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Volume 2, Number 5, 1999: Contents

Efficient Computational Algorithms for Docking and for Generating and Matching a Library of Functional Epitopes I. Rigid and Flexible Hinge-Bending Docking Algorithms. Pp. 249-259.
Ruth Nussinov and Haim J. Wolfson
[Abstract]

Efficient Computational Algorithms for Docking and for Generating and Matching a Library of Functional Epitopes II. Computer Vision-Based Techniques for the Generation and Utilization of Functional Epitopes. Pp. 261-269.
Ruth Nussinov and Haim J. Wolfson
[Abstract]

A High Throughput Platform for Systematic Evolution of Ligands by Exponential Enrichment (SELEX™). Pp. 271-278.
D. W. Drolet, R. D. Jenison, D. E. Smith, D. Pratt and B. J. Hicke
[Abstract]

Application of Homogeneous Time-Resolved Fluorescence (HTRF™) to Monitor Poly-ubiquitination of Wild-type p53. Pp. 279-287.
Nami Yabuki, Shin-ichi Watanabe, Tsutomu Kudoh, Shin-ichi Nihira and Chikara Miyamoto
[Abstract]

Identification of Synthetic By-products in Combinatorial Libraries Using High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Pp. 289-296.
J-L. Aubagnac, M. Amblard, C. Enjalbal, G. Subra, J. Martinez, P. Durand and P. Renaut
[Abstract]

Abstracts

[Back to top] Efficient Computational Algorithms for Docking and for Generating and Matching a Library of Functional Epitopes I. Rigid and Flexible Hinge-Bending Docking Algorithms. Ruth Nussinov and Haim J. Wolfson.
In this, and the next review article (1), we present highly efficient, computer-vision and robotics based algorithms for docking and for the generation and matching of epitopes on molecular surfaces. We start with descriptions of molecular surfaces, and proceed to utilize these in both rigid-body and flexible matching routines. These algorithms originate in the computer vision and robotics disciplines. Frequently used approaches, both in searches for molecular similarity and for docking, i.e., molecular complementarity, strive to obtain highly accurate correspondence of respective molecular surfaces. However, owing to molecular surface variability in solution, to mutational events, and to the need to use modeled structures in addition to high resolution ones, utilization of epitopes might prove to be a judicious approach to follow. Furthermore, through the deployment of libraries of epitopes which represent recurring features, or motifs in a given family of receptors or of enzymes, in principle we a priori focus on the more critical groups of atoms, or amino acids, essential for the binding of the two molecules. Utilization of recurring motifs may prove more robust than single molecule matchings. In addition, via utilization of epitopes one can make use of information derived from evolutionary related molecules. All of the above combine to represent an approach which may be highly advantageous. Combinatorial approaches have proven their immense utility in the wet laboratory. The combination of efficient computational approaches and the utilization of such libraries may well be particularly profitable. Our highly efficient techniques are amenable to such a task. In this review we focus on rigid and flexible docking algorithms. In the second review (1) we address the generation of epitopes in families of molecules. These may be used by the docking algorithms to identify the more likely bound interfaces.

[Back to top] Efficient Computational Algorithms for Docking and for Generating and Matching a Library of Functional Epitopes II. Computer Vision-Based Techniques for the Generation and Utilization of Functional Epitopes. Ruth Nussinov and Haim J. Wolfson.
This is the second review in a two-part series. In the first review (1) we described the computational complexity involved in the docking of a ligand onto a receptor surface. In particular, we focused on efficient algorithms designed to handle this computational task. Such a procedure results in a large number of potential, geometrically feasible solutions. The difficulty is to pinpoint which of these is the more likely candidate. While there exists a number of approaches to rank these solutions according to different criteria, such as the size of the interface or some approximation of their binding energetics, none of the existing methods has been shown to be consistently successful in this endeavor. If the binding site is unknown a priori, the magnitude of the task is awesome. Here we propose one way of addressing this problem, i.e., via derivation and utilization of binding epitopes. If a library of such epitopes is available, particularly for a large number of protein families, it may be used to predict more likely binding sites for a given ligand. We describe an efficient, computer-vision based method to construct binding epitopes focusing on two ways through which such a library can be generated, (i) molecular surface-based, or (ii) residue-based. Alternatively, the two can be combined. We further describe how such a library may be used efficiently in the matching/docking procedure.

[Back to top] A High Throughput Platform for Systematic Evolution of Ligands by Exponential Enrichment (SELEX™). D. W. Drolet, R. D. Jenison, D. E. Smith, D. Pratt and B. J. Hicke.
The systematic evolution of ligands by exponential enrichment (SELEX') process is a combinatorial chemistry method for the isolation of nucleic acid ligands (aptamers) that bind to a desired target molecule with high affinity. In order to increase throughput via automation, we have adapted the SELEX process for protein targets to a robotics-compatible microtiter plate format. A remarkable feature of the platform is that targets are immobilized by hydrophobic adsorption onto the plate surface. Hydrophobic immobilization procedures are simple and require no specialized modification of the protein target. This format was tested by manually performing four independent SELEX experiments. All were concluded within 8 rounds of selection and yielded aptamers that bind in solution to their respective protein target, calf intestinal alkaline phosphatase, human a-thrombin or human platelet derived growth factor, with equilibrium dissociation constants below 3 x 10-10 M.

[Back to top] Application of Homogeneous Time-Resolved Fluorescence (HTRF™) to Monitor Poly-ubiquitination of Wild-type p53. Nami Yabuki, Shin-ichi Watanabe, Tsutomu Kudoh, Shin-ichi Nihira and Chikara Miyamoto.
Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRF™). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRF™ or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.

[Back to top] Identification of Synthetic By-products in Combinatorial Libraries Using High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry. J-L. Aubagnac, M. Amblard, C. Enjalbal, G. Subra, J. Martinez, P. Durand and P. Renaut.
High performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI) and high performance liquid chromatography coupled to mass spectrometry (LC-MS) were used to analyze randomly chosen samples from parallel syntheses carried out on derivatized polypropylene crowns compatible with a Multipin™ solid support system. Side-reactions and by-products were clearly identified, and the yields of the expected molecules were unexpectedly low for most samples. LC-MS was superior to HPLC with absorbance detection or electrospray mass spectrometry alone for determining the identity and purity of each desired combinatorial compounds.