毛细管区带电泳法测定4种抗HIV-1活性的化合物与牛血清白蛋白的结合常数

采用毛细管区带电泳紫外检测方法,在pH 8.0、浓度50 mmol/L的磷酸盐缓冲溶液及运行电压15 kV的条件下,测定了4种新合成的具有抗HIV-1活性的化合物(IG3,iso-C3,C3,MC3)与牛血清白蛋白(BSA)相互作用的结合常数。在缓冲溶液中加入不同浓度的BSA,通过测定化合物迁移时间的变化,计算得到了上述4种化合物与BSA的结合常数分别为1.07×104, 1.34×104, 8.51×103和9.45×103 L/mol。该方法简单、快捷,可用于研究结合比为1∶1的小分子与生物大分子的     

Design and Synthesis of Guanidinoglycosides Directed against the TAR RNA of HIV‐1

Replication of human immunodeficiency virus type 1 (HIV‐1) requires specific interactions of the Tat protein with the transactivation responsive region (TAR) RNA. Tat‐TAR RNA Interaction is mediated by a short arginine‐rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. Here, four novel 6‐amino‐6‐deoxytrehalose guanidinoglycoside derivatives ( 10 and 13 – 15 ) as target molecules have been designed to bind to TAR RNA for blocking the interaction of Tat‐TAR RNA. They were obtained by coupling 6‐amino‐6‐deoxy‐α,α‐trehalose ( 6 ) with the protected amino acids, deprotection by catalytic hydrogenation, followed by guanidinylated with S‐methylisothiourea sulfate. Their abilities to inhibit Tat‐TAR RNA interaction were determined by a Tat‐dependent HIV‐1 long terminal repeats (LTR)‐driven chloramphenicol acetyltransferase (CAT) assays.     

Structural mimicry of retroviral tat proteins by constrained beta-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements

An approach is described to the design of beta-hairpin peptidomimetic ligands for bovine immunodeficiency virus (BIV) Tat protein, which inhibit binding to its transactivator response element (TAR) RNA. A library of peptidomimetics was derived by grafting onto a hairpin-inducing d-Pro-l-Pro template sequences related to the RNA recognition element in Tat. One hairpin mimetic was identified that binds tightly (K(d) approximately 150 nM) to BIV TAR, and another that binds also to HIV-1 TAR RNA (K(d) approximately 1-2 microM). (In the same assay, the wild-type BIV Tat(65-81) peptide binds to BIV TAR with K(d) approximately 50 nM.) The high-affinity BIV-Tat mimetic was shown to adopt a stable beta-hairpin conformation in free solution by NMR methods. Amino acid substitutions in this mimetic were shown to impact on the hairpin structure and to disrupt binding to the RNA. This family of conformationally constrained peptidomimetics affords insights into the structural requirements for binding to TAR RNA and provides a basis for the design of new ligands with increased inhibitory activity and specificity to both BIV and HIV TAR RNAs.     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

Design and synthesis of á, á-trehalose derivatives bearing guanidino groups as inhibitors for the Tat Protein-TAR RNA interaction of HIV-1

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6' -dideoxy-α,α-trehalose was obtained from selective bromination of α,α-trehalose at C-6,6', fo…     

Differentiate RNA Single‐Stranded Region of the Branched Structures and Hairpin Loops by an Octahedral Cobalt(II) Complex

Single‐stranded RNA molecules usually include secondary structural elements such as bulges, internal loops, and hairpin loops. These RNA secondary structural elements are often essential for the biological activity and functions of the RNA molecule. Chemical probe Co^II(HAPP)(TFA)₂ in the presence of H₂O₂ is found to differentiate single‐stranded RNA from branched structures and hairpin loops. This study uses Co^II(HAPP)(TFA)2 to analyze the structures of two RNA molecules: a fragment of HIV TAR RNA (TAR‐27) and the catalytic domain 5 of group II intron (D5‐29). The electrophoretic mobility of TAR‐27 does not shift in the presence of Co^II(HAPP)(TFA)₂, suggesting that the reagent does not change the conformation of RNA substrate. Cleavage of the RNA substrates by Co^II(HAPP)(TFA)₂ unambiguously differentiated RNA internal looped structures from hairpin loops. The results show that Co^II(HAPP)(TFA)2 is a sensitive, informative and convenient tool for analysis of RNA secondary structures.     

