Deconvolving Passive and Active Targeting of Liposomes Bearing LDL Receptor Binding Peptides Using the Zebrafish Embryo Model.

Improving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces is a key approach but has limited clinical success. A potential issue is the integration of targeting ligands also changes the physicochemical properties of nanoparticles (passive targeting). Direct studies to understand the mechanisms of active targeting and off-targeting in vivo are limited by the lack of suitable tools. Here, the biodistribution of a representative active targeting liposome is analyzed, modified with an apolipoprotein E (ApoE) peptide that binds to the low-density lipoprotein receptor (LDLR), using zebrafish embryos. The ApoE liposomes demonstrated the expected liver targeting effect but also accumulated in the kidney glomerulus. The ldlra-/- zebrafish is developed to explore the LDLR-specificity of ApoE liposomes. Interestingly, liver targeting depends on the LDLR-specific interaction, while glomerular accumulation is independent of LDLR and peptide sequence. It is found that cationic charges of peptides and the size of liposomes govern glomerular targeting. Increasing the size of ApoE liposomes can avoid this off-targeting. Taken together, the study shows the potential of the zebrafish embryo model for understanding active and passive targeting mechanisms, that can be used to optimize the design of nanoparticles.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

Liquid-Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length.

Phase transitions have an essential role in the assembly of nature's protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair. We show that the three silk proteins all undergo a phase transition from homogeneous solution to assembly formation. In the assembly phase, a length- and concentration-dependent transition between two distinct assembly morphologies, one forming aggregates and another coacervates, exists. The coacervates showed properties that were dependent on the protein size. Computational modeling of the proteins by a bead-spring model supports the experimental results and provides us a possible mechanistic origin for the assembly transitions based on architectures and interactions.

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up-regulating α1 integrin expression.

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR+ and p75NTR- hPDLSCs by fluorescence-activated cell sorting. Differences in osteogenic differentiation among p75NTR+ , p75NTR- and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR+ and p75NTR- hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1-overexpressing adenovirus were used to transfect p75NTR+ and p75NTR- hPDLSCs. The results showed that p75NTR+ hPDLSCs demonstrated superior osteogenic capacity than p75NTR- and unsorted hPDLSCs. Differentially expressed genes between p75NTR+ and p75NTR- hPDLSCs were highly involved in the extracellular matrix-receptor interaction signalling pathway, and p75NTR+ hPDLSCs expressed higher ITGA1 levels than p75NTR- hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR+ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR- hPDLSCs. These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up-regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.

Effects of a Comprehensive Reminder System Based on the Health Belief Model for Patients Who Have Had a Stroke on Health Behaviors, Blood Pressure, Disability, and Recurrence From Baseline to 6 Months: A Randomized Controlled Trial.

BACKGROUND: The health behaviors and blood pressure control of patients with hypertension who have had a stroke are unsatisfactory. A protocol of a comprehensive reminder system has been published, and the results of 3 months of implementation have demonstrated improved patient health behaviors and blood pressure control. The continuity of the intervention effect on these variables after 3 months was not clear. OBJECTIVE: The aim of this study was to determine the impact of a comprehensive reminder system intervention on health behaviors, medication adherence, blood pressure, disability, and stroke recurrence in patients with hypertension who have had a stroke from baseline to 6 months after discharge. METHOD: A multicenter, assessor-blinded, randomized controlled trial was conducted with 174 patients with hypertension who have had a stroke. The intervention consisted of health belief education, a calendar handbook, weekly short message services, and telephone interviews. Data were collected at baseline and at 3 and 6 months after discharge. RESULTS: Repeated-measures analysis of variance and single-effect analysis revealed that, compared with the control group, improvements of health behaviors, medication adherence, blood pressure, and disability of participants in the intervention group were superior. From 3 to 6 months after discharge, these trends remained or continually improved, whereas a downward trend was observed in the control group. There were only 2 stroke recurrences within 6 months, and no statistically significant difference between groups was found. CONCLUSION: The comprehensive reminder system improved patients' health behaviors and medication adherence and reduced blood pressure and disability; the effect extended to 6 months after discharge.

Stabilizing G-quadruplex DNA by methylazacalix[n]pyridine through shape-complementary interaction.

It is found that G-quadruplexes have important functions in biological systems, such as gene expression. Molecules which can stabilize the G-quadruplex structure may have potential application in regulating the expression of gene. A series of methylazacalix[n]pyridine (n=4, 6, 7, 8, 9) has been tested to stabilize the intermolecular human telomeric G-quadruplex (T12 and H12), intramolecular TBA, c-kit and bcl-2 G-quadruplex by CD denaturation experiments. The results showed that only methylazacalix[6]pyridine (MACP6) can stabilize the intermolecular G-quadruplex formed from the 12bp human telomere. Further studies evidenced that the shape-complementary binding mode was what contributed to the interaction between MACP6 and T12 G-quadruplex.

Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes.

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite the development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report the design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N-myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions in the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein 1 (FSP1) and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome without the requirement for genetic modification, which may prove broadly applicable to other PTMs.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

[Screening and clinical phenotype analysis of microdeletions of azoospermia factor region on Y chromosome in 1011 infertile men].

