Combinatorial Chemistry & High Throughput Screening - Volume 11, Issue 2, 2008

Throughout its first ten years of publication, Combinatorial Chemistry & High Throughput Screening has occupied a unique position among the peer reviewed literature by specializing in the publication of review articles and original research papers in combinatorial chemistry, high throughput screening, and the interface of these related fields. Papers published in CCHTS are highly visible and are abstracted and indexed by the major services including Current Contents/Life Sciences, Biological Abstracts, BIOBASE, BIOSIS, BIOSIS Previews, CAB Abstracts, Chemical Abstracts, Chemistry Citation Index, EMBASE, Google, Google Scholar, JCR/Science Edition, MEDLINE, Reaction Citation Index, Science Citation Index Expanded, and SCI Expanded. During the last year, CCHTS reached another milestone with its highest impact ever of 2.55 (according to the ISI Web of Knowledge, Journal Citation Reports, produced by the Thomson Corporation; Philadelphia, PA). In addition, CCHTS is ranked 4th out of 58 journals by Journal Citation Reports in the field of Applied Chemistry. After ten years of outstanding service to CCHTS, our Regional Editor for Asia, Prof. Takenori Kusumi of the Faculty of Pharmaceutical Sciences at the University of Tokushima, Japan, has decided to retire from this role at CCHTS. I would like to recognize Prof. Kusumi for his help when CCHTS was launched as a new journal ten years ago and then for his tireless commitment to make it a continuing success. When CCHTS began publication in 1998, combinatorial chemistry was so new that few manuscripts were being submitted from researchers in Asia. Thanks at least in part to the efforts of Prof. Kusumi, numerous manuscripts are now being submitted each year from authors in Asia. Taking his place as the new Regional Editor for Asia is one of the original members of the CCHTS Editorial Board, Prof. Ikuo Fujii of Osaka Prefecture University. Since Prof. Fujii brings ten years of experience on our Editorial Board to this position, the transition between Regional Editors for Asia should go unnoticed by authors awaiting referees' reviews and editorial decisions from this office. In addition to a new Regional Editor for Asia, there are several new members joining our Editorial Board as well as others who are retiring after many years of service. In particular, I would like to thank the retiring members of the Editorial Board of CCHTS for ten years of peer review of manuscripts submitted for publication and support of the journal. These distinguished Editorial Board members include Dr. Koshi Arai (Taisho Pharmaceutical; Saitama), Dr. Nobuyuki Okajima (Japan Tobacco; Osaka), Prof. Shigeki Sasaki (Kyushu University; Fukuoka), and Dr. Jun-ya Tamura (Yamanouchi Pharmaceutical; Ibaraki). For a complete list of the current Editorial Board members and Regional Editors of CCHTS, please visit our web site at the following Internet address: http://www.bentham.org/cchts. Information for authors, an index of papers published in CCHTS and free access to sample issues may also be found at our website. As we begin our 11th year of publication, Combinatorial Chemistry & High Throughput Screening remains a unique and essential scientific journal defining the intersection of both combinatorial chemistry and screening. We will continue to publish 10 issues per year, which is the most frequent publication rate in this field. This effort is made possible by the distinguished members of the Editorial Board, the Regional Editors, the production staff and management of Bentham Science publishers, the guest editors of special issues, the authors, and of course you, the readers. Thank you for ten years (and counting) of support for CCHTS.

