Vividion Therapeutics: Creating a New Era in Drug Discovery
The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.
Drug targets & disease targets glossary & taxonomy
N2 - A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/α-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an l-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound >40-fold, going from the starting ~60mg/L to 2.5g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy.
Enzymes capable of performing dehalogenating reactions have attracted tremendous contemporary attention due to their potential application in the bioremediation of anthropogenic polyhalogenated persistent organic pollutants. Nature, in particular the marine environment, is also a prolific source of polyhalogenated organic natural products. The study of the biosynthesis of these natural products has furnished a diverse array of halogenation biocatalysts, but thus far no examples of dehalogenating enzymes have been reported from a secondary metabolic pathway. Here we show that the penultimate step in the biosynthesis of the highly brominated marine bacterial product pentabromopseudilin is catalyzed by an unusual debrominase Bmp8 that utilizes a redox thiol mechanism to remove the C-2 bromine atom of 2,3,4,5-tetrabromopyrrole to facilitate oxidative coupling to 2,4-dibromophenol. To the best of our knowledge, Bmp8 is first example of a dehalogenating enzyme from the established genetic and biochemical context of a natural product biosynthetic pathway.
have led to a renewed interest in natural products in drug discovery
Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic -thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered -thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.
Nature Reviews Drug Discovery 14, ..
Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.