OBJECTIVE: To investigate the prevalence and subtypes of microdeletions in azoospermia factor (AZF) region in infertile men from Sichuan in order to correlate genotypes with phenotypes. METHODS: Multiplex-PCR was used to detect sequence tagged sites (STS) of AZF microdeletions in 1011 infertile men including 713 cases of non-obstructive azoospermia and 298 cases of severe oligospermia. RESULTS: The overall prevalence of microdeletions was 10.48% (106/1011), and the deletion rates were 11.08% (79/713) in non-obstructive azoospermia and 9.06% (27/298) in severe oligospermia. Complete AZFa or AZFb deletions were associated with azoospermia, whereas AZFc deletion (60.38%) was the most frequent deletion. The deletions were associated with variable spermatogenic phenotypes, and 37.50% of the patients with a deletion had sperms in the ejaculate. A mild decline in sperm concentration was found in two cases with partial AZFb deletion and one case with partial AZFb-c deletion. CONCLUSION: Deletions of the AZFc region were most commonly found in our patients. All cases with complete AZFa or AZFb deletions and a proportion of cases with AZFc deletion were associated with azoospermia. Our study has provided more insight into the genotype-phenotype correlation, and confirmed that Yq microdeletion screening has a significant value for the diagnosis for male infertility.

Association between chronic illness resources and health behaviors in hypertensive stroke patients at 6 months after discharge: a cross-sectional study.

Background: The adoption of appropriate health behaviors can prevent the recurrence of stroke. Previous research found a downward trend in hypertensive stroke patients' health behaviors from 3 to 6 months after discharge. The provision of appropriate support by chronic illness resources has been shown to predict patients' engagement in appropriate health behaviors in other chronic illness populations. This study sought to explore the association between chronic illness resources and health behaviors in hypertensive stroke patients in order to provide a foundation for the secondary prevention of stroke. Methods: Using convenience sampling method, we enrolled 133 hypertensive stroke patients at 6 months after discharge in Guangzhou, China. All the patients completed a demographic and disease-specific questionnaire, the Health Behavior Scale for Stroke Patients (HBS-SP) and the Chronic Illness Resources Survey (CIRS). A multiple stepwise regression analysis was conducted to test the association of chronic illness resources with health behaviors. Results: The total scores of the HBS-SP and CIRS were (2.89±0.38) and (2.94±0.66), respectively. The correlation coefficient for chronic illness resources and health behaviors was 0.517 (P<0.001). The positive association between chronic illness resources and health behaviors remained statistically significant after controlling for gender, education level, and the Barthel Index (unstandardized coefficient: 0.317, P<0.001). Conclusions: The chronic illness resources has positive association with health behaviors in hypertensive stroke patients at 6 months after discharge. A good support provided by chronic illness resources may contribute to promote positive health behaviors, and thus prevent the recurrence of stroke.

Generating Heterokaryotic Cells via Bacterial Cell-Cell Fusion.

Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs. IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion.

Structure-activity study on a series of α-glutamic acid scaffold based compounds as new ADAMTS inhibitors.

A series of α-glutamic acid scaffold based 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl) butanoic acids were designed and synthesized as new ADAMTS inhibitors. The compounds dose-dependently inhibited the enzymatic activities of ADAMTS-4 and ADAMTS-5. One of the most active compound 2h potently inhibited ADAMTS-4 and ADAMTS-5 with IC(50) values of 1.2 and 0.8 µM, respectively. These inhibitors may serve as new lead compounds for further development of therapeutics to treat osteoarthritis.

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides.

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from K F ≈ 108 to 104 M-1), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting 'K' peptides and homodimer-forming 'E' peptides. Furthermore, a new and highly fusogenic CC pair (E3/P1K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2E/P2K and P3E/P3K. This E3/P1k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.

Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity.

Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts.