High throughput screening (HTS) has become an indispensable tool in modern drug development and has been extended from the screening of chemical libraries to the screening of biological molecules or even whole organisms. Libraries of proteins and peptides can be screened for diverse applications including enzyme optimisation, functional genomics, or therapeutic purposes like drug development or targeted delivery. Mainly for technical difficulties in generating libraries covering large repertoires of polypeptide variants platform organisms were previously restricted to the prokaryotic kingdom. The scope of this edition is to review recent developments in display technologies beyond the well known and often used phage display technology. During the past years more and more library and screening systems have been described that are based on viruses and eukaryotic cells as platforms. This way, many sophisticated screening systems have been established. In this issue of Combinatorial Chemistry & High Throughput Screening, we have assembled a collection of articles describing the state of the art for different eukaryotic display platforms and for specific screening strategies performed in eukaryotic expression systems. The first two reviews focus on the use of two enveloped viruses as screening platform, baculovirus and retrovirus (reviews one and two). Both are used in many laboratories as vectors to produce large amounts of recombinant protein in a eukaryotic cell system or to genetically modify mammalian cells. Several unique properties of these viruses make them an ideal platform also for protein display and screening. Small peptides and also large polypeptides can be displayed on the viral surface or, using the virus as vector, on the cell membrane. In each case full processing and glycosylation of the displayed protein is possible. Screening strategies using retroviruses and baculoviruses include, but are not limited to, the identification of single-chain antibodies, protein interaction partners, antigenic epitopes or protease substrate peptides. Besides screening, display of polypeptides can also be applied for other purposes such as evoking an efficient immune response, identifying ligand receptor interaction partners, or modifying the tropism of viral vectors. HTS is also being used to improve viral gene transfer vectors. Libraries of vial variants of retroviruses and adeno-associated viruses are being generated by random peptide insertions, error prone PCR, DNA shuffling or transposon-based approaches (reviews three and four). Vectors based on the natural occurring virions have been used in many clinical gene therapy trials worldwide. However, to become more efficient in gene delivery in patients, properties like particle stability, nuclear entry into quiescent cells, escape from an inactivating immune response, or receptor choice and thus biodistribution are being improved by library screening. Yeast cells have become increasingly popular as display platform during the past years. Large repertoires can be covered and screening can be facilitated through fluorescent cell sorting techniques. Yeast based display libraries have been screened for different purpose including epitope mapping, the identification of interacting protein partners, or single-chain antibodies recognising specific molecules on the surface of human cells and tissue (review five). In peptide aptamers and Znfinger domains, which are in the focus of review six and seven, amino acid residues available for random diversification and library generation are well defined. Libraries of peptide aptamers are often screened in eukaryotic cell systems to identify peptides that interfere with certain cellular pathways by binding to a target protein whereas Zn-finger domains can be engineered to bind selectively to specific sequences of DNA. By combining them with effector domains of transcription factors, designer transcription factors can be generated. Effective “designer” transcription factors can be obtained through library screening for optimal binding and activation of a defined DNA sequence or a gene of interest. We conclude this issue with a review describing a novel approach that allows the screening for gene functions based on reversely transfected cell arrays. Effects on the cell phenotype resulting from the transfection of cDNA or siRNA can be screened in multi-well dishes. Applications of this technique in functional genomics are described and a laboratory protocol is provided. In summary, we have collected several articles that discuss the most important display platforms and screening strategies in eukaryotic expression systems. Only they enable the screening for properties, that exclusively manifest in mammalian cells or that require processing of the displayed polypeptide in a eukaryotic environment. Moreover, translational problems as they occur when molecules selected in a prokaryotic expression system are transferred to mammalian or human cells are avoided. It is therefore well conceivable that especially for therapeutic purposes HTS based on eukaryotic expression systems will become more and more popular in near future.

High throughput screening is a core technology in drug discovery. During the past decade, several strategies have been developed to screen (poly)peptide libraries for diverse applications including disease diagnosis and profiling, imaging, as well as therapy. The recently established baculovirus display vector system (BDVS) represents a eukaryotic screening platform that combines the positive attributes of both cell and virus-based display approaches, allowing presentation of complex polypeptides on cellular and viral surfaces. Compared to microbial display systems, the BDVS has the advantage of correct protein folding and post-translational modifications similar to those in mammals, facilitating expression and analysis of proteins with therapeutic interest. The applicability of the system is further expanded by the availability of genetically engineered insect cell lines capable of performing e.g. mammalianized glycosylation in combination with high level of expression. In addition to insect cells, baculovirus can mediate delivery and expression of heterologous genes in a broad spectrum of primary and established mammalian cells. Currently, a variety of baculovirus-based assays aiming at routine high throughput identification of agents targeting cell surface receptors or studies on ligand-receptor interactions are under construction. Here, the advancements and future prospects of the baculovirus display technologies with emphasis on molecular screening and drug delivery applications using insect cell display, mammalian cell display, and virion display are described.

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.

Retroviral and lentiviral based gene delivery vectors have been used in numerous pre-clinical studies and clinical trials due to their advantages, including stable and prolonged expression of therapeutic transgenes and minimal immune responses against the vector. Despite such advantages, however, retroviral vectors also have several limitations for gene therapy applications. For example, they can suffer from a lack of efficient or targeted gene delivery to key cell types. In addition, retroviral vector stability can be compromised by their envelope proteins. This review briefly describes how such limitations have been overcome by recently developed library selection approaches that borrow a lesson from nature: the ability of evolution to generate biomolecules with novel function. These library selection approaches are based on the construction of retroviral libraries where the sequences encoding natural viral components are partially randomized using a variety of methods in order to generate diverse libraries that can be selected to create improved or novel functions. These high throughput, library-based approaches provide a strong complement to rational engineering of viral components for the rapid development of efficient and safe retroviral and lentiviral vector systems for gene therapy.

