A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity.

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration.

In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase.

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in vivo whole-organism screens of zebrafish identified compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogues to define the structure-activity relationship (SAR) of DUSP6 inhibition. In vivo high-content analysis in transgenic zebrafish, coupled with cell-based chemical complementation assays, identified structural features of the pharmacophore of 1 that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses.

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen.

BACKGROUND: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS: Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS: Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.

Substituent-induced switch of the role of charge-transfer complexes in the Diels-Alder reactions of o-chloranil and styrenes.

Addition of p-substituted styrenes, XSty (X = H, Me, MeO, or Cl) to the solutions of o-chloranil, oCA, in dichloromethane resulted in the transient formation of the charge-transfer complexes, [XSty, oCA], followed by the Diels-Alder reaction. At low temperatures, these reactions led to formation of essentially pure endocycloadducts. As expected for the inverse-electron-demand Diels-Alder reaction, the rate constants of the cycloaddition rose with the increase of the donor strength. However, while facile cycloaddition took place in the neat mixtures of the o-chloranil with p-methyl, p-chloro-, or unsubstituted styrenes at low temperatures, a similar system involving the strongest MeOSty donor was surprisingly persistent. X-ray structural measurements and quantum-mechanical computations indicated that this anomaly is related to the fact that the diene/dienophile orientation in the charge-transfer [MeOSty, oCA] complex is opposite to that in the endocycloadduct and in the lowest-energy transition state leading to this isomer. Thus, the proceeding of the cycloaddition requires dissociation of the (dead-end) complex. For the systems involving the oCA diene and either the HSty, ClSty, or MeSty dienophile, the donor/acceptor arrangements in the charge-transfer complexes apparently are consistent with that in the corresponding products, and the formation of these complexes does not hinder the Diels-Alder reaction.

Antimalarial activity of 4-amidinoquinoline and 10-amidinobenzonaphthyridine derivatives.

Chloroquine (CQ) has been used as first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug due to its being long-acting and having a high potency against blood stage P. falciparum (Pf). However, serious CNS toxicity of MQ has compromised its clinical value as a prophylaxis drug. Therefore, new and inexpensive antimalarial drugs with no cross-resistance to CQ or CNS toxicity are urgently needed to combat this deadly human disease. In this study, a series of new 4-amidinoquinoline (4-AMQ) and 10-amidinobenzonaphthyridine (10-AMB) derivatives were designed, prepared, and assessed to search for new therapeutic agents to replace CQ and MQ. The new derivatives displayed high activity in vitro and in vivo, with no cross-resistance to CQ, and none were toxic in mice up to 160 mpk × 3. The best compound shows IC50 < 1 ng/mL against D6, W2 and C235 Pf clones, low inhibitory activity in hERG K(+) channel blockage testing, negativity in the Ames test, and 5/5 cure @ <15 mpk × 3 in mice infected with Plasmodium berghei. In addition to these desirable pharmacological profiles, compound 13b, one of the most active compounds, is metabolically stable in both human and mouse liver microsomal preparations and has a plasma t(1/2) of 50 h in mice, which made it a good MQ replacement candidate.