Ralf Thiericke

Drug discovery from nature

The Impact of Natural Products on Drug Discovery.- Recent Developments in Drug Discovery Technologies.- A Central Natural Product Pool: New Approach in Drug Discovery Strategies.- Automation Strategies in Drug Discovery.- Synergistic Use of Combinatorial and Natural Product Chemistry.- Supercritical Fluid Extraction - Novel Strategies in the Processing of Biomaterials.- Secondary Metabolites: From Past to Present.- The Chemical Screening Approach.- Myxobacteria as Producers of Secondary Metabolites.- Trends in Marine Biotechnology.- Structure Modification via Biological Derivation Methods.- Molecular Biological Aspects of Antibiotic Biosynthesis.- Combinatorial Biosynthesis of Antibiotics.- Impact of Natural Products on Cell Biology: Low-Molecular Mass Effectors of Folding Helper Enzyme.- Novel Antibacterial Drugs from Microorganisms.- New Antibiotics with Novel Mode of Action.- Synthetic Combinatorial Libraries: A New Tool for Antimicrobial Agent Discovery.- Strobilurins and Oudemansins.

Bioactive agents from natural sources: trends in discovery and application.

About 30% of the worldwide sales of drugs are based on natural products. Though recombinant proteins and peptides account for increasing sales rates, the superiority of low-molecular mass compounds in human diseases therapy remains undisputed mainly due to more favorable compliance and bioavailability properties. In the past, new therapeutic approaches often derived from natural products. Numerous examples from medicine impressively demonstrate the innovative potential of natural compounds and their impact on progress in drug discovery and development. However, natural products are currently undergoing a phase of reduced attention in drug discovery because of the enormous effort which is necessary to isolate the active principles and to elucidate their structures. To meet the demand of several hundred thousands of test samples that have to be submitted to high-throughput screening (HTS) new strategies in natural product chemistry are necessary in order to compete successfully with combinatorial chemistry. Today, pharmaceutical companies have to spend approximately US

Maremycins C and D, New Diketopiperazines, and Maremycins E and F, Novel Polycyclicspiro-Indole Metabolites Isolated fromStreptomycessp.

350 million to develop a new drug. Currently, approaches to improve and accelerate the joint drug discovery and development process are expected to arise mainly from innovation in drug target elucidation and lead finding. Breakthroughs in molecular biology, cell biology, and genetic engineering in the 1980 s gave access to understanding diseases on the molecular or on the gene level. Subsequently, constructing novel target directed screening assay systems of promising therapeutic significance, automation, and miniaturization resulted in HTS approaches changing the industrial drug discovery process drastically. Furthermore, elucidation of the human genome will provide access to a dramatically increased number of new potential drug targets that have to be evaluated for drug discovery. HTS enables the testing of an increasing number of samples. Therefore, new concepts to generate large compound collections with improved structural diversity are desirable.

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

New diketopiperazines, named maremycins C1/C2 (1a/1b) and D1/D2 (2a/2b), as well as the novel spiro-indoles, maremycins E (3) and F (4), have been detected alongside the known maremycin B (6) in the culture broth of Streptomyces sp. (strain GT 051237) by chemical screening. The structures have been determined by detailed NMR spectroscopic investigations of the isolated metabolites. Maremycins C1/C2 (1a/1b) as well as D1/D2 (2a/2b) are diastereomers and were identified as mixtures. Structurally, maremycins C1/C2 (1a/1b) are the diastereomers of the sulfur oxidation products of maremycin B (6), while D1/D2 (2a/2b) are the demethylmercapto analogues of maremycins A (5) and B (6), respectively. Maremycins E and F possess a novel structural skeleton, where a spiro moiety is formed between the 6-position of the cyclopenta[f]quinoxaline moiety and the 3′-position of the indol-2-one moiety of the initial diketopiperazine product.

Evaluation of Liquid Handling Conditions in Microplates

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

Gabosines L, N and O: New Carba-Sugars fromStreptomyces with DNA-Binding Properties

Liquid handling in higher density microplates (e.g., 1536-well microplates) for more efficient drug screening necessitates carefully selected and optimized parameters. The quality of a liquid handling procedure is dependent on the carryover rate of residual liquids during the pipetting process, the mixing behavior in the wells, foam and bubble formation, and evaporation. We compared and optimized these parameters in 96-, 384-, and 1536-well microplates, and herein we critically evaluate the performance of the CyBi™-Well 96/384/1536 automated micropipetting device, which formed the basis of our evaluation studies.

Miniaturization of a Functional Transcription Assay in Yeast (Human Progesterone Receptor) in the 384- and 1536-Well Plate Format

In addition to the known gabosines A (4), B (5) and C (6), three new gabosines L (1), N (2) and O (3) were detected by chemical screening as secondary metabolites of Streptomyces (strains GT 041230, GT 051024 and S 1096). The constitutions of 1, 2 and 3 were established by spectroscopic techniques and derivatization reactions. The absolute stereochemistry of 1 and 2 was determined by Helmchens method and has been verified in the case of gabosine N (2) by X-ray analysis. The DNA-binding properties of the gabosines were investigated and analyzed by binding studies using a recently developed thin-layer chromatography technique (bimolecular-chemical screening).

Acyl α-L-Rhamnopyranosides, a Novel Family of Secondary Metabolites fromStreptomyces sp.: Isolation and Biosynthesis

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (β-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligands dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi™-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Rakicidin C, A New Cyclic Depsipeptide from Streptomyces sp.

In the course of our chemical screening program, the novel acyl α-L-rhamnopyranosides (1-6) were detected as metabolites from five different strains of Streptomycetes. The structures of all these compounds were elucidated by chemical and spectroscopic methods. The biosynthesis of 1 and 3 was established by feeding 13C-labelled acetate, glycerol, and D-glucose to Streptomyces griseoviridis (strain Tu 3634), and resulted in a complete labelling pattern of the 2,4-dimethyl-3-furanylcarbonyl and benzoyl residues, as well as the rhamnose moiety. These results reveal biosynthetic pathways of general importance and give an insight into the generation of the hexose phosphates, from which deoxysugars are formed. The acyl rhamnosides are members of a novel family of microbial metabolites and are considered as rhamnoconjugates from Streptomycetes.

Biosynthesis of Gabosines A, B, and C, Carba Sugars from Streptomyces cellulosae

Chemical screening of extracts of Actinomycetes strains has led to the detection, isolation, and structure elucidation of the new cyclic depsipeptide rakicidin C (1). The secondary metabolite from Streptomyces sp. (strain GT 61042) is built-up from glutamine, N-methyl glycine, 4-amino-(2E,4)-pentadienoic acid, and 3-hydroxy-2,4,6,8-tetramethylnonanoic acid.