After attracting the attention of the scientific community due to a number of favourable characteristics that make it an attractive vector for human gene therapy [1, 2], AAV has been thoroughly investigated in the past two decades. Standard technologies for the manipulation of the viral genome and for efficient packaging and purification protocols have paved the road for trial and error manipulation by educated guesses to study viral infectious biology by reverse genetics and to generate improved vectors for human gene transfer. However, despite remarkable progress, our limited knowledge of molecular mechanisms implicated in virus-cell interactions has been a limiting factor. Combinatorial engineering and high-throughput selection techniques hold the potential to boost technological improvement by offering the possibility to screen large numbers of randomly generated clones by appropriate selection protocols. These approaches not only require lesser knowledge of viral biology, but can also be employed as valuable tools to investigate molecular mechanisms that drive the infection process. In this review we recapitulate the rationale for employment of combinatorial methods in AAV vector development and the accomplishments achieved so far, discussing current limitations and interesting developments that are in sight.

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Recent advances have greatly expanded the capability of yeast surface display, and are highlighted by cellbased selections, epitope mapping, cDNA library screening, and cell adhesion engineering. In this review, we discuss the state-of-the-art yeast display methodologies and the rapidly expanding set of applications afforded by this technology.

Peptide aptamers are molecules that bind to protein targets and are able to interfere with their functions. In the past, important achievements have been made using such peptide aptamers in different approaches and for various purposes. Peptide aptamers are comprised of a variable peptide region of 8 to 20 amino acids in length, which is displayed by a scaffold protein. An overview of the numerous scaffold proteins that have been investigated for their suitability to present peptide aptamers will be given. To identify peptide aptamers efficiently and specifically binding to a predetermined target, two eukaryotic systems have been used in multiple studies: a modified version of the Gal4 yeast-two-hybrid system and the optimized LexA interaction trap system. The two yeast systems are compared and the design of high-complexity peptide aptamer libraries for these systems is described. Although the yeast-two-hybrid system is based on intracellular interactions mammalian screens, performed in cell culture experiments, are sometimes preferred or required. We will give an overview of the mammalian selection systems available, which are based on the expression of peptide aptamers in retroviral or lentiviral vectors. We will show that the isolation and use of peptide aptamers as inhibitors of individual signaling components represents a new challenge for drug development.

Artificial Transcription Factors (ATFs) are engineered DNA-binding proteins designed to bind specific sequences of DNA. ATFs made of Zinc Finger (ZF) domains have been developed to regulate specific genes and phenotypes both in cells and whole organisms. Recently, an emerging application of engineered DNA-binding domains include the specific editing of the genome, the ability to specifically cut, recombine, modify DNA and image protein-nucleic acid interactions in living cells. In this review we will summarize the techniques used for the rational design, screening and functional selection of ZF proteins in mammalian cell systems and their applications in areas of biotechnology, functional genomics and molecular therapeutics. The in vivo specificity of the engineered ATFs will be discussed, with particular emphasis on epigenetic modifications influencing ATF-DNA interactions.

Reversely transfected cell microarrays (RTCM) have been introduced as a method for parallel high throughput analysis of gene functions in mammalian cells. Hundreds to thousands of different recombinant DNA or RNA molecules can be transfected into different cell clusters at the same time on a single glass slide with this method. This allows either the simultaneous overexpression or - by using the recently developed RNA interference (RNAi) techniques - knockdown of a huge number of target genes. A growing number of sophisticated detection systems have been established to determine quantitatively the effects of the transfected molecules on the cell phenotype. Several different cell types have been successfully used for this procedure. This review summarizes the presently available knowledge on this technique and provides a laboratory protocol.

