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HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.

Resonance Raman spectra of myoglobins reconstituted with hemes isotopically substituted at the central iron atom or the pyrrole nitrogen atoms have been recorded to address the issue of whether the strong line at ~220 cm"1 is the iron-histidine stretching mode or the iron-pyrrole nitrogen stretching mode. The frequency of the line at 220 cm'1 is 1.7 cm"1 lower in myoglobin reconstituted with the 57Fe heme than it is in the 54Fe-substituted heme. No large shifts were detected in any other Raman lines. When myoglobin reconstituted with 15N-substituted pyrrole nitrogens in the heme is compared to the unsubstituted myoglobin no large change is detected in the line at 220 cm"1, but the frequency of the line at 243 cm"1 is 1.5 cm"1 lower. In comparing myoglobin buffered in D20 to that buffered in H20 only the line at 220 cm"1 changes frequency (1.4 cm"1). From these isotopic substitution studies, we conclude that the line at ~220 cm"1 in myoglobin is the iron-histidine stretching mode. The mode at ~243 cm"1 has a significant contribution from the pyrrole nitrogens, and it is likely an out-of-plane pyrrole tilting mode. The 54Fe-57Fe isotope shift of 1.7 cm"1 in the 220-cm"1 line is smaller than predicted for a diatom oscillator of the iron and the histidine. We conclude that the iron-histidine stretching mode is either mixed with an internal mode of the histidine and/or mixed with skeletal modes of the porphyrin macrocycle.Resonance Raman scattering has been applied extensively to 7 AT&T Bell Laboratories.

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