Screening Of Natural Products

The potential of combinatorial biology was evaluated in a pharmaceutical discovery environment [27,28] with 34 actinomycete strains. The natural products produced by each of these donor strains under defined culture conditions were well characterized with respect to their chemical properties and biological activities.

DNA from each donor species was isolated, pooled, cloned, and transferred into the expression host S. lividans. Both natural (vide supra) and combinatorial (vide supra) pathway expression clones were constructed for the project. A total of 10,400 isolated combinatorial biology expression clones representing both natural pathway (8,200 clones) and combinatorial pathway (2,200 clones) formats were generated and screened using a traditional approach.

Each clone was screened independently and individually cultivated on a small scale for approximately two weeks, the culture medium was extracted with butanol, butanol solution was removed, and the resulting residue was redis-solved in a small volume of DMSO. The resulting DMSO solution was screened for activity in antimicrobial plate assays against a panel of eight target microorganisms. Of the 8,200 natural pathway clones screened 205 (2.5%) clones produced metabolite(s) that had significant biological activity when compared to control cultures of S. lividans harboring cloning vector without donor inserts. Initial dereplication analysis of an early subset of natural pathway clones indicated the presence of at least 5 known natural products produced by several donor strains. This early result supported the original project premise for facile heterologous expression of biosynthetic pathways in these closely related microorganisms.

For the combinatorial pathway format set of clones, 71 (3.2%) of 2,200 clones produced biologically active compound(s) relative to controls. Many of the active clones from both the natural and the combinatorial pathway sets of clones produced potentially novel natural products based upon their biological activity profiles and chemical properties. Twenty-one natural pathway clones and 19 combinatorial pathway clones were selected for dereplication and chemical analysis based upon specific criteria. The results from dereplication and structural elucidation included two novel compounds, BMS-246784 and BMS-240777, a known but unusual metabolite (BMS-246781), and two known but unusual pro-digiosins.

The effort to screen the combinatorial biology clones for this early project required considerable resources. The 10,400 250-mL flasks needed for the initial-culture of this number of clones fill a space of 10 cubic meters. Furthermore, more than 1000 liters of medium and 500 liters of butanol were required for the primary fermentations and extractions. Butanol solvent had to be removed from each of the 10,400 from each extraction. Considerable additional resources and effort were required for scale-up and analysis of selected active clones.

Over the years, pharmaceutical companies have developed an encompassing traditional process to screen fermentation broths [28]. Of necessity, this process reflects an expected diversity of growth and production requirements learned from screening soil isolates composed of organisms that have distinct inherently unpredictable biological properties. The natural products screening ap proach used reflected this historical experience. Combinatorial biology library clones do not share these characteristics, as each clone is genetically identical except for the relatively small amount of donor DNA that has been introduced. This important property of these approaches can be taken advantage of to develop high throughput for screening combinatorial biology library clones. At Cubist, an encapsulation approach to primary biological activity screening was developed to exploit the uniform growth properties and temporal production characteristics of clones and facilitate the natural products drug discovery process. In this HTS system recombinant expression library clones are encapsulated to generate individual assay units that serve as culture flask, medium, and extract on a millimeter scale.

0 0

Post a comment