Yamazaki, Satoko: High Throughput Screening and mRNA-Display Selection for the Identification of Biologically Functional Molecules. - Bonn, 2006. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5N-08697
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5N-08697,
author = {{Satoko Yamazaki}},
title = {High Throughput Screening and mRNA-Display Selection for the Identification of Biologically Functional Molecules},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2006,
note = {Biologically functional molecules with desired properties have proven to be invaluable tools for investigating biological systems. Moreover, the identification of those functional molecules is a crucial task in modern drug discovery. These goals can be achieved by two major approaches: gscreeningh and gselectionh.
Chapter 2 introduces one attractive method for gscreeningh to identify small molecule inhibitors of a given protein target from a small organic chemical library.
The reverse transcriptase (RT) plays a crucial role in the early steps of the life cycle of HIV. Therefore HIV-1 RT serves as one of the major drug targets in anti-HIV therapy. Although current therapy for HIV-infected patients involving a combination of three or more of the following drugs: RT inhibitors, protease inhibitors, and/or viral entry inhibitors, has been highly successful, its long-term efficacy is severely limited by the emergence of drug-resistant variants of HIV. One way to address this problem is to identify new type of drugs with different inhibitory mechanisms to combat HIV. Thus, HIV-1 RT served a valuable target in the herein depicted screening approach.
Protein-dependent alosteric ribozymes (or reporter ribozymes) are powerful tools which are able to sense molecular interactions, e.g. protein-protein, protein-nucleic acid, or protein-metabolite interactions in real-time. The expedience of this assay format makes it applicable to and suitable for high throughput screening in order to search for new molecules which disrupt such interactions. Reporter ribozyme FK-1 is a rationally engineered hammerhead ribozyme in a fusion construct with anti-RT aptamer, which can detect the presence of HIV-1 RT selectively in a domain specific manner. Thus, FK-1 enables to identify compounds via FRET-based fluorescence readout, which compete for the binding to HIV-1 RT with aptamer sequences inserted in the ribozyme construct.
Through screening of 2500 small molecules using the reporter ribozyme (FK-1)-based assay, three potential inhibitors (28F6, 3E4 and 2E10) of HIV-1 RT were identified with in vitro inhibitory concentration that gave half-maximal activity (IC50) of 2-5 µM on DNA-dependent DNA polymerase activity. The three compounds were re-synthesized and further investigated to evaluate their selectivity and to elucidate the inhibitory mechanism. The selectivity was determined by testing inhibitory effect of the compound on other RTs (AMV and MMLV) and DNA polymerases from both prokaryotic (Klenow Fragment) and eukaryotic (human DNA polymerase ƒÀ) sources. Among the three compounds, 3E4 showed not only the lowest IC50 value for HIV-1 RT (2.1 µM) but the highest discrimination from all the other RT and DNA polymerases (10~50 fold) in terms of DNA-dependent DNA polymerase activity. The selectivity of 3E4 with the cognate RT from HIV-2 was a factor of approximately 4.5. In addition, the interaction between 3E4 and HIV-1 RT was observed by a surface plasmon resonance-based biosensor.
Moreover, a cell-based assay for phenotypic analysis of sensitivity of HIV-1 to the three inhibitors using a self-inactivating virus vector system demonstrated that 3E4 inhibited marker gene expression in concentration dependent manner with a IC50 value of 5.3 µM. The effect of 3E4 was further confirmed with the similar reduction of HIV-1 replication and infectivity in vivo using a HIV wild type strain.
In summary, the data strongly indicates that the small molecule 3E4 identified by reporter ribozyme-based screening is a highly specific and cellular active HIV-1 RT inhibitor, which has significant potentiality as novel type of anti-HIV drug. Furthermore, the approach using a reporter ribozyme assay could be broadly applicable as screening format.
In chapter 3, the challenge of a gselectionh approach has been performed with the aim to evolve functional proteins from a random sequence protein library.
Prior to the selection, the protein library consisting of a random 88 amino acid sequence was constructed by short cassettes ligations to avoid stop codons and frameshift mutations. The design of secondary structural patterns like ƒ¿-helix and ƒÀ-strand was introduced into the random regions.
A display technology called mRNA-display was exploited to prepare protein library, where each protein sequence was covalently linked through its carboxy terminus to the 3f end of its encoding mRNA. Starting with a library that contained 1 ~ 1011 - 5 ~ 1012 different proteins, three in vitro selections were attempted to identify protein binders to small molecules such as cibacron blue 3GA and coenzymes (NADP and CoA). The selection against cibacron blue 3GA yielded enriched fractions retaining on the column matrix after the ninth cycle. These sequences could not be eluted by NAD, the structural analogue of cibacron blue 3GA, even at higher concentration. The small fractions eluted with NAD from this column have been cloned and sequenced, resulting in unexpectedly high frequency of frameshifts and internal stop codons. The alignment of full length intact sequences exhibited no conserved motif but the most appearance of ƒ¿ƒÀƒ¿ƒÀ type of cassette. The remaining column fractions were able to be eluted with free cibacron blue 3GA, implying those sequences might be specific cibacron blue binders. Other selections against both NADP and CoA did not enrich protein binders.
The observed problems through the experiment concerning poor solubility and fold instability of mRNA-displayed proteins should be improved for further investigations.},

url = {http://hdl.handle.net/20.500.11811/2675}

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