Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy.

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4-5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL.

Discovery of a potent inhibitor of replication protein a protein-protein interactions using a fragment-linking approach.

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA transactions. The RPA N-terminal domain (RPA70N) is a recruitment site for proteins involved in DNA-damage response and repair. Selective inhibition of these protein-protein interactions has the potential to inhibit the DNA-damage response and to sensitize cancer cells to DNA-damaging agents without affecting other functions of RPA. To discover a potent, selective inhibitor of the RPA70N protein-protein interactions to test this hypothesis, we used NMR spectroscopy to identify fragment hits that bind to two adjacent sites in the basic cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N-ligand complexes revealed how these fragments bind to RPA and guided the design of linked compounds that simultaneously occupy both sites. We have synthesized linked molecules that bind to RPA70N with submicromolar affinity and minimal disruption of RPA's interaction with ssDNA.

Identification and optimization of small molecules that restore E-cadherin expression and reduce invasion in colorectal carcinoma cells.

E-cadherin is a transmembrane protein that maintains intercellular contacts and cell polarity in epithelial tissue. The down-regulation of E-cadherin contributes to the induction of the epithelial-to-mesenchymal transition (EMT), resulting in an increased potential for cellular invasion of surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors as a result of somatic mutations, chromosomal deletions, silencing of the CDH1 gene promoter, and proteolytic cleavage. To date, no compounds directly targeting E-cadherin restoration have been developed. Here, we report the development and use of a novel high-throughput immunofluorescent screen to discover lead compounds that restore E-cadherin expression in the SW620 colon adenocarcinoma cell line. We confirmed restoration of E-cadherin using immunofluorescent microscopy and were able to determine the EC(50) for selected compounds using an optimized In-Cell Western assay. The profiled compounds were also shown to have a minimal effect on cell proliferation but did decrease cellular invasion. We have also conducted preliminary investigations to elucidate a discrete molecular target to account for the phenotypic behavior of these small molecules and have noted a modest increase in E-cadherin mRNA transcripts, and RNA-Seq analysis demonstrated that potent analogues elicited a 10-fold increase in CDH1 (E-cadherin) gene expression.

Synthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine dual EGFR/ErbB-2 kinase inhibitors.

Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compounds containing the reference aniline.