Human Amniotic Mesenchymal Stem Cell Sheets Encapsulating Cartilage Particles Facilitate Repair of Rabbit Osteochondral Defects.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are being widely applied in various fields. Therefore, hAMSCs represent a promising candidate to facilitate cartilage regeneration. Nonetheless, no studies have investigated the application of hAMSC sheets to repair cartilage defects in vivo. PURPOSE: To evaluate hAMSC sheets encapsulating cartilage particles to promote repair of rabbit osteochondral defects. STUDY DESIGN: Controlled laboratory study. METHODS: hAMSC sheets were constructed with passage 3 hAMSCs. The phenotypic and structural characteristics of hAMSC sheets were evaluated by flow cytometry and scanning electron microscopy, respectively. The potential for chondrogenic differentiation of hAMSC sheets was assessed by cartilage-specific marker staining, immunohistochemistry, and mRNA and protein expression (SOX9, COLII, and ACAN). Osteochondral defects (diameter, 3.5 mm; depth, 3 mm) were created in the left patellar grooves of 20 New Zealand White rabbits (female or male). The defects were treated with hAMSC sheet/cartilage particles (n = 5), cartilage particles (n = 5), hAMSC sheets (n = 5), or fibrin glue (n = 5). Macroscopic and histological evaluations of the regenerated tissue were conducted after 3 months. The survival time and differentiation of transplanted hAMSCs in the defect area were evaluated by immunofluorescence. RESULTS: hAMSC sheets had a multilayered structure, with cells stacked layer by layer. Importantly, hAMSC sheets highly expressed phenotypic markers of mesenchymal stem cells. Cartilage-specific marker staining and immunohistochemistry were positive, and mRNA and protein expression was higher in the chondrogenically induced hAMSC sheet group than in the hAMSC sheet group (P < .05). hAMSC sheet/cartilage particles formed a large amount of hyaline-like cartilage in the defect area. In addition, macroscopic and histological scores were significantly higher than those in the other groups. Integration with surrounding normal cartilage and subchondral bone regeneration in the hAMSC sheet/cartilage particles group were better when compared with the other groups. A large number of human nuclear-specific antigen-positive cells were observed in the defect area of hAMSC sheet/cartilage particles and hAMSC sheet groups. Moreover, some positive cells expressed SOX9. CONCLUSION: hAMSC sheets encapsulating cartilage particles facilitate osteochondral defect repair. CLINICAL RELEVANCE: Delivery of cells in the form of a cell sheet in conjunction with cartilage particles provides a novel approach for cell-based cartilage regeneration.

High-resolution snapshots of human N-myristoyltransferase in action illuminate a mechanism promoting N-terminal Lys and Gly myristoylation.

The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.

Synthetic, Zwitterionic Sp1 Oligosaccharides Adopt a Helical Structure Crucial for Antibody Interaction.

The zwitterionic Streptococcus pneumoniae serotype 1 polysaccharide (Sp1) is an important anchor point for our immune system to act against streptococcal infections. Antibodies can recognize Sp1 saccharides, and it has been postulated that Sp1 can elicit a T-cell-dependent immune reaction as it can be presented by MHC-II molecules. To unravel the molecular mode of action of this unique polysaccharide we here describe the chemical synthesis of a set of Sp1 fragments, ranging from 3 to 12 monosaccharides in length. We outline a unique synthetic approach to overcome the major synthetic challenges associated with the complex Sp1 structure and provide a stereoselective route of synthesis for the oligosaccharide backbone as well as a strategy to introduce the carboxylic acid functions. Molecular dynamics (MD) simulations together with NMR spectroscopy studies reveal that the oligosaccharides take up helical structures with the nona- and dodecasaccharide completing a full helical turn. The 3D structure of the oligosaccharides coincides with the topology required for good interaction with anti-Sp1 antibodies, which has been mapped in detail using STD-NMR. Our study has revealed the Sp1 nona- and dodecasaccharides as promising synthetic antigens, displaying all (3D) structural elements required to mimic the natural polysaccharide and required to unravel the molecular mode of action of these unique zwitterionic polysaccharides.

[Analysis of benzoic acid content in Radix Paeoniae Alba treated by different process].

OBJECTIVE: To analyze the benzoic acid content in the Radix Paeoniae Alba treated by three kinds of process. METHODS: After harvesting, the fresh roots were processed as follows: stored in low temperature, dried after boiling a few minutes and dried immediately. Then the content of benzoic acid was determined by UV spectrophotometry. RESULTS: The content of benzoic acid in the above material without outer part of the cortex was at the higher level than those with outer part of the cortex. In material without outer part of the cortex, the content of benzoic acid was 0.0635% in stored sample, 0.0248% in poaching sample, 0.0639% in dried sample, 0.6635% in part of the cortex. CONCLUSION: Benzoic acid was mostly distributed in the root cortex, Radix Paeoniae Alba treated by boiling can reduce the benzoic acid content. Fresh roots without cortex are comparatively economical and reasonable in distilling Paeoniflorin.

G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch.

Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and (1)H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

Novel thiazole amine class tyrosine kinase inhibitors induce apoptosis in human mast cells expressing D816V KIT mutation.

Gain-of-function mutations of receptor tyrosine kinase KIT play a critical role in the pathogenesis of systemic mastocytosis (SM) and gastrointestinal stromal tumors. D816V KIT mutation, found in ∼80% of SM, is resistant to the currently available tyrosine kinase inhibitors (TKIs) (e.g. imatinib mesylate). Therefore, development of promising TKIs for the treatment of D816V KIT mutation is still urgently needed. We synthesized thiazole amine compounds and chose one representative designated 126332 to investigate its effect on human mast cells expressing KIT mutations. We found 126332 inhibited the phosphorylation of KIT and its downstream signaling molecules Stat3 and Stat5. 126332 inhibited the proliferation of D816V KIT expressing cells. 126332 induced apoptosis and downregulated levels of Mcl-1 and survivin. Furthermore, 126332 inhibited the tyrosine phosphorylation of ß-catenin, inhibited ß-catenin-mediated transcription and DNA binding of TCF. Moreover, 126332 also exhibited in vivo antineoplastic activity against cells harboring D816V mutation. Our findings suggest thiazole amine compounds may be promising agents for the treatment of diseases caused by KIT mutation.