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

Hwang, Sangwon; Tamilarasu, Natarajan; Kibler, Karen V.; Cao, Hong; Ali, Akbar; Ping, Yueh Hsin; Jeang, Kuan Teh; Rana, Tariq M.

doi:10.1074/jbc.m301749200

Cited by 68 publications

(54 citation statements)

References 49 publications

(41 reference statements)

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“…It has been established that the TAT protein has a key role in the progression of AIDS and is a potential target for anti-HIV vaccines (4). For the TAT protein to carry out its biological functions, it needs to be readily imported into the cell.…”

mentioning

confidence: 99%

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

Rao

Kwok

Lombardi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson-Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants K d for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC K d was found to be 7.5 ± 2 μM, and for POPG K d was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from −57 mV to −37 mV. A numerical calculation of K d , which included all terms obtained from application of the Poisson-Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution.Gouy-Chapman model | hyper-Rayleigh scattering | biomolecules/water interface | colloid/water interface | nonlinear optical spectroscopy T he HIV type 1 (HIV-1) transacting activator of transduction (TAT) is an important regulatory protein for viral gene expression (1-3). It has been established that the TAT protein has a key role in the progression of AIDS and is a potential target for anti-HIV vaccines (4). For the TAT protein to carry out its biological functions, it needs to be readily imported into the cell. Studies on the cellular internalization of TAT have led to the discovery of the TAT peptide, a highly cationic 11-aa region (protein transduction domain) of the 86-aa full-length protein that is responsible for the TAT protein translocating across phospholipid membranes (5-8). The TAT peptide is a member of a class of peptides called cell-penetrating peptides (CPPs) that have generated great interest for drug delivery applications (ref. 9 and references therein). The exact mechanism by which the TAT peptide enters cells is not fully understood, but it is likely to involve a combination of energy-independent penetration and endocytosis pathways (8, 10). The first step in the process is high-affinity binding of the peptide to phospholipids and other components on the cell surface such as proteins and glycosaminoglycans (1, 9).The binding of the TAT peptide to liposomes has been investigated using a variety of techniques, each...

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mentioning

confidence: 99%

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

Rao

Kwok

Lombardi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Consequently the development and use of antiviral drugs capable of preventing or abrogating HPIV3 infection becomes a strong alternative approach. In the recent past antiviral discovery programs have been initiated towards targeting many pathogenic viruses using various mechanism-based screening methods (Hwang et al, 2003;Mason et al, 2004). Random chemicals and natural product mixtures are being tested for their ability to block replication of a variety of viruses in cell culture systems.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of human parainfluenza virus type 3 infection by novel small molecules

Mao¹,

Thakur²,

Chattopadhyay³

et al. 2008

Antiviral Research

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Human parainfluenza virus type 3 (HPIV3) is an important respiratory tract pathogen of infants and children. There are no vaccines or antivirals currently approved for prevention or treatment of HPIV3 infection. Towards developing an antiviral therapy to combat HPIV3 infection, we have established a green fluorescent protein (GFP)-tagged HPIV3 infected-cell assay and used it for screening of a small molecule library obtained from ChemBridge Diver. Two novel small molecules (C5 and C7) which shared structural similarities were identified and their inhibitory effects on HPIV3 were confirmed in CV-1 and human lung epithelium A549 cells by plaque assay, Western blot and Northern blot analyses. C5 and C7 effectively prevented the cytopathic effect in cells infected with HPIV3, achieving IC 50 values of 2.36μM and 0.08μM, respectively, for infectious virus production. The inhibition appears to be at the primary transcriptional level of HPIV3 life cycle based on sequential time course test, binding and internalization assays, and finally by a minigenome transcription assay in cells as well as measuring viral transcripts in cells in the presence of anisomycin. Interestingly, vesicular stomatitis virus (VSV), another member of mononegavirales order, was also inhibited by these compounds, whereas poliovirus-a picornavirus was not. Use of these inhibitors has a strong potential to develop novel antiviral agents against this important human pathogen.

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“…Indeed, on-bead screening with RNA probes have resulted in the identification of ligands for the TAR RNA sequence (Hwang et al, 2003). Similarly, small molecule microarrays might be useful to identify molecules that interact with DNA in a sequence-specific manner.…”

Section: Identification Of Dna Rna and Carbohydrate Ligandsmentioning

confidence: 99%

“…This and related approaches have been used successfully to find peptide ligands for several proteins such as streptavidin (Lam et al, 1991) and the src SH3 domain , ligands for RNAs (Hwang et al, 2003), and peptoid ligands for mdm2 and glutathione S-transferase (Alluri et al, 2003). Up to 1 to 2 million compounds per day can be queried on beads.…”

Section: On-bead Screeningmentioning

confidence: 99%

Ligand Discovery Using Small Molecule Microarrays

He¹,

Gerona‐Navarro²,

Jaffrey³

2004

J Pharmacol Exp Ther

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Small molecule microarrays have recently been used to identify ligands for several proteins, and several themes regarding screening strategies and limitations have emerged. In this review, some of the technical issues related to the manufacture and screening of small molecule microarrays, as well as prospects for small molecule microarrays in several areas of drug discovery and chemistry, are discussed.A major advance in drug discovery has been the development of techniques to generate large libraries of chemical compounds using combinatorial chemistry. Several combinatorial chemistry techniques exist to generate large libraries, including the split-pool (or "one bead-one compound") approach. These techniques have increased the number of compounds that are available for screening purposes. However, with the large number of different types of libraries available, methods to rapidly screen these libraries for molecules that have biologically relevant activity are needed. In this review, we will discuss one new approach for improving library screening-the small molecule microarray. In this review, we will use the term "small molecule" to refer to non-DNA molecules that are prepared using synthetic chemistry, rather than molecules that are obtained from cells or biochemical reactions, such as proteins. The small molecule microarray is a library of compounds that is immobilized on a two-dimensional surface, such as a glass slide, and is spatially addressable and miniaturized. Small molecule microarrays have recently been used to identify ligands for several proteins, and several themes regarding screening strategies and limitations have emerged. In this review, we will discuss the manufacture and use of microarrays as well as its potential for contributing to the lead identification process. We will also discuss prospects for small molecule microarrays in other areas of drug discovery and chemistry. Historical Foundation of Small Molecule Microarrays On-Bead ScreeningSmall molecule microarrays merge the relatively mature technology of on-bead library screening with microarray technology, the latter of which has become an especially powerful tool used primarily by molecular biologists in the context of DNA microarrays. Screening immobilized libraries of small molecules on beads was described in one of the original reports that described the technique of split-pool library synthesis (Lam et al., 1991). The split-pool approach (also known as "portioning-mixing" and related names) involves dividing polymeric supports (i.e., the "beads") into portions for coupling to modular units (i.e., "diversity elements") and then recombining the beads (Furka et al., 1991;Houghten et al., 1991;Lam et al., 1991). Once combined, the beads are washed, deprotected, etc., and the process of splitting the beads and pooling the beads is repeated for several cycles to make a large collection of molecules. Thus, each bead will contain molecules whose structure is a consequence of the history of the diversity element coupling steps to w...

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

Cited by 68 publications

References 49 publications

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

Inhibition of human parainfluenza virus type 3 infection by novel small molecules

Ligand Discovery Using Small Molecule Microarrays

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