Discovery of selective, small-molecule inhibitors of RNA complexes—1. The tat protein/TAR RNA complexes required for HIV-1 transcription

Mei, Houng‐Yau; Mack, David P.; Galan, Adam; Halim, Nadia S.; Heldsinger, Andrea; Loo, Joseph A.; Moreland, D.W.; Sannes‐Lowery, Kristin A.; Sharmeen, Lamia; Truong, Hoa N.; Czarnik, Anthony W.

doi:10.1016/s0968-0896(97)00064-3

Cited by 140 publications

(136 citation statements)

References 37 publications

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“…Tat 40 and TAR 31 ( Figure 1) were chemically synthesized and purified as described previously (19). RNA was labeled either at the 3′-end with [5′-32 P]pCp using T4 RNA ligase or at the 5′-end with [γ-32 P]ATP using T4 polynucleotide kinase.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously reported a drug discovery approach (19) designed to identify small organic molecules that inhibit HIV-1 replication by blocking Tat-TAR interaction. As shown in Figure 1, the sequences of TAR 31 (containing residues 18-44 of HIV-1 mRNA) and Tat 40 peptide (residues 47-86), each containing the key element for protein-RNA recognition, were used in these studies.…”

Section: Identification Of Three Small Molecule Tat-tar Inhibitorsmentioning

confidence: 99%

“…Under similar conditions, no complexes with a Tat 40 peptide were found for any of these small molecules. Furthermore, from ESI-MS measurements, addition of these small molecules to preformed the Tat- IC 50 values were obtained using gel mobility shift assays as described previously (19). All inhibition studies were performed in a buffer solution containing 10 mM Tris-HCl (pH 7.5), 70 mM NaCl, 0.2 mM EDTA, 0.01% Nonidet P-40, 5% glycerol, and ∼100 pM 3′-end 32 P-labeled TAR.…”

Section: Identification Of Three Small Molecule Tat-tar Inhibitorsmentioning

confidence: 99%

“…Deletion of either bulge or loop residues of TAR impairs the transcription and, subsequently, the replication of HIV-1 (5, 12). HIV-1 TAR RNA and its cellular functions, therefore, represent a potential target for therapeutic intervention (17)(18)(19).In this paper, we report that multiple small molecule inhibitors can be identified for the HIV-1 Tat-TAR system, each sufficiently potent to evoke interest in initiating a structure-activity relationship (SAR) study. Furthermore, these three small molecule inhibitors bind to TAR at three distinct sites, as characterized by footprinting methods.…”

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confidence: 99%

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Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Mei¹,

Cui²,

Heldsinger³

et al. 1998

Biochemistry

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TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model. Drug discovery has traditionally involved a search for inhibitors of protein complexation to small molecules (e.g., substrates or ligands) or macromolecules (e.g., other proteins, nucleic acids, or polysaccharides). While agonists or antagonists of macromolecular receptors that become useful drugs must display a wide range of other attributes, including the appropriate physicochemical properties, pharmacokinetic and pharmacodynamic properties, stability, etc., they must first be protein ligands. The vast majority of available drugs act by binding noncovalently to a protein, preventing or (less often) stimulating that protein's complexation to a complement (1).Complexes of nucleic acid and proteins are key intermediates in all transcriptional and translational processes. Some nucleic acids (e.g., ribozymes) even form functional complexes with small molecules (2). Nonetheless, nucleic acids are widely viewed as ineffective targets for the discovery of low-molecular weight inhibitors. One reason is that the linear motif in single-stranded DNA and the repetitive motif in double-stranded DNA provide attractive targets for large, linear binding molecules (3), but unattractive targets for the small molecules that lead to orally available medications. Another reason is that the lack of tertiary structure in DNA does not afford the diverse topology associated with folded proteins.However, single-stranded RNA often folds into welldefined tertiary structures (4). Furthermore, such structures serve as docking sites for transcriptional activators (5) and substrates for self-splicing reactions (6, 7). Can such structured nucleic acids display sufficient shape diversity to permit the complexation of a small molecule at one site in a virtual sea of nucleic acid material? This is the essential question whose presumed negative answer hinders drug discovery at the nucleic acid level.Certain cis-acting RNA elements are essential for the gene expression of human immunodeficiency virus type 1 (HIV-1) (8). The functions and sequences of these RNA molecules have been well characterized (9-11). A segment of HIV-1 mRNA (residues 1-59), identified as the transactivation responsive element (TAR), adopts a stem-loop secondary structure consisting of a highly cons...

