Mark R. Hansen

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.

[15] Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions

Publisher Summary This chapter describes the application of filamentous bacteriophage as a versatile tool for generating partially ordered solutions of nucleic acids and proteins. The Pfl phage system described in the chapter is an ideal method for obtaining a tunable degree of alignment of RNA and DNA oligomers and some proteins in solution, which then allows the measurement of homonuclear and heteronuclear dipolar coupling interactions. The Pfl filamentous phage system is ideally suited for obtaining a tunable degree of alignment in RNA and DNA molecules and acidic proteins for the observation of dipolar couplings. The phage particles are fully aligned at magnetic fields as low as 300 MHz and are stable indefinitely at a range of temperatures and buffer conditions. These dipolar couplings provide long-range structural information that is presently unavailable by standard solution nuclear magnetic resonance (NMR) techniques; therefore, in the future, dipolar coupling data may prove to be as indispensable as 1 H– 1 H nuclear Overhauser effect (NOE) in the solution structure determinations of macromolecules. This should be especially true for structure determinations of RNA and DNA oligomers where the low density of protons compared to proteins leads to fewer useful NOE distance constraints, and therefore generally less well-defined structures. In addition, the current solution NMR techniques are generally unable to determine global structural features, such as bending in nucleic acids, due because of the local nature of the NOE distance constraints.

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.

Computational Techniques in Fragment Based Drug Discovery

Fragment based drug discovery is gaining acceptance as a complement to other more established techniques to identify leads and optimize drug candidates. In this review we illustrate areas where fragment based drug discovery has had an impact and point to some examples that show how fragment based analysis is being applied to new arenas. The traditional uses of computational methods in fragment based for lead discovery and optimization and for risk assessment are briefly summarized. The application of fragment analysis for the definition of bioisosteric replacements are discussed together with techniques to characterize the diversity of chemical libraries based on fragment distribution.

Design of chemical libraries for screening

Importance of the field: The ultimate goal of discovery screening is to have a fast and cost-effective strategy to meet the demands of producing high-content lead series with improved prospects for clinical success. While high-throughput screening (HTS) dominates the drug discovery landscape, other processes and technologies have emerged, including high-content screening and fragment-based design to provide alternatives that may be more suitable for certain targets. There has been a growing interest in reducing the number of compounds to be screened to prevent the escalation in the costs, time and resources associated with HTS campaigns. Library design plays a central role in these efforts. Areas covered in this review: This opinion provides a survey of some recent developments in the diversity based library design process, but within a historical context. In particular, the importance of chemotyping and substructure analysis and the challenges presented by novel lead discovery technologies that require the design of libraries for screening are discussed. What the reader will gain: Readers will gain an appreciation of some developments in the field of library design and the factors that are driving the development of new library design technologies; specifically, challenges presented for chemoinformatics with the novel screening technologies in diversity based screening and compound filtering. Take home message: Chemotyping and substrutural analysis are techniques that have been underutilized in the process of library design. However, they offer a direct way to evaluate libraries and have been successfully used to develop predictive methodologies. Tools are available to this end, but the full power of the approach has not been realized yet.

A path from primary protein sequence to ligand recognition.

A novel method to organize protein structural information based solely on sequence is presented. The method clusters proteins into families that correlate with the three‐dimensional protein structure and the conformation of the bound ligands. This procedure was applied to nicotinamide adenine dinucleotide [NAD(P)]‐utilizing enzymes to identify a total of 94 sequence families, 53 of which are structurally characterized. Each of the structurally characterized proteins within a sequence family correlates to a single protein fold and to a common bound conformation of NAD(P). A wide range of structural folds is identified that recognize NAD(P), including Rossmann folds and β/α barrels. The defined sequence families can be used to identify the type and prevalence of NAD(P)‐utilizing enzymes in the proteomes of sequenced organisms. The proteome of Mycobacterium tuberculosis was mined to generate a proteome‐wide profile of NAD(P)‐utilizing enzymes coded by this organism. This enzyme family comprises approximately 6% of the open reading frames, with the largest subgroup being the Rossmann fold, short‐chain dehydrogenases. The preponderance of short‐chain dehydrogenases correlates strongly with the phenotype of M. tuberculosis, which is characterized as having one of the most complex prokaryotic cell walls. Proteins 2003;50:589–599.

Substructural Analysis in Drug Discovery

The dominant paradigm in drug discovery emphasizes techniques that generate large amounts of data. What was possible by simple inspection in the past, nowadays cannot be effectively achieved without the aid of informatics techniques. In this context substructural analysis techniques are increasing their role in the organization and management of information generated. Advances in the field of substructure analysis have expanded the applicability of substructural analysis in multiple fronts in early lead discovery and optimization. It can be applied beyond the management of information, including compound library design and virtual screening to structure activity relationships. The relationships between chemical substructures and drug-like properties also aid in developing more robust rationales for fragment-based approaches for lead discovery, predictive toxicology, and elucidation of pharmacokinetic properties.A review of recent developments in substructure analysis in a broad range of areas in drug discovery is presented. The focus is on the application of substructural analysis in computational chemistry for drug design and the methods used to identify substructures in a chemical database, as well as their relation to fragment-based drug discovery. The discussion shows the benefits of substructural analysis to the drug discovery process and gives impetus to further advancement of substructure analysis techniques.

Pf1 filamentous phage as an alignment tool for generating local and global structural information in nucleic acids.

Abstract Pf1 filamentous phage represent a simple versatile method for generating partially ordered macromolecules in solution. The phage allow tunable degrees of alignment of macromolecules under a wide range of temperature and solvent conditions. The negatively charged phage are ideal for aligning negatively charged nucleic acids and these phage-nucleic acid solutions are stable indefinitely. We have used Pf1 phage to align various DNA and RNA molecules in solution for measurement of dipolar coupling interactions. These dipolar couplings can be used to improve the local structure of nucleic acids. More importantly they also contain information on the global structure, such as DNA bending, which presently cannot be obtained by standard NMR methods. The principles involved in using Pf1 phage to generate solutions of partially order macromolecules will be discussed. The use of 1H-1H, 1H-13C and 1H-15N dipolar couplings for generating angle constraints for structure refinement of nucleic acids will also be discussed.

Development of an Informatics Platform for Therapeutic Protein and Peptide Analytics

The momentum gained by research on biologics has not been met yet with equal thrust on the informatics side. There is a noticeable lack of software for data management that empowers the bench scientists working on the development of biologic therapeutics. SARvision|Biologics is a tool to analyze data associated with biopolymers, including peptides, antibodies, and protein therapeutics programs. The program brings under a single user interface tools to filter, mine, and visualize data as well as those algorithms needed to organize sequences. As part of the data-analysis tools, we introduce two new concepts: mutation cliffs and invariant maps. Invariant maps show the variability of properties when a monomer is maintained constant in a position of the biopolymer. Mutation cliff maps draw attention to pairs of sequences where a single or limited number of point mutations elicit a large change in a property of interest. We illustrate the program and its applications using a peptide data set collected from the literature.