CCR5 (C-C chemokine receptor type 5). Adaptavir inhibits the binding of gp120/sCD4 complex to CCR5 with IC50 values: 55 ± 0.08 pM for HIV-1 Bal gp120 and 0.32 ± 0.03 nM for HIV-1 CM235 gp120 on Cf2Th/synR5 cells. On GHOST CD4-CCR5 cells, the IC50 for gp120CM235/sCD4 binding inhibition is 51 ± 0.09 pM. [2]

In monocytes/macrophages (M/M), DAPTA (1 nM) reduces HIV-1 multiplication by more than 90%. DAPTA inhibits the entrance of HIV and stops HIV-1 infection. DAPTA decreases human primary macrophage CCR5 mAb binding. Neuronal apoptosis induced by R5 gp120 is potently blocked by DAPTA. When it comes to stopping neuronal apoptosis, DAPTA is even more effective than TAK-779, a CCR5 antagonist[1]. Specific CD4-dependent binding of gp120 Bal (IC50 = 0.06 nM) and CM235 (IC50 = 0.32 nM) to CCR5 is potently inhibited by DAPTA. The formation of the gp120/sCD4 complex with CCR5 is inhibited by DAPTA (1 nM). With an IC50 of 55 ± 0.08 pM, DAPTA blocks the binding of gp120BaL/sCD4 to CCR5 (Cf2Th/synR5) cells[2].

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In monocytes/macrophages (M/M) infected with R5 HIV-1 strains (BaL and 81A), Adaptavir at a concentration of 10^{-9} M inhibited HIV-1 replication by >90% (p24 antigen production measured at 14 and 21 days post-infection). The protective effect was confirmed by a reduction in cytopathic effect, syncytia formation, and cell aggregation. [1]
In M/M, Adaptavir (at 10^{-7} M and 10^{-10} M) reduced HIV-1 DNA formation by 64% and 70%, respectively, compared to untreated cells, as measured by semi-quantitative inverse/nested PCR for a conserved gag region. This inhibition of HIV-1 DNA formation was greater than that of the specific anti-CCR5 antibody 2D7 (~39% inhibition). [1]
Adaptavir treatment (10^{-12} M) reduced CCR5 detection on M/M by 73% (from 35% CCR5-positive in mock-treated to 9% in treated), as measured by flow cytometry using FITC-labeled anti-CCR5 mAb (2D7), indicating that DAPTA masks the CCR5 binding site. [1]
In differentiated SK-N-SH neuronal cells, Adaptavir at 10^{-13} M and 10^{-12} M reduced CCR5 expression by 68.5% and 72%, respectively, compared to unexposed cells. Furthermore, DAPTA completely (100%) inhibited R5 HIV-1 BaL-induced apoptosis in these cells, as assessed by propidium iodide staining and flow cytometry. In comparison, the CCR5 antagonist TAK-779 (1.8×10^{-6} M) only provided 60% inhibition of apoptosis. [1]
Adaptavir (1 nM) blocks the formation of the gp120/sCD4 complex with CCR5 in co-immunoprecipitation studies using solubilized CCR5 from Cf2Th/synR5 cells or membrane-bound CCR5 on HOS CD4-CCR5 cells. [2]
Adaptavir potently and substantially inhibits CD4-dependent binding of FITC-labeled gp120 (from Bal and CM235 strains) to CCR5-expressing cells (Cf2Th/synR5 and GHOST CD4-CCR5) in a rapid filtration binding assay. The binding inhibition is dose-dependent. [2]
Confocal microscopy shows that FITC-labeled Adaptavir co-localizes with CCR5 on CCR5-positive Cf2Th/synR5 cells, but not on CCR5-negative Cf2Th cells, confirming that CCR5 is a direct receptor for DAPTA. [2]

In a randomized, double-blind, placebo-controlled trial for HIV-associated cognitive impairment, a blind analysis of frozen stored plasma samples found a significant reduction (0.54 log10, P = 0.037) in viral load between baseline and month 6 in patients treated with Adaptavir. [1]
An uncontrolled clinical trial reports that Adaptavir substantially suppressed virus in persistently infected cellular reservoirs in both HAART-experienced and treatment-naïve patients. [2]