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Section: Methodsmentioning

confidence: 99%

Section: Identification Of Three Small Molecule Tat-tar Inhibitorsmentioning

confidence: 99%

Section: Identification Of Three Small Molecule Tat-tar Inhibitorsmentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Mei¹,

Cui²,

Heldsinger³

et al. 1998

Biochemistry

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158

140

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“…On-bead Selection of TAR RNA-binding Unnatural Small Molecules-Combinatorial chemistry has been previously used to identify new ligands that block the Tat-TAR interaction, but previous studies have relied on a variety of complex methods that are labor-intensive or require expensive robotic equipment (25). For the most part, these methods originated in the study of individual protein-nucleic acid interactions.…”

Section: Small Molecule Tat-tar Antagonist Inhibits Hiv-1 Replicationmentioning

confidence: 99%

Discovery of a Small Molecule Tat-trans-Activation-responsive RNA Antagonist That Potently Inhibits Human Immunodeficiency Virus-1 Replication

Hwang

Tamilarasu

Kibler

et al. 2003

Journal of Biological Chemistry

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Antiretroviral therapy to treat AIDS uses molecules that target the reverse transcriptase and protease enzymes of human immunodeficiency virus, type 1 (HIV-1).A major problem associated with these treatments, however, is the emergence of drug-resistant strains. Thus, there is a compelling need to find drugs against other viral targets. One such target is the interaction between Tat, an HIV-1 regulatory protein essential for viral replication, and trans-activation-responsive (TAR) RNA. Here we describe the design and synthesis of an encoded combinatorial library containing 39,304 unnatural small molecules. Using a rapid high through-put screening technology, we identified 59 compounds. Structure-activity relationship studies led to the synthesis of 19 compounds that bind TAR RNA with high affinities. In the presence of a representative Tat-TAR inhibitor (5 M TR87), we observed potent and sustained suppression of HIV replication in cultured cells over 24 days. The same concentration of this inhibitor did not exhibit any toxicity in cell cultures or in mice. TR87 was also shown to specifically disrupt Tat-TAR binding in vitro and inhibit Tat-mediated transcriptional activation in vitro and in vivo, providing a strong correlation between its activities and inhibition of HIV-1 replication. These results provide a structural scaffold for further development of new drugs, alone or in combination with other drugs, for treatment of HIV-1-infected individuals. Our results also suggest a general strategy for discovering pharmacophores targeting RNA structures that are essential in progression of other infectious, inflammatory, and genetic diseases.

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Application of mass spectrometry for target identification and characterization

Loo¹,

DeJohn²,

Du³

et al. 1999

Med. Res. Rev.

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Mass spectrometry‐based methodologies span the vast expanse of drug discovery. Both electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) support proteomics‐based research projects by identifying proteins separated and isolated by polyacrylamide gel electrophoresis. MALDI‐MS‐based surface scanning of one‐dimensional isoelectric focusing gels, “virtual 2‐D gel electrophoresis,” represents a potentially high throughput means to map proteins and to determine protein profiles. Mass spectrometry can also be used to directly study the covalent and noncovalent interactions of drug molecules and biomolecule targets. Drug binding examples discussed include the binding of covalent and noncovalent inhibitors to src SH2 domain protein, and the interaction of aminoglycoside antibiotic neomycin and HIV Tat peptide‐TAR RNA. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 4, 307–319, 1999.

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Discovery of selective, small-molecule inhibitors of RNA complexes—1. The tat protein/TAR RNA complexes required for HIV-1 transcription

Cited by 140 publications

References 37 publications

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Inhibitors of Protein−RNA Complexation That Target the RNA: Specific Recognition of Human Immunodeficiency Virus Type 1 TAR RNA by Small Organic Molecules

Discovery of a Small Molecule Tat-trans-Activation-responsive RNA Antagonist That Potently Inhibits Human Immunodeficiency Virus-1 Replication

Application of mass spectrometry for target identification and characterization

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