Fluorescent protein sensor to diagnose HIV at low cost Fluorescent protein sensor to diagnose HIV at low cost It is estimated that over 33 million individuals are at present infected with HIV (http://www.unaids.org). The treatment of HIV is moving towards chronic management of the disease, e.g., by combing three-drug regimens to reduce the number of dosing units. To date, six classes of antiretroviral drug (nucleoside and nonnucleoside reverse transcriptase, protease, fusion, entry, and integrase strand transfer inhibitors) have been discovered including 35 drugs that have been approved by the Food and Drug Administration (FDA) for the treatment of HIV infection (http://www.fda.gov/). However, these drugs are inadequate for the treatment, since they only reduce the number of HIV virus below the detection limit instead of clearing the HIV infection. On the other hand, more than 60 Phase I, II and III vaccine trials have been developed around the world. However, none of them has been developed into a truly effective HIV vaccine. Therefore, the early detection of HIV infection is extremely important to prevent the spread of the epidemic. The most widely used HIV tests are antibody tests, which detect the antibodies against HIV in human body. However, it takes some time for human immune system to produce detectable antibodies against HIV and this time period varies from person to person. The average time to produce enough antibodies for the antibody test is 25 days. But in some cases, it may need 6 months to develop detectable antibodies. Another type of HIV test is to detect the RNA of HIV virus directly. The time between RNA detection and HIV infection is typically nine to eleven days. However, the RNA tests are more costly than antibody tests and used less often. We propose to develop a novel approach: design and prepare a fluorescent sCD4 protein that can specifically bind with HIV gp120 protein. Once bound, the changed color will be used to monitor the HIV infection. The progress of HIV-1 entry into cells is mediated by the trimeric spikes of glycoproteins gp120 on the surface of the virion. The entry of HIV-1 into host cells begins with the binding of gp120 envelope protein to cellular CD4 receptors, which is primarily expressed on the surface of macrophages and T cells.1 In the CD4-gp120 binding complex, the Phe43 of CD4 plugs the entrance of a conserved deep hydrophobic pocket, which was denoted as the Phe43 cavity in gp120.2 The interaction between Phe43 and the cavity plays a vital role for stabilizing interactions between the CD4 and HIV gp120. One single phenyl extension of this phenylalanine (biphenylalanine) into the conserved hydrophobic interfacial pocket enhances neutralization capability of a CD4 mimic by roughly 10-fold.3 Previous study has reported a series of fluorescent solvatochromic dyes (Dapoxyl dye), whose fluorescence wavelength and fluorescence quantum yield are highly sensitive to solvent polarity.4 The fluorescence maximum wavelength of a representative Dapoxyl dye (compound 1) is 636 nm (red color) in 1:4 acetonitrile/water aqueous solvent. The maximum wavelength of compound 1 in organic solvent is 516 nm (green color) in ethyl acetate and 509 nm (green color) in chloroform, respectively. The fluorescence quantum yield of compound 1 is 0.04 in 1:4 acetonitrile/water, 0.77 in ethyl acetate, 0.77 in chloroform, respectively.4 In this study, we will couple the fluorophore of compound 1 with glycine to produce fluorescent amino acid (compound 2). Then we will incorporate the compound 2 into the position 43 of sCD4, which is human CD4 recombinant, to obtain mutant fluorescent sCD4. According to the X-ray structure (PDB code 1YYL), the hydrophobic pocket of gp120 has enough space to accommodate the side chain of compound 2. In aqueous solution, the free fluorophore of mutant sCD4 will have a red color. Once the mutant sCD4 binding with HIV gp120, the fluorophore will dock into the Phe43 cavity of gp120, which forms a hydrophobic environment, and will have a green color. At the same time, the intensity of green fluorescence will be increased about twenty times, since the fluorescence quantum yield of the fluorophore is much higher in hydrophobic environment than in aqueous environment. The fluorescent alteration from a weak red color to a high intense green color will be used to detect the HIV infection.
|Effective start/end date||5/1/12 → 4/30/14|
- Gates (Bill and Melinda) Foundation: $100,000.00
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