Discovery and development of drug cocktails evolved by Nature - PROJECT SUMMARY Discovery and development of new pharmaceuticals and therapeutic alternatives is required to address rapid emergence of drug resistance. An attractive approach is to maximize the potential of currently available drugs through combination therapy. However, de novo design of effective drug cocktails has suffered from the limited understanding of their mechanisms of action or the tedious trial- and-error methods. Moreover, drug resistance can even be aggravated by irrational and abusive usage of noneffective drug cocktails. Therefore, a design strategy for effective cocktail recipe is needed. Numerous observations have suggested that microbes have evolved a large collection of effective drug cocktail recipes to fight against drug resistance after billions of years of natural selection. However, besides a few known examples, co-produced natural products and their combinatorial potential have been overlooked for decades. This is primarily due to the focus of the field on the biological activity of the purified individual compounds, rather than the mixture itself. In addition, it is challenging to predict co- produced natural products based on the genomes of the microbes that synthesize them. Moreover, the synthesis of co-produced and synergistic natural products remains difficult. This MIRA grant will address these challenges. The first research direction is to identify the synergies between intermediates and the final antimicrobial natural products co-produced by single biosynthetic pathways. The structural similarity between these compounds is expected to allow their binding towards the same targets, which can lead to synergistic inhibition of drug-resistant microbes. By characterizing compounds biosynthesized through the same pathways, it is expected to directly provide effective drug cocktail recipe against resistance and to deepen the understanding of the design rules for combination therapy. The second research direction is to address the challenge during the production of the synergistic compounds by dividing their biosynthesis into two bacterial hosts, with one host producing the intermediate, the other producing the final product. It is expected to have three advantages compared to the conventional biosynthesis using a single bacterial host. First, this strategy will reduce the metabolic burden that each bacterial population will experience, and thereby improve the overall production of the final products. Second, the titer ratio of the synergistic intermediates and final products can be fine-tuned by manipulating only one bacterial host, providing modularity during optimization process. Third, the system will produce the compounds with the titer ratio showing the optimal synergistic effect, and then alleviate extensive compound isolation and purification. Overall, the synergy of the two proposed research directions will lead to the discovery of effective drug cocktail recipes against drug resistance, provide fundamental insights for the design rules of combination therapy, and address challenges in the production of these compounds.
