Synthesis and biological evaluation of novel FiVe1 derivatives as potent and selective agents for the treatment of mesenchymal cancers.

Epithelial-mesenchymal transition (EMT) endows stem cell-like properties to cancer cells. Targeting this process represents a potential therapeutic approach to overcome cancer metastasis and chemotherapy resistance. FiVe1 was identified from an EMT-based synthetic lethality screen and was found to inhibit the stem cell-like properties and proliferation of not only cancer cells undergoing EMT, but also more broadly in mesenchymal cancers that include therapeutically intractable soft tissue sarcomas. FiVe1 functions by directly binding to the type III intermediate filament protein vimentin (VIM) in a mode that induces hyperphosphorylation of Ser56, which results in selective disruption of mitosis and induced multinucleation in transformed VIM-expressing mesenchymal cancer cell types. Cell-based potency (IC50 = 1.6 µM, HT-1080 fibrosarcoma), poor solubility (<1 µM) and low oral bioavailability limits the direct application of FiVe1 as an in vivo probe or therapeutic agent. To overcome these drawbacks, we performed structure-activity relationship (SAR) studies and synthesized a set of 35 new compounds, consisting of diverse modifications of the FiVe1 scaffold. Among these compounds, 4e showed a marked improvement in potency (IC50 = 44 nM, 35-fold improvement, HT-1080) and cell type selectivity (19-fold improvement), when compared to FiVe1. Improvements in the potency of 4e, in terms of overall cytotoxicity, directly correlate with VIM Ser56 phosphorylation status and the oral bioavailability and pharmacokinetic profiles of 4e in mouse are superior to FiVe1. Successful optimization also resulted in potent and selective derivatives 11a, 11j and 11k, which exhibited superior pharmacological profiles, in terms of metabolic stability and aqueous solubility. Collectively, these optimization efforts have resulted in the development of promising FiVe1 analogs with potential applications in the treatment of mesenchymal cancers, as well as in the study of VIM-related biology.

A vimentin binding small molecule leads to mitotic disruption in mesenchymal cancers.

Expression of the transcription factor FOXC2 is induced and necessary for successful epithelial-mesenchymal transition, a developmental program that when activated in cancer endows cells with metastatic potential and the properties of stem cells. As such, identifying agents that inhibit the growth of FOXC2-transformed cells represents an attractive approach to inhibit chemotherapy resistance and metastatic dissemination. From a high throughput synthetic lethal screen, we identified a small molecule, FiVe1, which selectively and irreversibly inhibits the growth of mesenchymally transformed breast cancer cells and soft tissue sarcomas of diverse histological subtypes. FiVe1 targets the intermediate filament and mesenchymal marker vimentin (VIM) in a mode which promotes VIM disorganization and phosphorylation during metaphase, ultimately leading to mitotic catastrophe, multinucleation, and the loss of stemness. These findings illustrate a previously undescribed mechanism for interrupting faithful mitotic progression and may ultimately inform the design of therapies for a broad range of mesenchymal cancers.

Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody-drug Conjugate.

Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.

Transition states. Trapping a transition state in a computationally designed protein bottle.

The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.

Synthetic studies towards Zetekitoxin AB: preparation of 4,5-epi-11-hydroxy-saxitoxinol.

A concise synthesis of 4,5-epi-11-hydroxy-saxitoxinol utilizing D-ribose to direct an asymmetric Mannich reaction. This approach allows many modes of reactivity, which can be used to access various analogs of saxitoxin.