A Zebrafish Chemical Suppressor Screening Identifies Small Molecule Inhibitors of the Wnt/β-catenin Pathway

SummaryGenetic screening for suppressor mutants has been successfully used to identify important signaling regulators. Using an analogy to genetic suppressor screening, we developed a chemical suppressor screening method to identify inhibitors of the Wnt/β-catenin signaling pathway. We used zebrafish embryos in which chemically induced β-catenin accumulation led to an “eyeless” phenotype and conducted a pilot screening for compounds that restored eye development. This approach allowed us to identify geranylgeranyltransferase inhibitor 286 (GGTI-286), a geranylgeranyltransferase (GGTase) inhibitor. Our follow-up studies showed that GGTI-286 reduces nuclear localization of β-catenin and transcription dependent on β-catenin/T cell factor in mammalian cells. In addition to pharmacological inhibition, GGTase gene knockdown also attenuates the nuclear function of β-catenin. Overall, we validate our chemical suppressor screening as a method for identifying Wnt/β-catenin pathway inhibitors and implicate GGTase as a potential therapeutic target for Wnt-activated cancers

Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance1–5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors 6. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2010: General view of the pathogens\u27 antibacterial susceptibility

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from patients in Japan, was conducted by Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases and Japanese Society for Clinical Microbiology in 2010.The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period from January and April 2010 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical and Laboratory Standard Institutes using maximum 45 antibacterial agents.Susceptibility testing was evaluable with 954 strains (206 Staphylococcus aureus, 189 Streptococcus pneumoniae, 4 Streptococcus pyogenes, 182 Haemophilus influenzae, 74 Moraxella catarrhalis, 139 Klebsiella pneumoniae and 160 Pseudomonas aeruginosa). Ratio of methicillin-resistant S.aureus was as high as 50.5%, and those of penicillin-intermediate and -resistant S.pneumoniae were 1.1% and 0.0%, respectively. Among H.influenzae, 17.6% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant, 33.5% to be β-lactamase-non-producing ABPC-resistant and 11.0% to be β-lactamase-producing ABPC-resistant strains. Extended spectrum β-lactamase-producing K.pneumoniae and multi-drug resistant P.aeruginosa with metallo β-lactamase were 2.9% and 0.6%, respectively.Continuous national surveillance of antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis

Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers

YAP and TAZ oncoproteins confer malignancy and drug resistance to various cancer types. We screened for small molecules that inhibit the nuclear localization of YAP/TAZ. Dasatinib, statins and pazopanib inhibited the nuclear localization and target gene expression of YAP and TAZ. All three drugs induced phosphorylation of YAP and TAZ, and pazopanib induced proteasomal degradation of YAP/TAZ. The sensitivities to these drugs are correlated with dependence on YAP/TAZ in breast cancer cell lines. Combinations of these compounds with each other or with other anti‐cancer drugs efficiently reduced cell proliferation of YAP/TAZ‐dependent breast cancer cells. These results suggest that these drugs can be therapeutics and chemosensitizers for YAP/TAZ‐dependent breast cancers

Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents.

Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs

Synthesis and evaluation of azalamellarin N and its A-ring-modified analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant

Azalamellarin N, a synthetic lactam congener of the marine natural product lamellarin N, and its A-ring-modified analogues were synthesized and evaluated as potent and non-covalent inhibitors of the drug-resistant epidermal growth factor receptor T790M/L858R mutant. An in vitro tyrosine kinase assay indicated that the inhibitory activities of the synthetic azalamellarin analogues were higher than those of the corresponding lamellarins.The azalamellarin analogue bearing two 3-(dimethylamino)propoxy groups at C20- and C21-positions exhibited the highest activity and selectivity against the mutant kinase [IC50 (T790M/L858R) = 1.7 nM; IC50 (WT) = 4.6 nM]. The inhibitory activity was attributed to the hydrogen bonding interaction between the lactam NH group of the B-ring and carbonyl group of a methionine residue

Design, synthesis, and evaluation of A-ring-modified lamellarin N analogues as noncovalent inhibitors of the EGFR T790M/L858R mutant

A series of A-ring-modified lamellarin N analogues were designed, synthesized, and evaluated as potential noncovalent inhibitors of the EGFR T790M/L858R mutant, a causal factor in the drug-resistant non-small cell lung cancer. Several water-soluble ammonium- or guanidinium-tethered analogues exhibited good kinase inhibitory activities. The most promising analogue, 14f, displayed an excellent inhibitory profile against the T790M/L858R mutant [IC50 (WT)?=?31.8?nM; IC50 (T790M/L858R)?=?8.9?nM]. The effects of A-ring-substituents on activity were rationalized by docking studies

ROCK inhibitors override SAC.

<p>(A) Fucci-based small molecule screening for overriding SAC. HeLa. S-Fucci2 cells were arrested by single thymidine block (STA) and released in medium containing 1 μM nocodazole for 16 hr. Most cells showed green fluorescence, and nocodazole-arrested cells (left) were treated with the MPS1 inhibitor, reversine for 5 hr. Cells show red fluorescence due to overriding SAC. (B) Identification of ROCK inhibitors as small molecules bypassing SAC. 30 μM Y-27632, 1 μM H-1152, and 30 μM fasudil overrode SAC as cells show red fluorescence. The population of the cells in G1 (red) was counted after 5 hr treatment. Data is mean and standard error from three independent experiments. (C) ROCK inhibitors bypassed SAC by a microtubule-dependent mechanism, not by inhibiting the SAC directly. (i) Immunofluorescent images of microtubules at low or high concentration of nocodazole. At 0.3 μM nocodazole, microtubules still remained, whereas they were completely disrupted at 3.3 μM nocodazole. Bar represents 10 μm. (ii) Reversine triggered cyclin B1 degradation in both conditions, whereas ROCK inhibitors failed to trigger cyclin B1 degradation at 3.3 μM nocodazole. Relative amount of Cyclin B1 was measured by densitometry.</p