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author:("Shi, hairbin")
1.  Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function 
PLoS ONE  2015;10(7):e0133590.
The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.
PMCID: PMC4519180  PMID: 26222440
2.  Simple quantitative measurement based on DWI to objectively judge DWI-FLAIR mismatch in a canine stroke model 
Diffusion-weighted imaging (DWI) - fluid attenuated inversion recovery (FLAIR) mismatch was proven useful to time the onset of wake-up stroke; however, identifying the status of FLAIR imaging has been mostly subjective. We aimed to evaluate the value of relative DWI signal intensity (rDWI), and relative apparent diffusion coefficient (rADC) in identifying the FLAIR status in the acute period.
Autologous clot was used to embolize left middle cerebral artery in 20 dogs. Magnetic resonance imaging was performed 3–6 hours and 24 hours after embolization. DWI-FLAIR mismatch was defined as hyperintense signal detected on DWI, but not on FLAIR. The mean values of rDWI or rADC of FLAIR− and FLAIR+ lesions were compared and the critical cutoff values of rDWI and rADC for identifying the FLAIR status were determined.
Stroke models were successfully established in all animals. DWI+ lesions were found in all 20 dogs from three hours, while FLAIR+ lesions were found in three, 11, 16, 19, and 20 dogs at five time points after embolization, respectively. The mean rDWI values were significantly different between FLAIR− and FLAIR+ lesions (P < 0.001), but rADC values were not (P = 0.73). Using rDWI=1.90 as the threshold value, excellent diagnostic efficacy was achieved (AUC, 0.88; sensitivity, 0.77; specificity, 0.88). However, rADC appeared not useful (AUC, 0.48; sensitivity, 0.52; specificity, 0.58) in identifying the FLAIR status.
In our embolic canine stroke model, rDWI was useful to identify FLAIR imaging status in the acute period, while rADC was not.
PMCID: PMC4498432  PMID: 26038954
3.  Comparison of Treatment Safety and Patient Survival in Elderly versus Nonelderly Patients with Advanced Hepatocellular Carcinoma Receiving Sorafenib Combined with Transarterial Chemoembolization: A Propensity Score Matching Study 
PLoS ONE  2015;10(2):e0117168.
This retrospective study was carried out to compare the outcomes between elderly (≥70 years of age) and nonelderly patients (<70 years of age) with advanced hepatocellular carcinoma (HCC) who received sorafenib combined with transarterial chemoembolization (TACE).
88 patients with a confirmed diagnosis of advanced HCC were enrolled in this study. Of these, 24 elderly patients were matched with 48 nonelderly patients at a 1:2 ratio using propensity score matching to minimize selection bias. The related adverse events and survival benefits were compared between the two groups.
Sorafenib combined with TACE was equally well tolerated in both age groups, and grade 3 or 4 adverse events were similarly observed in 54.2% of elderly and 50.0% of nonelderly patients (P = 0.739). There were no significant differences in survival time between the elderly and nonelderly patients (P = 0.876). Significant prognostic factors for overall survival as identified by multivariate analysis were the Child–Pugh score and portal vein invasion.
Sorafenib combined with TACE may be well tolerated and effective in elderly patients with advanced HCC. Age alone is not a parameter for the treatment of advanced HCC patients.
PMCID: PMC4331363  PMID: 25689846
4.  PET/CT evaluation of response to chemotherapy in non-small cell lung cancer: PET response criteria in solid tumors (PERCIST) versus response evaluation criteria in solid tumors (RECIST) 
Journal of Thoracic Disease  2014;6(6):677-683.
18F-FDG PET/CT is increasingly used in evaluation of treatment response for patients with non-small cell lung cancer (NSCLC). There is a need for an accurate criterion to evaluate the effect and predict the prognosis. The aim of this study is to evaluate therapeutic response in NSCLC with comparing PET response criteria in solid tumors (PERCIST) to response evaluation criteria in solid tumors (RECIST) criteria on PET/CT.
Forty-four NSCLC patients who received chemotherapy but no surgery were studied. Chemotherapeutic responses were evaluated using 18F-FDG PET and CT according to the RECIST and PERCIST methodologies. PET/CT scans were obtained before chemotherapy and after 2 or 4-6 cycles’ chemotherapy. The percentage changes of tumor longest diameters and standardized uptake value (SUV) (corrected for lean body mass, SUL) before and after treatment were compared using paired t-test. The response was categorized into 4 levels according to RECIST and PERCIST: CR (CMR) =1, PR (PMR) =2, SD (SMD) =3, PD (PMD) =4. Pearson chi-square test was used to compare the proportion of four levels in RECIST and PERCIST. Finally the relationship between progression-free survival (PFS) and clinicopathologic parameters (such as TNM staging, percentage changes in diameters and SUL, RECIST and PERCIST results etc.) were evaluated using univariate and multivariate Cox proportional hazards regression method.