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For the rapid filtration binding assay, a fluorescently labeled tracer was prepared from soluble gp120 proteins. Binding assays were performed in binding buffer in a final volume of 100 μl. Binding was carried out for 1 hour at 37°C in 96-well filter plates. Unbound labeled proteins were removed by rapid vacuum filtration and washing five times with 0.2 ml of cold washing buffer per well. Filters were counted with a fluorescent plate reader at 495/530 nm. Specificity of gp120 binding was shown by competition with non-labeled HIV-1 Bal, MIP-1β (a CCR5 selective ligand), or anti-CCR5 antibody 2D7. [2]
For the immunoprecipitation assay, cell lysates from Cf2Th/synR5 cells (CCR5+) were incubated with an anti-CCR5 antibody (1D4) and protein A/G agarose to capture solubilized CCR5 receptors. The beads were then incubated with a pre-formed gp120/sCD4 complex in the presence or absence of Adaptavir (1 or 10 nM). The bead-associated complexes were eluted, run on SDS-PAGE, transferred to PVDF membranes, and detected with human HIVIg or anti-CCR5 antibody. A related procedure was used for membrane-bound receptors on HOS CD4-CCR5 cells, where cells were pre-treated with DAPTA, incubated with gp120, lysed, and immunoprecipitated with anti-CD4 or anti-CCR5 antibodies. [2]

A novel FITC-labeled tracer from soluble gp120 proteins (25 g/mL) is prepared using a Fluorescent protein labeling kit, according to the manufactures instructions. Uncoupled FLUOS is removed by Sephadex G-10 column filtration. The molar ratio between FLUOS-labeling molecules and protein is from 3.5 to 4.5 fluorescence molecules per molecule of gp120. The concentration of fluorescent-labeled proteins is measured by Bradford assay and Western blotting by using calibrating amounts of soluble molecules with known concentration. Binding assays are performed in binding buffer, in final volume 100l. Binding is carried out for 1 h at 37°C in 96-well filter plates. Unbound-labeled proteins are removed by rapid vacuum filtration and ishing using a 96-well plates manifold. Each binding mix is washed five times with 0.2 mL (total volume of 1.0 mL/well) cold ishing buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM CaCl2). Filters are counted with a fluorescent plate reader at 495/530 nm.

Monocytes/macrophages (M/M) were prepared from healthy HIV-seronegative donors. M/M were exposed to various concentrations of Adaptavir (10^{-14} to 10^{-10} M) for 20 minutes and then challenged with R5 HIV-1 strains BaL or 81A (2,000 pg/ml of p24 gag). After 2 hours, cells were washed and cultured in the presence of DAPTA. Supernatants were collected at 7, 14, and 21 days, and virus production was assessed by HIV p24 gag ELISA. [1]
For HIV-1 DNA analysis, M/M were infected with HIV-1 BaL (30 pg/ml) in the presence or absence of Adaptavir (10^{-9} M and 10^{-7} M) or anti-CCR5 mAb 2D7. After 18 hours, genomic DNA was isolated. Inverse/nested PCR specific for a conserved region within the gag gene was performed using 1×10^6 to 1.25×10^5 cell equivalents. The 115 bp PCR products were detected by oligomer hybridization. Amplification of the β-actin gene was used as a control. Band density was measured by software. [1]
For flow cytometric analysis of CCR5 expression, M/M or differentiated SK-N-SH neuronal cells were incubated with Adaptavir at several doses for 20-30 minutes at 4°C, then stained with FITC-labeled anti-CCR5 mAb (2D7) for 30 minutes at 4°C in the dark. Stained cells were washed and analyzed with a FACScan flow cytometer (10,000 events per sample). [1]
For neuronal apoptosis analysis, differentiated SK-N-SH cells were exposed to 8,000 pg/ml of p24-gag of HIV-1 BaL grown in M/M cultures and Adaptavir (at 10^{-13} and 10^{-12} M) or TAK-779 (1.8×10^{-6} M) for 5 days. Cells were then detached, permeated with ice-cold 70% ethanol, incubated with propidium iodide (PI) and RNase at 4°C for 2 hours in the dark, and analyzed by flow cytometry (10,000 events per sample). [1]
Binding assays on cell lines (Cf2Th/synR5, GHOST CD4-CCR5, HOS CD4-CCR5) were performed by incubating cells with FITC-labeled gp120 in the presence of sCD4 and varying concentrations of Adaptavir. Cells were incubated for 1 hour at 37°C, then unbound material was removed by vacuum filtration and washing, and fluorescence was measured. [2]
For double-color immunofluorescence analysis, Cf2Th/synR5 (CCR5+) and Cf2Th (CCR5-) cells grown on glass coverslips were incubated with FITC-labeled Adaptavir (10 nM) for 1 hour at 37°C. After washing, cells were incubated with anti-CCR5 antibody (1D4) overnight at 4°C, then with anti-mouse IgG-rhodamine for 2 hours at 4°C. Coverslips were fixed and analyzed by confocal laser scanning microscopy with a 40× oil immersion objective. Images were acquired sequentially for each fluorochrome. [2]

