infectious sites. The β1-integrin-activating properties of IDR-1002 may have implications at other stages of monocyte recruitment. VLA-4, a β1-integrin member, is a well-known receptor for VCAM-1 on endothelial cells and plays a major role in cellular rolling, firm adhesion and transmigration across the endothelia. In addition, with many similarities between the regulation of β1-integrins and other integrin family members, it is conceivable that IDR peptides may affect other stages of integrin-mediated adhesion and migration. Indeed we have observed that IDR-1002 promotes THP-1 adhesion to ICAM-1 [unpubl. data], a β2-integrin-mediated interaction essential for monocyte arrest and adhesion on the endothelial layer.

In summary, we established that IDR peptides can promote the adhesion and migration of monocytes to fibronectin through enhanced β1-integrin function via activation of the PI3K-Akt pathway. This study identifies novel functions of IDR peptides in promoting integrin-mediated monocyte adhesion to fibronectin and synergistically enhancing monocyte migration towards host chemokines, further expanding the repertoire of immune-regulating effects of IDR peptides. Monocyte adhesion not only has implications in monocyte recruitment, but far-reaching regulatory functions in many aspects of the host immune response, including wound healing, cellular differentiation and tissue homeostasis, among others. Understanding how IDR peptides regulate monocyte migration, adhesion and integrin function through its effect on cellular signaling networks will aid in the development and optimization of novel agents with improved anti-infective and immunomodulatory functions.[1]

C79H132N26O13

1654.05979537964

1651.031

C, 57.43; H, 7.93; N, 22.04; O, 12.59

1224095-25-3

166603919

H-Val-Gln-Arg-Trp-Leu-Ile-Val-Trp-Arg-Ile-Arg-Lys-NH2
Val-Gln-Arg-Trp-Leu-Ile-Val-Trp-Arg-Ile-Arg-Lys-NH2
L-valyl-L-glutaminyl-L-arginyl-L-tryptophyl-L-leucyl-L-isoleucyl-L-valyl-L-tryptophyl-L-arginyl-L-isoleucyl-L-arginyl-L-lysinamide

VQRWLIVWRIRK-NH2
VQRWLIVWRIRK
H-VQRWLIVWRIRK-[NH2]

White to off-white solid powder

0.2

23

18

54

118

3370

14

CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](C(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N

SEBZWKNTKRJFDZ-BRVBZNILSA-N

InChI=1S/C79H130N26O13/c1-11-44(9)63(75(117)99-54(28-20-34-91-78(86)87)66(108)95-52(65(83)107)26-17-18-32-80)104-69(111)55(29-21-35-92-79(88)89)97-70(112)59(38-47-40-94-51-25-16-14-23-49(47)51)102-74(116)62(43(7)8)103-76(118)64(45(10)12-2)105-72(114)57(36-41(3)4)100-71(113)58(37-46-39-93-50-24-15-13-22-48(46)50)101-67(109)53(27-19-33-90-77(84)85)96-68(110)56(30-31-60(81)106)98-73(115)61(82)42(5)6/h13-16,22-25,39-45,52-59,61-64,93-94H,11-12,17-21,26-38,80,82H2,1-10H3,(H2,81,106)(H2,83,107)(H,95,108)(H,96,110)(H,97,112)(H,98,115)(H,99,117)(H,100,113)(H,101,109)(H,102,116)(H,103,118)(H,104,111)(H,105,114)(H4,84,85,90)(H4,86,87,91)(H4,88,89,92)/t44-,45-,52-,53-,54-,55-,56-,57-,58-,59-,61-,62-,63-,64-/m0/s1

(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-N-[(2S)-5-(diaminomethylideneamino)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-(diaminomethylideneamino)-1-[[(2S,3S)-1-[[(2S)-5-(diaminomethylideneamino)-1-[[(2S)-1,6-diamino-1-oxohexan-2-yl]amino]-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopentan-2-yl]pentanediamide

IDR 1002; 1224095-25-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)