Christian Buchholz studied microbiology at the University of Munich. He obtained his Ph.D. at the Max-Planck-Institute for Biochemistry in 1993, where he worked on the molecular mechanism of viral RNA-dependent RNA polymerases under supervision of Prof. Wolfgang Neubert. He then became a postdoctoral fellow in the Institute for Molecular Biology in Zurich where he elucidated the role of the measles virus receptor in membrane fusion in Prof. Roberto Cattaneo's laboratory. As an EMBO fellow he joined the Centre for Protein Engineering in Cambridge (UK) and established the retrovirus based screening and display library technology together with Prof. Stephen J. Russell. Since 1999 he is head of the Section “Viral Gene Transfer Medicinal Products” at the Paul-Ehrlich-Institut, and later on became Associate Professor for Biochemistry at the University of Frankfurt. SELECTED PUBLICATIONS [1] Buchholz, C.J., Peng, K.W., Morling, F.J., Zhang, J., Cosset, F.L., and Russell, S.J. (1998) In vivo selection of protease cleavage sites from retrovirus display libraries. Nature Biotechnology 16: 951-954. [2] Schneider, R.M., Medvedovska, Y., Hartl, I., Voelker, B., Chadwick, M., Russell, S.J., Cichutek, K., and Buchholz, C.J. (2003) Directed evolution of retroviruses activatable by tumour associated matrix metalloproteases. Gene Therapy 10: 1370-1380. [3] Merten, C.A., Stitz, J., Braun, G., Poeschla, E.M., Cichutek, K. and Buchholz, C.J. (2005) Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles. Journal of Virology 79: 834-840. [4] Hartl, I., Schneider, R.M., Sun, Y., Medvedovska, J., Chadwick, M.P., Russell, S.J., Cichutek, K. and Buchholz, C.J. (2005) Library-based selection of retroviruses selectively spreading through matrix metalloprotease-positive cells. Gene Therapy 12: 918-926. [5] Urban, J.H., Schneider, R.M., Compte, M., Finger, C., Cichutek, K., Álvarez-Vallina, L., and Buchholz, C.J. (2005) Selection of functional human antibodies from retroviral display libraries. Nucleic Acids Research 33: e35. [6] Merten, C.A., Stitz, J., Braun, G., Medvedovska, J., Cichutek, K., Buchholz, C.J. (2006) Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein. Nucleic Acids Research 34: e41. Hildegard Büning studied biology at the Universities of Münster and Munich. She received her Ph.D. at the Institute for Biochemistry of the University of Munich in 1997, where she worked on protein-DNA interaction under supervision of Prof. Haralabos Zorbas and Prof. Ernst-Ludwig Winnacker. She then worked as a postdoctoral fellow and later as Principal Investigator at the Gene Center in Munich on AAV vector development in the laboratory of Prof. Michael Hallek. In 2004 she started her own research group at the Clinic I for Internal Medicine of the University of Cologne. One year later, she became a member of the Center for Molecular Medicine Cologne, and is currently the Scientific Secretary of the German Society of Gene Therapy. Her work focuses on AAV vector targeting and on the study of AAV biology. SELECTED PUBLICATIONS [1] Nicklin, S.A., Buning, H., Dishart, K.L., de Alwis, M., Girod, A., Hacker, U., Thrasher, A.J., Ali, R.R., Hallek, M. and Baker, A.H. (2001) Efficient and selected AAV2-mediated gene transfer directed to human vascular endothelial cells. Molecular Therapy 4, 174-181. [2] Seisenberger, G., Ried, M.U., Endress, T., Buning, H., Hallek, M., Brauchle, C. (2001) Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science. 294, 1929-1932. [3] Perabo, L., Buning, H., Kofler, D.M., Ried, M.U., Girod, A., Wendtner, C.M., Enssle, J., Hallek, M. (2003) In vitro selection of viral vectors with modified tropism: the adeno-associated virus display. Molecular Therapy 8, 151-157. [4] Lux, K., Goerlitz, N., Schlemminger, S., Perabo, L., Goldnau, D., Endell, J., Leike, K., Kofler, D.M., Finke, S., Hallek, M., Buning, H. (2005) Green Fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking. Journal of Virology 79: 11776-11787. [5] Perabo, L., Endell, J., King S., Lux, K., Goldnau, D., Hallek, M., Buning, H. (2006) Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus. The Journal of Gene Medicine 8: 155-162. [6] Work, L.M., Buning, H., Hunt, E., Nicklin, S.A., Denby, L., Britton, N., Leike, K, Odenthal, M., Drebber, U., Hallek, M, Baker, A.H. (2006) Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses. Molecular Therapy 13: 683-693. [7] Perabo, L., Goldnau, D., White, K., Endell, J., Boucas, J., Humme, S., Work, L.M., Janicki, H., Hallek, M., Baker, A.H., Buning, H. (2006) Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism. Journal of Virology 80: 7265-7269.