Anti-HIV-assay

Bioassay-guided fractionation of an aqueous extract from a Philippine Islands collection of the soft coral Lohophytum spp. yielded cembranoid diterpenes, Fig. (3), which exhibited moderate HIV-inhibitory activity in a cell-based in vitro anti-HIV assay [44], while new isomalabaricane triterpenes. Fig. (4), have been isolated from the sponge Stelletta spp. [45]. Other anti-HIV diterpenes also included the dolabellane diterpenes isolated from the Brazilian brown algae Dictyota pfaffi [46] and Dictyota menstrualis [47]. To investigate the effect of these diterpenes in the reverse transcription of the viral genomic RNA, the recombinant HIV-1 RT was assayed in vitro in the presence of each compound. All compounds inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT and consequently virus replication. 

Figure 2 Anti-HIV results of vaginal formulations via viral binding assay.

In 2000, Boyd et al. reported for the first time an anti-HIV active carbazole alkaloid, siamenol (89) (see Scheme 2.17). This prenylated carbazole alkaloid inhibited HIV-1 induced cytopathic inhibitor activity in an XTT-tetrazolium assay with EC50 2.6 pg/mL (85,449). Recently, Kongkathip et al. reported anti-HIV-1 activity for O-methylmukonal (glycosinine) (38) (see Scheme 2.9), 7-methoxy-O-methylmu-konal (2,7-dimethoxy-3-formylcarbazole) (48) (see Scheme 2.10), and clausine K (clausazoline-J) (51). These studies showed strong anti-HIV-1 activity for... 

Detecting exposure to HIV ELISA assays and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient s blood sample. ELISA assays are used as the primary screening tool, because they are very sensitive. These assays sometimes give false-posi-tives, however, so western blots, which are more specific, are often used as a confirmatory test (Figure 32.23). [Note ELISA and western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]... 

The chiral sulfoxide 354 was shown to be one of a number of potent CC chemokine receptor 5 antagonists (IC50 1.9 nM) and a potent inhibitor (ICS0 1.0nM) in the HIV-1 envelope mediated membrane fusion assay. This compound also exhibited favorable oral absorption characteristics in rats making it a promising lead for further development as an anti-HIV agent <2005BMC363>. 

Polysaccharides are another class of macromolecules that can interfere with cellular and enzyme assays, as demonstrated during the NCI campaign to discover anti-HIV agents from natural sources. Anionic polysaccharides show anti-HIV activity and, to avoid a too high hit rate, had to be removed by 50% aqueous ethanol precipitation.72... 

Table 1 Anti-HIV-1 activity of selected experimental nucleosides used in the HIV-1 Q-RT-PCR assay in human PBM cells. All concentrations are expressed in micromolar units. H NMR and elemental analysis of experimental agents of selected intermediates supplied by author...

Anti-HIV-1 Activity of Calanolides in Hollow Fiber Mouse Evaluation of (-l-)-calanolide A (1) in a hollow fiber culture-based in a SCID mouse assay of antiviral efficacy indicated that (+)-calanolide A exhibited significant anti-HIV-1 activity after oral or parenteral administration on a once-daily (200mg/kg/ dose) or twice-daily (150 mg/kg/dose) treatment. Furthermore, a synergistic effect was observed in the combination of (-l-)-calanolide A and AZT. ... 

HIV-2 HIV-2, which is closely related to HIV-1, was first reported to be associated with AIDS in 1986 in West Africa, where it is believed to be endemic. Several cases of HIV-2 infection have been reported among Europeans and West Africans residing in Europe and in the USA. The spectrum of disease and modes of transmission of HIV-2 seem to be similar to those of HIV-1 (182). However, there have been only relatively few reports of HIV-2 transmission in blood. The anti-HIV-1 El A tests currently used for screening blood donors are estimated to detect from 42 to 92% of HIV-2 infections (183). Anti-HIV-2 assays are available and are used routinely in blood donor screening. 

Examples of other anti-HIV compounds also included four phlorotannin derivatives, eckol, 8,8 -dieckoI, 8,4 -dieckoI and phlorofucofiiro-eckol A, isolated from the brown alga Ecklonia cava [65]. Among these compounds, 8,8 -dieckol and 8,4 -dieckol exhibited an inhibitory effect on HIV-1 RT and protease. An enzyme kinetic assay... 

Pauwels, R., Balzarini, J., Baba, M., Snoeck, R., Schols, D Herdewijn, P., Desmyter J., and De Clercq, E. (1988) Rapid and automated tetrazolium-basd colorimetric assay for the detection of anti-HIV compounds. J. Virol. Methods 20,309-321. 

This assay was developed and used in anti-HIV screening programs (Pauwels et al. 1988). The tetrazolium salt MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide] is converted to dark blue formazan by living cells but not by dead cells or culture medium. These assays are carried out in 96-well plates. 

In Subheading 5. a few genotypic tests will be described. Using phenotypic assays, clinical isolates that display various levels of resistance to particular drugs have been identified. Often the researcher wants to know what mutations are responsible for this phenotypic resistance, in an attempt to understand and possibly to anticipate the development of the resistance. Because present-day clinical anti-HIV drugs are RT or protease inhibitors, sequencing protocols for clinical HIV-1 RT and protease genes will be described. Once researchers have established a stable relationship between resistance and particular mutations, tests that identify specific mutations may be very valuable and much faster than any other method. In this respect, the protocol for two amplification refractory mutation systems (ARMS) will be described, which use mutation-specific PCRs. Other useful assays are the point mutation assay (10) and LiPA (see also Chapter 10). 

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