α-Helix-Mimetic Foldamers for Targeting HIV-1 TAR RNA

An oligopyridylamide-based foldamer approach has been employed to target HIV TAR RNA-TAT assembly as a model system to study RNA-protein interactions. The oligopyridylamide scaffold adopts a constrained conformation which presents surface functionalities at distinct spatial locations and mimic the chemical features of the secondary structure of proteins. We have designed a library of oligopyridylamides containing diverse surface functionalities which mimic the side chain residues of the TAT protein domain. The interaction of TAR RNA and TAT plays a pivotal role in facilitating HIV replication. The library was screened using various fluorescent based assays to identify antagonists of the TAR RNA-TAT complex. A tricationic oligopyridylamide ADH-19, possessed the highest affinity towards TAR and efficiently inhibited the TAR RNA-TAT interaction with apparent K_d of 4.1±1.0 μm . Spectroscopic studies demonstrated that ADH-19 interacts with the bulge and the lower bulge regions of TAR RNA, the domains important for TAT interaction. ADH-19 demonstrated appreciable in vivo efficacy (IC₅₀=25±1 μm ) by rescuing TZM-bl cells infected with the pseudovirus HIV-1HXB-2.     

A distance ruler for RNA using EPR and site-directed spin labeling

As a basic model study for measuring distances in RNA molecules using continuous wave (CW) EPR spectroscopy, site-directed spin-labeled 10-mer RNA duplexes and HIV-1 TAR RNA motifs with various interspin distances were examined. The spin labels were attached to the 2'-NH2 positions of appropriately placed uridines in the duplexes, and interspin distances were measured from both molecular dynamics simulations (MD) and Fourier deconvolution methods (FD). The 10-mer duplexes have interspin distances ranging from 10 A to 30 A based on MD; however, dipolar line broadening of the CW EPR spectrum is only observed for the RNAs for predicted interspin distances of 10-21 A and not for distances over 25 A. The conformational changes in TAR (transactivating responsive region) RNA in the presence and in the absence of different divalent metal ions were monitored by measuring distances between two nucleotides in the bulge region. The predicted interspin distances obtained from the FD method and those from MD calculations match well for both the model RNA duplexes and the structural changes predicted for TAR RNA. These results demonstrate that distance measurement using EPR spectroscopy is a potentially powerful method to help predict the structures of RNA molecules.     

Target-Directed Azide-Alkyne Cycloaddition for Assembling HIV-1 TAR RNA Binding Ligands

The highly conserved HIV-1 transactivation response element (TAR) binds to the trans-activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat–TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV-1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin-tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole-linked thiazole peptidomimetic products have been isolated from the biotin-tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat–TAR interactions.     

Mapping the interaction forces between TAR RNA and TAT peptides on GaAs surfaces using chemical force microscopy

The complexation of the HIV transactivation response element (TAR) RNA with the viral regulatory protein TAT is of enormous interest for the design of new sensing and therapeutic strategies. In this work, we anchored TAT peptides on GaAs surfaces using microcontact printing. Atomic force microscopy was used to quantify the interaction between TAR RNA and model TAT peptide sequences. Different pH conditions were utilized in order to assess specific vs nonspecific interactions. AFM tips functionalized with TAR RNA molecules were used to collect adhesion maps that displayed stronger interaction with peptide sequences that contained a greater number of arginine residues. All of the studies consistently showed a pH dependence of the interaction between the surface bound peptides and the TAR RNA on the AFM tips. This work quantifies the TAR RNA/TAT peptide interaction after one of the molecules is anchored on a surface. The conclusions in this paper are consistent with previous work and demonstrate that cationic residues are responsible for the polyelectrolyte-like affinity of TAT peptides for TAR RNA.     

Target‐Directed Azide‐Alkyne Cycloaddition for Assembling HIV‐1 TAR RNA Binding Ligands

The highly conserved HIV‐1 transactivation response element (TAR) binds to the trans‐activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat–TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV‐1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin‐tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole‐linked thiazole peptidomimetic products have been isolated from the biotin‐tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat–TAR interactions.