The difference of percentage changes between diameters and SUL was not significant using paired t-test (t=–1.69, P=0.098). However the difference was statistically significant in the 40 cases without increasing SUL (t=–3.31, P=0.002). The difference of evaluation results between RECIST and PERCIST was not significant by chi-square test (χ2=5.008, P=0.171). If RECIST evaluation excluded the new lesions which could not be found or identified on CT images the difference between RECIST and PERCIST was significant (χ2=11.759, P=0.007). Reduction rate of SULpeak (%), RECIST and PERCIST results were significant factors in univariate Cox analysis. But Multivariate Cox proportional hazards regression analysis demonstrated that only PERCIST was a significant factor for predicting DFS [hazard ratio (HR), 3.20; 95% (CI), 1.85-5.54; P<0.001].
PERCIST and RECIST criteria have good consistency and PERCIST (or PET) is more sensitive in detecting complete remission (CR) and progression. PERCIST might be the significant predictor of outcomes. The combination of PERCIST and RECIST would provide clinicians more accurate information of therapeutic response in earlier stage of treatment.
PMCID: PMC4073366  PMID: 24976990
Non-small cell lung cancer (NSCLC); treatment response; response evaluation criteria in solid tumors (RECIST); PET response criteria in solid tumors (PERCIST); 18F-FDG PET/CT
5.  Sorafenib Combined with Transarterial Chemoembolization versus Transarterial Chemoembolization Alone for Advanced-Stage Hepatocellular Carcinoma: A Propensity Score Matching Study 
PLoS ONE  2014;9(5):e96620.
The purpose of the present study was to compare the efficacies of transarterial chemoembolization (TACE) combined with sorafenib versus TACE monotherapy for treating patients with advanced hepatocellular carcinoma (HCC).
We enrolled 321 patients and selected 280 with advanced HCC (Barcelona Clinic Liver Cancer stage C) who underwent TACE therapy between February 2009 and February 2013. TACE alone (monotherapy group) was administered to 198 patients (70.7%), and the remaining 82 (29.3%) underwent repeat combined TACE and sorafenib therapy (combined group). To minimize selection bias, these latter 82 patients were matched using propensity-score matching at a 1∶2 ratio with 164 patients who received TACE monotherapy. The primary endpoints were overall survival (OS) and related subgroup analysis. The secondary endpoints were time to progression (TTP) and treatment-related adverse events.
Of the respective patients in the combined and monotherapy groups, 64.6% and 49.2% had vascular invasion, 87.8% and 91.1% had extrahepatic metastasis, and 54.3% and 47.1% had both. In the propensity-score–matched cohort, the OS survival of the combined group was significantly higher compared with the monotherapy group (7.0 months vs. 4.9 months, respectively, P = 0.003). The TTP was significantly longer in the combined group (2.6 months vs. 1.9 months, respectively, P = 0.001). Subgroup analysis showed that the outcomes of patients with advanced HCC without main portal vein invasion who were treated with combined therapy were significantly better compared with those who received monotherapy (P<0.05). Univariate and subsequent multivariate analyses revealed that the addition of sorafenib was an independent predictor of favorable OS and TTP (adjusted hazard ratios, 0.63 and 0.62, respectively; P<0.05 for both).
Sorafenib plus TACE was more effective than TACE monotherapy for treating patients with advanced HCC without main portal vein invasion. Future trials with larger samples are required to validate these preliminary findings.
PMCID: PMC4016022  PMID: 24817002
6.  Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties 
ACS nano  2008;2(10):2121-2134.