Adaptavir is suggested to penetrate the central nervous system (CNS) based on observed improvements in cognition in HIV-1-infected humans. [1]

[1]. Profound anti-HIV-1 activity of DAPTA in monocytes/macrophages and inhibition of CCR5-mediated apoptosis in neuronal cells. Antivir Chem Chemother. 2007;18(5):285-95.

[2]. Chemokine receptor-5 (CCR5) is a receptor for the HIV entry inhibitor peptide T (DAPTA). Antiviral Res. 2005 Aug;67(2):83-92.

Adaptavir (DAPTA) is a viral entry inhibitor that targets CCR5, blocking HIV-1 entry into monocytes/macrophages and preventing gp120-induced apoptosis in neuronal cells. It is more potent than the non-peptidic CCR5 antagonist TAK-779 in inhibiting apoptosis in a neuroblastoma cell line. The mechanism of action is competitive binding with HIV gp120 on the CCR5 receptor. DAPTA is 34 to 180 times more potent than Maraviroc in inhibiting gp120-sCD4 complex binding to CCR5 (IC50 0.06-0.32 nM vs. 11 nM for Maraviroc). [1]
Adaptavir inhibits CD4-dependent binding of gp120 to CCR5, as shown by rapid filtration binding and co-immunoprecipitation assays. Direct binding of FITC-labeled DAPTA to CCR5-positive cells was visualized by confocal microscopy, confirming CCR5 as a receptor for DAPTA. The peptide is active against R5-tropic HIV-1 isolates but not X4-tropic isolates. Resistance development was not evident in vitro or in vivo. The DAPTA epitope in gp120 plays an important role in co-receptor binding. [2]

C35H56N10O15

856.888

856.393

106362-34-9

184644

White to off-white solid powder

1.415g/cm3

1514.3ºC at 760mmHg

869.6ºC

0mmHg at 25°C

1.596

-6.9

16

16

24

60

1530

12

C[C@H]([C@@H](C(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@@H](C)N)O

AKWRNBWMGFUAMF-ZESMOPTKSA-N

InChI=1S/C35H56N10O15/c1-13(36)29(54)41-22(12-46)32(57)43-26(16(4)49)34(59)45-27(17(5)50)35(60)44-25(15(3)48)33(58)40-21(11-23(37)52)30(55)39-20(10-18-6-8-19(51)9-7-18)31(56)42-24(14(2)47)28(38)53/h6-9,13-17,20-22,24-27,46-51H,10-12,36H2,1-5H3,(H2,37,52)(H2,38,53)(H,39,55)(H,40,58)(H,41,54)(H,42,56)(H,43,57)(H,44,60)(H,45,59)/t13-,14-,15-,16-,17-,20+,21+,22+,24+,25+,26+,27+/m1/s1

L-Threoninamide, D-alanyl-L-seryl-L-threonyl-L-threonyl-L-threonyl-L-asparaginyl-L-tyrosyl-

RAP-101DAPTA mDAPTAAdaptavir D-Ala-1-peptide T-NH-2 Monomeric (D-Alanine-1) Peptide T amide

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, avoid exposure to moisture.

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

H2O : ~50 mg/mL (~58.35 mM)

Solubility in Formulation 1: 10 mg/mL (11.67 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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