Doug E. Frantz

Cardiogenic small molecules that enhance myocardial repair by stem cells

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.

Allosteric inhibition of hypoxia inducible factor-2 with small molecules

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of many protumorigenic pathways. Though transcription factors are typically considered “undruggable”, the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors such as renal cell carcinomas.

A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth

The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signaling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) which specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumors in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumor burden and prolongs survival. Importantly, we find that patients with breast tumors that overexpress Jumonji demethylases have significantly lower survival. Thus JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance.

Small-molecule activation of neuronal cell fate

We probed an epigenetic regulatory path from small molecule to neuronal gene activation. Isoxazole small molecules triggered robust neuronal differentiation in adult neural stem cells, rapidly signaling to the neuronal genome via Ca(2+) influx. Ca(2+)-activated CaMK phosphorylated and mediated nuclear export of the MEF2 regulator HDAC5, thereby de-repressing neuronal genes. These results provide new tools to explore the epigenetic signaling circuitry specifying neuronal cell fate and new leads for neuro-regenerative drugs.

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.

Genetic and Molecular Basis of Drug Resistance and Species-Specific Drug Action in Schistosome Parasites

Blood Fluke Resistance The larval stages of the blood fluke Schistosoma mansoni are disseminated via a replicative cycle in freshwater snails. When people come into contact with contaminated water, the larvae attach to and penetrate the skin. The resulting disease, bilharzia or schistosomiasis, afflicts approximately 67 million people in Africa and South America. Unfortunately, the parasite is showing resistance to one of the available therapeutic drugs, oxamniquine, which means that schistosome control relies on a single drug, praziquantel. Valentim et al. (p. 1385, published online 21 November) analyzed the genetic and molecular basis of resistance to oxamniquine through a combination of genetic linkage mapping, genome sequencing, functional genomics analysis, and x-ray crystallography. Mutations in a distinctive sulfotransferase are responsible for oxamniquine resistance in a human blood fluke. Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally derived second-generation progeny, and identified a single quantitative trait locus (logarithm of odds = 31) on chromosome 6. A sulfotransferase was identified as the causative gene by using RNA interference knockdown and biochemical complementation assays, and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Pd-catalyzed asymmetric β-hydride elimination en route to chiral allenes.

We wish to report our preliminary results on the discovery and development of a catalytic, asymmetric β-hydride elimination from vinyl Pd(II)-complexes derived from enol triflates to access chiral allenes. To achieve this, we developed a class of chiral phosphite ligands that demonstrate high enantioselectivity, allow access of either allene enantiomer, and are readily synthesized. The methodology is demonstrated on over 20 substrates, and application to the formal asymmetric total synthesis of the natural product, (+)-epibatidine, is also provided.

Synthesis of Substituted Pyrazoles via Tandem Cross-Coupling/Electrocyclization of Enol Triflates and Diazoacetates

The synthesis of 3,4,5-trisubstituted pyrazoles via a tandem catalytic cross-coupling/electrocyclization of enol triflates and diazoacetates is presented. The initial scope of this methodology is demonstrated on a range of differentially substituted acyclic and cyclic enol triflates as well as an elaborated set of diazoacetates to provide the corresponding pyrazoles with a high degree of structural complexity.

Development of inhibitors of the PAS-B domain of the HIF-2α transcription factor

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity relationship study of small molecules designed to inhibit HIF-2α-ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor-protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α-ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action.

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.