Develop a High-Throughput Screening Method to Identify C-P4H1 (Collagen Prolyl 4-Hydroxylase 1) Inhibitors from FDA-Approved Chemicals.
Develop a High-Throughput Screening Method to Identify C-P4H1 (Collagen Prolyl 4-Hydroxylase 1) Inhibitors from FDA-Approved Chemicals.
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DOI：
10.3390/ijms21186613
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发表时间：
2020-09-10
影响因子：
5.6
通讯作者：
Xu R
中科院分区：
文献类型：
--
作者：
Wang S;Lee KH;Araujo NV;Zhan CG;Rangnekar VM;Xu R
胶原蛋白脯氨酰4-羟化酶1（C-P4 H1）是一种α-酮戊二酸（α-KG）依赖性双加氧酶，催化胶原蛋白上脯氨酸的4-羟基化。C-P4 H1诱导的脯氨酰羟基化是适当的胶原沉积和癌症转移所必需的。因此，靶向C-P4 H1被认为是胶原相关癌症进展和转移的潜在治疗策略。然而，没有C-P4 H1抑制剂可用于临床测试，并且高含量测定目前不可用于C-P4 H1抑制剂筛选。在本研究中，我们开发了一种高通量的筛选测定定量琥珀酸，C-P4 H催化羟基化的副产物。C-P4 H1是胶原蛋白脯氨酰4-羟化酶（CP 4 H）的主要亚型，其贡献了大部分脯氨酰4-羟化酶活性。使用从真核表达系统中纯化的C-P4 H1四聚体，我们表明，与羟脯氨酸比色法相比，琥珀酸-GloTM羟化酶测定法用于测量C-P4 H1活性更敏感。接下来，我们用FDA批准的药物库进行了高通量筛选，并鉴定了几种新的C-P4 H1抑制剂，包括Silodosin和Ticlopidine。Silodosin和噻氯匹定以剂量依赖性方式抑制C-P4 H1活性，并抑制3D组织培养中的胶原分泌和肿瘤侵袭。这些C-P4 H1抑制剂提供了新的药物来测试靶向C-P4 H1抑制癌症进展和转移的临床潜力。
Collagen prolyl 4-hydroxylase 1 (C-P4H1) is an α-ketoglutarate (α-KG)-dependent dioxygenase that catalyzes 4-hydroxylation of proline on collagen. C-P4H1-induced prolyl hydroxylation is required for proper collagen deposition and cancer metastasis. Therefore, targeting C-P4H1 is considered a potential therapeutic strategy for collagen-related cancer progression and metastasis. However, no C-P4H1 inhibitors are available for clinical testing, and the high content assay is currently not available for C-P4H1 inhibitor screening. In the present study, we developed a high-throughput screening assay by quantifying succinate, a byproduct of C-P4H-catalyzed hydroxylation. C-P4H1 is the major isoform of collagen prolyl 4-hydroxylases (CP4Hs) that contributes the majority prolyl 4-hydroxylase activity. Using C-P4H1 tetramer purified from the eukaryotic expression system, we showed that the Succinate-GloTM Hydroxylase assay was more sensitive for measuring C-P4H1 activity compared with the hydroxyproline colorimetric assay. Next, we performed high-throughput screening with the FDA-approved drug library and identified several new C-P4H1 inhibitors, including Silodosin and Ticlopidine. Silodosin and Ticlopidine inhibited C-P4H1 activity in a dose-dependent manner and suppressed collagen secretion and tumor invasion in 3D tissue culture. These C-P4H1 inhibitors provide new agents to test clinical potential of targeting C-P4H1 in suppressing cancer progression and metastasis.
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