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Hit Validation in Drug Discovery: Application to Gcn2 and Skp2
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Hit Validation in Drug Discovery: Application to Gcn2 and Skp2. |
| 개인저자 | Lough, Lea Cassandra. |
| 단체저자명 | New York University. Basic Medical Science. |
| 발행사항 | [S.l.]: New York University., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 169 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-02B. Dissertation Abstract International |
| ISBN | 9781085678902 |
| 학위논문주기 | Thesis (Ph.D.)--New York University, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-02, Section: B.
Advisor: Cardozo, Timothy J. |
| 이용제한사항 | This item must not be sold to any third party vendors. |
| 요약 | This dissertation focuses on the process of small molecule development, hit validation, and evaluates the biological potential of hit compounds against two different protein targets, Gcn2 and Skp2. First, Gn2 is a stress response kinase of the integrated stress response (ISR) and the amino acid response (AAR) pathways. These pathways converge at the phosphorylation of eIF2慣, a key element of the translation machinery, by activating the kinases, Gcn2, PERK, and PKR. Drug-like, potent and selective chemical inhibitors (valid chemical probes) targeting major ISR kinases have been previously identified, with the exception of Gcn2. We synthesized and evaluated a series of Gcn2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited Gcn2 in vitro and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds inhibited phosporylation of eIF2慣 in HEK293T cells with an IC50 < 150 nM, validating them as chemical probes for cellular studies. These probes were screened against the National Cancer Institute NCI-60 human cancer cell line panel. Uniform growth inhibition was observed in the leukemia group of cell lines. Growth inhibition in the most sensitive cell lines coincided with high GCN2 and asparagine synthetase (ASNS) mRNA expression levels. Oncomine analysis revealed high GCN2 expression accompanied by lower ASNS expression in patient-derived acute lymphoblastic leukemias with B-Cell origins (B-ALL) as well. Notably, asparaginase, which depletes amino acids and triggers GCN2 activity, is a licensed, first-line B-ALL treatment. Thus, we hypothesize that leukemias exhibiting high GCN2 expression and low ASNS expression may be susceptible to pharmacologic GCN2 inhibition.Second, Skp2 is a member of the F-box family of proteins that serve as substrate-specific adaptors in Skp1-CUL1-ROC1-F-box (SCF) E3 ubiquitin ligases. Skp2 (Fbxl1) directly binds to the tumor suppressor p27 in the context of the SCFSkp2 E3 ubiquitin ligase to ubiquitylate and target-phosphorylated p27 for proteasomal degradation. As p27 is a powerful suppressor of growth in a variety of cells, and as Skp2 is also overexpressed in many human cancers, Skp2 is considered an oncogene and an intriguing drug target. However, despite 20 years of investigation, a valid chemical inhibitor of Skp2-mediated degradation of p27 has not been identified. Recently, an increasing number of compounds designed to have this bioactivity have been reported. Here, we conduct a meta-analysis of the evidence regarding bioactivity, structure, and medicinal chemistry in order to evaluate and compare these Skp2 inhibitor compounds. Despite chemically diverse compounds with a wide array of Skp2-mediated p27 ubiquitylation inhibition properties reported by several independent groups, no current chemical probe formally qualifies as a validated pharmaceutical hit compound. This finding suggests that our knowledge of the structural biochemistry of the Skp2-p27 complex remains incomplete and highlights the need for novel modes of inquiry.We validated Gcn2 chemical probes, and performed a meta-analysis on data from available Skp2 compounds. The results proved that a validated hit produces a more robust phenotype and thus it is highly encouraged to work with validated compounds. Overall, we have provided a roadmap for kinase inhibitor validation and for meta-analysis compound validation. |
| 일반주제명 | Oncology. Pharmacology. Computational chemistry. |
| 언어 | 영어 |
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