The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.
When primary rabbit kidney cell cultures are treated with either polyadenylic acid-polyuridylic acid or polyadenylic acid-polyribothymidylic acid (poly(rT)) and then judiciously exposed to actinomycin D and cycloheximide, high titers of interferon are found in the extracellular medium ("superinduction") (Vilcek, J. (1970) Ann. N. Y. Acad. Sci. 173, 390-403; Tan, Y. H., Armstrong, J. A., Ke, Y. H., and Ho, M. (1970) Proc. Natl. Acad. Sci. U. S. A. 67, 464-471). If polyinosinic acid is added 1 hour prior to, simultaneously with, or 1 hour after the active interferon inducers, dramatic reductions in interferon production from the "superinduced" cells result. Based on experiments involving sucrose gradient ultracentrifugation, pancreatic ribonuclease A resistance, ultraviolet mixing curves, and ultraviolet absorbance-temperature profiles, the explanation for this phenomenon was determined to be the formation of polynucleotide triplexes in the following way: poly(A)-poly(U) + poly(I) yields poly(A)-poly(U)-poly(I)poly(A)-poly(rT) + poly(I) yields poly(A)-poly(rT)-poly(I). In addition, based on similar methodology, the following reactions involving these triplexes were demonstrated: poly(A)-2 poly(I) + poly(U) yields poly(A)-poly(U)-poly(I) + poly(I)poly(A)-2 poly(I) + poly(rT) yields poly(A)-poly(rT)-poly(I) + poly(I)POLY(A)-2 poly(I) + 2 poly(U) yields poly(A)-2 poly(U) + 2 poly(I) and POLY(A)-poly(U)-poly(I) + poly (U) yields poly(A)-2 poly(U) + poly(I).
The bicyclam AMD3100 (originally called JM3100), in which the two cyclam rings are tethered by an aromatic bridge, emanated from JM2763, where the two cyclam moieties are tethered by an aliphatic linker - JM2763 in turn originated from JM1657, where the cyclam rings are directly linked to one another via a C-C bridge, and which was identified as an impurity, showing anti-HIV activity, in a commercial cyclam preparation. AMD3100 proved very effective against HIV-1 and HIV-2, inhibiting virus replication within the nM range, without toxicity for the host cells at concentrations that were > 100,000-fold higher than those required to inhibit HIV replication. The anti-HIV activity of AMD3100 appeared to be confined to the T-lymphotropic (X4) HIV strains, i.e. those strains that use the CXCR4 receptor to enter their target cells, and AMD3100 as of today still stands as one of the most potent and selective CXCR4 antagonists ever discovered. Hence, AMD3100 was found to interfere with a number of (patho)physiological processes which depend on the interaction of CXCR4 with its natural ligand, stromal derived factor (SDF-1) and which play an important role in rheumatoid, allergic and malignant diseases. AMD3100 has been shown to mobilize CD34+ stem cells from the bone marrow into the bloodstream and has also been shown to augment migration of bone marrow-derived endothelial progenitor cells into sites of neovascularization after myocardial infarction. Currently, AMD3100 is actively pursued as a stem cell mobilizer for transplantation in patients with multiple myeloma and non-Hodgkin's lymphoma.
The HIV replicative cycle reveals several virus-specific events that could function as targets for chemotherapeutic intervention. The compounds that are presently available as anti-HIV drugs are targeted at either the substrate binding site of the reverse transcriptase (zidovudine, didanosine, zalcitabine, stavudine, lamivudine) or a non-substrate binding site of the reverse transcriptase (nevirapine, delavirdine), or the viral protease (saquinavir, ritonavir, indinavir, nelfinavir). Remarkable clinical efficacy has been observed with combinations of different reverse transcriptase inhibitors and protease inhibitors. It may be anticipated that with the advent of newer and more efficient compounds the effectiveness of HIV inhibition could still be improved upon and the prospects for a definitive cure of the disease may be accomplished. An account with 107 references.
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Studies on the interaction with cells of the double-stranded complex of polyriboinosinic acid and polyribocytidylic acid [poly(rI).poly(rC)] have shown that the polymer binds rapidly to the cell and that of this only a very small amount penetrates into the cell (Bausek & Merigan, 1969). It has been shown further that the interferon inducing capacity of synthetic polynucleotides is markedly enhanced upon pre-incubation of the polymers at 37° in tissue culture medium in the absence of polycations (De Clercq, Wells & Merigan, 1970; De Clercq & Merigan, 1971) and that the preheated polymers bind more rapidly to the cell and persist for a longer time at the outer cell membrane than do the unheated polymers (De Clercq et al. 1971). Although these studies do not indicate whether the interferon response is triggered by the bulk of cell-associated polymer that resides at the cell surface or by the minute amount that penetrates into the cell (Bausek & Merigan, 1969; De Clercq et al. 1971), they suggest that the cell surface may play an important role in the production of interferon by synthetic polynucleotides.
