Kehu Yuan

Small Molecules Blocking the Entry of Severe Acute Respiratory Syndrome Coronavirus into Host Cells

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-β-d-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 μM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.

Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein

Abstract Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC50 values of 0.14 and 1.19μM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.

Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

BackgroundAn antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein.ResultsBy using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application.ConclusionIn summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.

Nuclear entry of active caspase-3 is facilitated by its p3-recognition-based specific cleavage activity

As a critical apoptosis executioner, caspase-3 becomes activated and then enters into the nucleus to exert its function. However, the molecular mechanism of this nuclear entry of active caspase-3 is still unknown. In this study, we revealed that caspase-3 harbors a crm-1-independent nuclear export signal (NES) in its small subunit. Using reverse-caspase-3 as the study model, we found that the function of the NES in caspase-3 was not disturbed by the conformational changes during induced caspase-3 activation. Mutations disrupting the cleavage activity or p3-recognition site resulted in a defect in the nuclear entry of active caspase-3. We provide evidence that the p3-mediated specific cleavage activity of active caspase-3 abrogated the function of the NES. In conclusion, our results demonstrate that during caspase-3 activation, NES is constitutively present. p3-mediated specific cleavage activity abrogates the NES function in caspase-3, thus facilitating the nuclear entry of active caspase-3.

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.

Abstract 3921: A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein

Increased sensitivity and specificity of immunohistochemical (IHC) detection of HER2 expression is crucial as the role of Trastuzumab have expanded in the treatment of HER2 positive non-breast cancer patients such as gastric cancer patients. Non-specific nuclear and cytoplasmic staining of the HER2 antibody clone 4B5 has been reported by other labs. In this study, we evaluated the specificity of HER2 clone 4B5 and a new HER2 antibody clone UMAB36 using a suite of methods including protein lysate arrays, western blots, FISH and IHC correlation screens on 129 breast and 158 colon cancer cases for HER2 expression. The protein lysate array and western results revealed that clone 4B5 recognizes three proteins: HER2, HER4, and ZSCAN18, a nuclear transcription protein. In comparison, clone UMAB36 recognized only HER2 protein in the same protein lysate array and western screen as clone 4B5. False negative results, based on correlation of IHC with FISH HER2 positives, were generated using clone 4B5 in 1 breast and 5 gastric of the total 287 cancer cases screened. Comparatively, no false negative results were observed using clone UMAB36 in which there was a 100% correlation between IHC and FISH screen. In this study, areas of normal gastric tissue often stained positive by HER2 clone 4B5, so we performed further analysis of 470 normal gastric cases using Ventana9s BenchMark instrument. The results show that 278 normal gastric cases had positive stain with clone 4B5 compared to 3 cases with clone UMAB36. The high background by clone 4B5 may be due HER4 being upregulated in adjacent normal gastric tissue. Our results indicate clone UMAB36 has higher specificity and sensitivity than clone 4B5 in screening gastric tumors. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Ki, Min Zhou, Wei Fu, Xiaozheng Huang, Rachel M. Gonzalez, Youmin Shu, Yi Shen, Guangli Wang, Donghui Ma, Wei-Wu He, Jian Chen. A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3921.

Abstract 1706: Evaluation of LGR5 protein expression in colon cancer stem cells

Cancer stem cells are a subpopulation of cancer cells responsible for cancer initiation, development and metastasis. A number of studies demonstrated that Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) can drive cancer development through triggering canonical Wnt signaling and its downstream gene expression. LGR5 is an important biomarker specifically expressed on colon cancer stem cells. In this study, we have successfully developed an LGR5 antibody with high specificity to detect endogenous LGR5 expression in different immunoassay application. By screening multiple anti-LGR5 hybridoma clones, antibody generated by clone UMAB212 was proven to be highly specific on our high density protein microarray chip assay. Here our experimental data demonstrated that clone UMAB212 recognizes not only human LGR5 protein but also mouse LGR5 protein in both western blot and flow cytometry applications. No cross-reactivity was observed with the other two LGR family members, LGR4 and LGR6. Furthermore, this antibody also works great on immunohistochemistry application on FFPE tissue blocks. In summary, UMAB212 is great tool for us to study LGR5 protein in different immunoassay setting and it could also be a potential cancer diagnostic reagent. Citation Format: Wei Fu, Rachel Gonzalez, Kehu Yuan, Caiwei Chen, Haitao Wei, Hsiangmin Lu, Qi Ren, Evelin Logis, Sean Kelly, Wei-Wu He, Donghui Ma. Evaluation of LGR5 protein expression in colon cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1706.

Abstract 415: A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC

All non-small cell lung cancer (NSCLC) patients are recommended to be screened for anaplastic lymphoma kinase (ALK)-rearrangement despite its low occurrence ( Citation Format: Hsiangmin Lu, Rachel Gonzalez, Yi Shen, Mu-lan Jin, Yipan Wu, Yungang Zhang, Kehu Yuan, Boyang Chu, Lili Qi, Huibo Liu, Chenlin Wang, Guangli Wang, Youmin Shu, Julie McDowell, Donghui Ma, Wei-wu He, Jian Chen, Ray Lin. A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 415.

Abstract 3393: Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology

Programmed death-1 (PD-1) expresses in many tumors in response to inflammation. It is up-regulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands PD-L1 and PDL2 and serves as a key checkpoint of the immune system. Pembrolizumab is the first anti-PD-1 therapy approved in the United States and received FDA9s Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin-fixed paraffin-embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed. To develop the best monoclonal antibody for PD-1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono-specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross-reactivity tests were also explored. In summary, two newly generated monoclonal antibodies demonstrated ultra-specificity against PD-1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before pembrolizumab prescription. Citation Format: Caiwei Chen, Yanlin Tang, Haitao Wei, Kehu Yuan, Guiyin Wu, Jian Chen, Boyang Chu, Guangli Wang, Youmin Shu, Wei-Wu He, Donghui Ma. Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2015-3393

Abstract 3386: The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry

Ki-67 is a nuclear protein that is a useful marker of proliferation. Ki-67 antibodies are used to generate a Ki-67 labeling index, which is the percentage of cells with nuclear immunostaining in immunohistochemistry. The Ki-67 labeling index may be useful as a prognostic and predictive marker in cancer. The Ki-67 labeling index relies on the use of antibodies with a suitable level of sensitivity and specificity, and many Ki-67 antibodies have been developed over the last two decades. One of the most widely used Ki-67 antibodies is the mouse monoclonal MIB1. However, there have been many reports showing that in some cases MIB1 detects an unexpected membranous and cytoplasmic staining pattern in immunohistochemistry. Since Ki-67 is exclusively expressed in the nucleus this raises questions about the specificity of MIB1. We have created a nearly comprehensive microarray of about 17,000 human proteins to investigate the specificity of MIB1 and identify cross reacting proteins. The protein microarray was also used to screen new panels of mouse monoclonals against Ki-67. We have identified the antibody UMAB107 that in immunohistochemistry performs with similar sensitivity as MIB1, but unlike MIB1, UMAB107 is highly specific. Citation Format: Caiwei Chen, Kehu Yuan, Boyang Chu, Youmin Shu, Jian Chen, Joe Stafford, Wei Fu, Wei-Wu He, Ross Chambers, Donghui Ma. The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3386. doi:10.1158/1538-7445.AM2015-3386