Nanomaterials (NM) exhibit novel physicochemical properties that determine their interaction with biological substrates and processes. Three metal oxides nanoparticles that are currently being produced in high tonnage, TiO2, ZnO and CeO2, were synthesized by flame spray pyrolysis process and compared in a mechanistic study to elucidate the physicochemical characteristics that determine cellular uptake, subcellular localization, and toxic effects based on a test paradigm that was originally developed for oxidative stress and cytotoxicity in RAW 264.7 and BEAS-2B cell lines. ZnO induced toxicity in both cells, leading to the generation of reactive oxygen species (ROS), oxidant injury, excitation of inflammation and cell death. Using ICP-MS and fluorescent-labeled ZnO, it is found that ZnO dissolution could happen in culture medium and endosomes. Non-dissolved ZnO nanoparticles enter caveolae in BEAS-2B, but enter lysosomes in RAW 264.7 cells in which smaller particle remnants dissolve. In contrast, fluorescent-labeled CeO2 nanoparticles were taken up intact into caveolin-1 and LAMP-1 positive endosomal compartments, respectively, in BEAS-2B and RAW 264.7 cells, without inflammation or cytotoxicity. Instead, CeO2 suppressed ROS production and induced cellular resistance to an exogenous source of oxidative stress. Fluorescent-labeled TiO2 was processed by the same uptake pathways as CeO2 but did not elicit any adverse or protective effects. These results demonstrate that metal oxide nanoparticles induce a range of biological responses that vary from cytotoxic to cytoprotective and can only be properly understood by using a tiered test strategy such as we developed for oxidative stress and adapted to study other aspects of nanoparticle toxicity.
PMCID: PMC3959800  PMID: 19206459
Nanotoxicology; Nanoparticle; Reactive oxygen species; Oxidative stress; Dissolution; Nano-bio interface
7.  Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Anti-retroviral Activity 
Chemistry & biology  2013;20(1):82-91.
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
PMCID: PMC3559019  PMID: 23352142
8.  First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy 
Cancer discovery  2012;2(8):694-705.
Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as “undruggable”. We developed a decoy targeting STAT3 and performed a phase 0 trial. Expression levels of STAT3 target genes were decreased in the head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexa-ethyleneglycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development.
PMCID: PMC3668699  PMID: 22719020
STAT3; decoy oligonucleotide; phase 0; head and neck cancer
9.  HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck 
BMC Chemical Biology  2012;12:1.
Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.
To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.
These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
PMCID: PMC3328272  PMID: 22420777
10.  Structure of the HIV-1 Full-Length Capsid in a Conformationally-Trapped Unassembled State Induced by Small-Molecule Binding 
Journal of molecular biology  2010;406(3):371-386.
The capsid protein (CA) plays crucial roles in HIV-infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable functional substrate binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer (APD) configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in-situ during crystallization, is bound tightly at a hinge-site (“H-site”), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides 3D views of an assembly-defective conformation, a state targeted in and, thus, directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by ‘conformationally-trapping’ CA in assembly-incompetent conformational states, induced by H-site binding.
PMCID: PMC3194004  PMID: 21146540
native full-length HIV-1 capsid crystal structure; assembly inhibitor; hinge- binding site; conformational-trapping; alternative dimer states
11.  Usefulness of Stent Implantation for Treatment of Intracranial Atherosclerotic Stenoses 
Neurointervention  2012;7(1):27-33.
We evaluated the usefulness of intracranial stent implantation for treating patients with atherosclerotic stenosis and with recurrent, ischemic, neurological symptoms despite having undergone medical therapy.
Materials and Methods
Between March 2004 and April 2010, we attempted intracranial, stent-assisted angioplasty in 77 patients with 85 lesions (anterior circulation 73 cases, posterior circulation 12 cases) and who had ischemic neurological symptoms with more than 50% major cerebral artery stenosis. We analyzed the results regarding the technical success rate, complication rate, and restenosis rate during the mean 29.4 month follow-up period.
Intracranial stent implantation was successfully performed in 74 cases (87.1%). In nine cases among the 11, failed cases, stent implantation failure was due to the tortuosity of the target vessel. One patient experienced middle cerebral artery rupture during the procedure, and we embolized the vessel using a microcoil. Five patients developed cerebral infarction in three weeks after the procedure, three of whom improved using conservative management, although the other, two patients expired. The mean number of residual stenoses decreased from 72.3% to 14.7%. Three patients demonstrated significant in-stent restenosis, i.e. more than 50%, during the follow-up period.
As stent-assisted angioplasty in intracranial, atherosclerotic stenosis is effective and relatively safe, it can be considered as an alternative treatment for patients with recurrent, ischemic, neurologic symptoms despite having undergone medical therapy.
PMCID: PMC3299947  PMID: 22454782
Atherosclerosis, Intracranial; Stents; Angioplasty

Results 1-11 (11)