The chemical diversification of natural products provides a robust and general method for creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria 1-7. Here we report the development of a web application, called eNTRyway, which predicts compound accumulation (in E. coli) from its structure. eNTRyway, in conjunction with structure-activity relationship and x-ray data, was utilized to redesign Debio-1452, a Gram-positive-only antibiotic 8 , into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI as validated by in vitro enzyme assays and generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice, and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli, and a general blueprint for the conversion of Gram-positive-only compounds into broadspectrum antibiotics.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Glucose transporters and the glycolysis enzyme lactate dehydrogenase A (LDH-A) are both overexpressed in cancer cells, two proliferation tactics that underlie the phenomenon known as the Warburg effect. Herein we report the development and activity of a glucose-conjugated LDH-A inhibitor designed to target both of these tumor-promoting facets. In addition to the promise of this conjugate, dual targeting of the Warburg effect using glycoconjugation as an anticancer strategy could be applied to inhibitors of many of the enzymes involved in glycolysis or tumor metabolism.

Myotonic dystrophy is the most common form of the adult-onset muscular dystrophy, originating in a CTG repeat expansion in the DMPK gene. The expanded CUG transcript sequesters MBNL1 a key regulator of alternative splicing, leading to the misregulation of numerous pre-mRNAs. We report an RNA-targeted agent as a possible lead compound for the treatment of DM1 that reveals both the promise and challenges for this type of small molecule approach. The agent is a potent inhibitor of the MBNL1-rCUG complex with an inhibition constant, KI of 25 ± 8 nM, and is also relatively non-toxic to HeLa cells, able to dissolve nuclear foci, and correct the IR splicing defect in DM1 model cells. Moreover, treatment with this compound improves two separate disease phenotypes in a DM1 Drosophila model, the adult external eye degeneration and larval crawling defect. However, the compound has a relatively low maximum tolerated dose in mice and its cell uptake may be limited, providing insights into directions for future development.