Younho Lee

3D-QSAR method on indole and pyrrole inhibitors of monoamine oxidase type A

Monoamine oxidase A (MAO-A) converts norepinephrine and serotonin to an oxidative form. These monoamine neurotransmitters have important roles in depression. The MAO-A inhibitors have been discovered for neurodegenerative disease therapy. In order to design novel MAO-A inhibitors, in this study, the quantitative structure-activity relationships for the combined series of indoles and pyrroles were elucidated and the structural conditions to show good inhibitory effects on MAO-A were derived. This result can help us design new inhibitors irrespective of their specific moiety.

Discovery of a novel potent peptide agonist to adiponectin receptor 1

Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of in vitro and in vivo studies over last two decades. These findings suggest that AdipoRs’ agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs’ agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1’s peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions via surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.

High-Level Production of Human Papillomavirus (HPV) Type 16 L1 in Escherichia coli.

Human papillomavirus (HPV), a non-enveloped, double-stranded DNA tumor virus, is a primary etiological agent of cervical cancer development. As a potential tool for prophylactic vaccination, the development of virus-like particles (VLPs) containing the HPV16 L1 capsid protein is highly desired. In this study, we developed a high-level expression system of the HPV16 L1 in Escherichia coli for the purpose of VLP development. The native gene of HPV16 L1 has many rare codons that cause the early termination of translation and result in the production of truncated forms. First, we optimized the codon of the HPV16 L1 gene to the preferable codons of E. coli, and we succeeded in producing the full-size HPV16 L1 protein without early termination. Next, to find the best host for the production of HPV16 L1, we examined a total of eight E. coli strains, and E. coli BL21(DE3) with the highest yield among the strains was selected. With the selected host-vector system, we did a fed-batch cultivation in a lab-scale bioreactor. Two different feeding solutions (complex and defined feeding solutions) were examined and, when the complex feeding solution was used, a 6-fold higher production yield (4.6 g/l) was obtained compared with that with the defined feeding solution.

Abstract 941: Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Signal transduction in the Ras-Raf-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. The B-Raf mutants are found in various cancers, such as melanoma, colorectal, ovarian, and prostate cancer. The Ras mutants are also found in various carcinomas including colon cancer, pancreatic cancer, non-small-cell lung cancer, and liver cancer. The mutated K-Ras gene sends continuous signals to the colorectal cancer cells, telling them to grow independent of the way ERBITUX blocks the EGFR growth signals. Especially, over 90% of detected mutations in B-Raf are a V600E which leads to constitutive kinase activity 500-fold greater than B-Raf wild type and correlates with increased malignancy. Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the angiogenesis that is essential for growth and metastasis of solid tumors. Since formation of solid tumors are angiogenesis dependent, several strategies have been developed to inhibit VEGF signal transduction as part of anticancer therapy. The first VEGFR2 and Raf-1 inhibitor to enter clinical development was the multikinase inhibitor SORAFENIB, which is now approved for the treatment of patients with advanced renal cell carcinoma. More potent dual inhibitors against VEGFR2 and Raf-1 may be beneficial for patients suffering from various tumors. In this study, some of rational-designed compounds were synthesized and carried out the in vitro and in vivo studies to find more efficient VEGFR2 and Raf-1 inhibitors. Methods: About two hundreds of compounds were designed and synthesized. in vitro assays and in vivo studies were performed to characterize the potential of VEGFR2 and Raf-1 inhibitors for solid tumors. Results: Compound UI-162 showed strong kinase inhibition against VEGFR2 (12 nM), B-Raf V600E mutant (0.2 nM), B-Raf wildtype (2.0 nM) and Raf-1(0.6 nM). The compound showed good inhibitory effect in against malignant cells expressing K-Ras or B-Raf V600E mutant type of cell-line (SK-MEL-2 GI50: 133 nM / HCT-116 GI50: 229 nM / MDA-MB231 GI50: 278 nM / A375P GI50: 51 nM / HT29 GI50: 40 nM / COLO 205 GI50: 12 nM, respectively). In mechanism studies, the phosphorylation of MEK and ERK were suppressed by the UI-162 compound in HCT-116 cell-line. In addition, paradoxical effects were not detected compared with VEMURAFENIB. The compound had also a good anti-proliferative activity against HUVEC and showed a good oral bioavailability. Conclusions: The results of in vitro and in vivo studies suggests that dual inhibition of VEGFR2 and Raf-1 may provide strong antitumor efficacy and that UI-162 is a promising candidate for the treatment of various human cancers harboring the K-Ras and B-Raf V600E mutation. Citation Format: Seung Yong Kim, Younho Lee, Hyungtae Bang, Kihwan Eum, Sungpyo Hong, Ky-Youb Nam, Nam Song Choi, Ju Hee Kang, Hara Kang, Kil Won Kim, Michael Lee, Soon Kil Ahn. Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2013-941

Abstract 1803: Discovery of novel B-Raf kinase inhibitor with potent orally anticancer activity

Introduction: The B-Raf, one of serine/threonine kinase, is related to the Ras-Raf-MEK-ERK (MAPK) signal transduction. The signaling pathway plays a key role in cell survival, growth and proliferation. The B-Raf mutants are found in various cancer patients, such as melanoma, papillary thyroid, cisplatin-refractory testicular cancer, colorectal, ovarian, and prostate cancer. Especially, over 90% of detected mutations in B-Raf are a V600E which leads to constitutive kinase activity 500-fold greater than B-Raf wild type and correlates with increased malignancy and decreased response the chemotherapy. There are currently numerous efforts to develop therapeutic agents to target B-Raf or its downstream kinases. The first Raf inhibitor to enter clinical development was the multi-kinase inhibitor Nexavar (sorafenib tosylate), which is now approved for the treatment of patients with advanced renal cell carcinoma. However, the broad selectivity profile of this inhibitor makes it unsuitable for proof of concept evaluation of Raf kinase inhibition in tumors. Recently, more selective Raf inhibitors have been disclosed, and chemical inhibition of B-Raf enzymatic activity using these compounds can result in diminished proliferation and survival of tumor cells. However, very limited drug candidates with in vivo data have been published for B-Raf V600E . In this study, some of rational-designed compounds were synthesized and carried out the in vitro and in vivo studies to investigate more efficient B-Raf V600E inhibitors. Methods: About two hundreds of compounds were designed and synthesized. The in vitro assays and in vivo studies were performed to characterize the potential of B-Raf V600E inhibitors for solid tumors. Results: Compound UAI-201 had good inhibition effect in malignant cells expressing B-Raf V600E mutant type of cell-line (A375P GI 50 : 6nM / HT29 GI 50 : 5nM / COLO 205 GI 50 : 1nM), selectively. And the compound showed better tumor growth inhibition in xenograft mouse models (TGI(%): 61.87, 10mg/kg, PO, BID x 2weeks) compared with known inhibitors. In mechanism studies, the phosphorylation levels of MEK and ERK were effectively decreased by the UAI-201 compound in A375P cell-line. In addition, paradoxical effects were not detected compared with known compound. The compound had no toxicity in normal cell-line(HS-27 cell) and did not inhibit several CYP P450s. And also, UAI-201 showed good oral bioavailability with an acceptable clearance and half-life pharmacokinetic properties. Conclusions: The in vitro and in vivo studies of UAI-201 indicate that it is safe and efficacious, and suggests that it may good drug candidate for the preclinical study. Preclinical study our candidate, UAI-201, was started and the results will be disclosed in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1803. doi:1538-7445.AM2012-1803