Houng-Yau Mei

Inhibition of an HIV-1 Tat-derived peptide binding to TAR RNA by aminoglycoside antibiotics

Abstract Aminoglycoside antibiotics were found for the first time to inhibit an HIV-1 Tat-derived peptide binding to TAR RNA. The IC 50 values of neomycin, streptomycin, and gentamicin were determined as 0.92 μM, 9.5 μM, and 45 μM, respectively. In the absence of Tat peptide these antibiotics were found to cause mobility shifts of the TAR RNA on non-denaturing polyacrylamide gels. This is the first example, to our knowledge, of non-peptide or non-nucleotide like small molecules that bind to and induce a mobility shift of TAR RNA. It was further found that the antibiotics which demonstrate higher affinity for TAR are better inhibitors of the Tat/TAR interaction. Mutational and competition studies indicate that neomycin binds to the duplex domain of TAR RNA.

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

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.

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

Abstract 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.

Positive ion electrospray ionization mass spectrometry of oligonucleotides

Positive ion electrospray ionization mass spectra have been obtained of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), including transfer RNAs (77-mer, ∼ 25 kDa). For several different solution conditions, the charge state distributions of DNA and RNA molecules were determined. It is postulated that the production of the multiply charged positive ions results from gas phase dissociation of complexes between nitrogen-containing bases and oligonucleotides.

Discovery of selective, small-molecule inhibitors of RNA complexes--II. Self-splicing group I intron ribozyme.

Self-splicing group I intron RNA was chosen as a potential therapeutic target for small-molecule intervention. High-throughput screening methodologies have been developed to identify small organic molecules that regulate the activities of these catalytic introns. Group introns derived from pathogenic Pneumocystis carinii and phage T4 were used as model systems. Inhibitors identified from a library of approximately equal to 150,000 compounds were shown to regulate biochemical reactions including the two-step intron splicing and an RNA ligation catalyzed by the group I introns. These inhibitors provide a unique opportunity to understand small-molecule recognition of the self-splicing RNA. The methodologies developed for group I introns should be applicable to studies of other RNA systems.

Integrated drug discovery technologies

From gene to screen: target identification and validation Coupling Genotype to Phenotype functional genomics integrated proteomics technologies where science meets silicone - microfabrication techniques and their scientific applications SNP scoring for drug discovery applications protein display chips integrated proteomics technologies and in vitro validation of molecular tests. High throughput screening: high throughput screening as a discovery resource fluorescence correlation spectroscopy (FCS) and FCS-related confocal fluorimetric methods (FCS + plus) multiple read-out options or miniaturized screening homogeneous time-resolved fluorescence (HTRF) ADME-tox screening in drug discovery screening lead compounds in the post-genomic era - an integrated approach to knowledge building from living cells.

Studying Noncovalent Protein-RNA Interactions and Drug Binding by Electrospray Ionization Mass Spectrometry

The field of biochemical analysis by mass spectrometry is maturing at an ever-increasing pace. The race to develop so-called “soft” desorption/ionization methods to vaporize intact, large biomolecules consumed many research laboratories prior to 1990. As important milestones along the molecular weight path were reached, from small peptides, to insulin (5.7 kDa), to trypsin (23 kDa), new applications were developed. However, the explosive growth in the field did not occur until electrospray ionization (ESI) [1] and matrix-assisted laser desorption/ionization (MALDI) [2] in the late 1980’s-to-early-1990’s came onto the scene. Biological compounds with Mr greater than 150 kDa can be measured with sub-picomole sensitivity by ESI-MS and MALDI-MS. Now, much of mass spectrometry has been put into the hands of the non-mass spectrometrist.

Studying Noncovalent Small Molecule Interactions with Protein and RNA Targets by Mass Spectrometry

Developing methods for elucidating the mechanism of action of drugs and inhibitors on a structural level is an active area of biomedical and pharmaceutical research. To develop new therapies at an increasing pace, more new drugs need to be synthesized or discovered, more novel macromolecular targets need to be identified, and compounds need to be characterized, screened and evaluated at a faster rate. Advances and application of combinatorial chemistry address the need for more compounds [1, 2], whereas research endeavors such as genomics [3] and proteomics [4, 5] have shown great potential for uncovering novel targets. Methods for high-throughput screening using a variety of large-scale automated processes and robotics have been developed to further address some of these important needs [6]. However, biophysical techniques are still needed to fully elucidate the fundamental aspects of inhibitor action on its target.