CBR-5884

Phosphoglycerate dehydrogenase/PHGDH
3-phosphoglycerate dehydrogenase (PHGDH) [1]
IC50 = 33 ± 12 μM (in vitro enzymatic assay) [1]
Ki = 50 ± 20 μM (with respect to substrate 3-PG) [1]
Ki = 50 ± 3 μM (with respect to cofactor NAD+) [1]

CBR-5884（15 或 30 μM；3-5 天）专门防止具有强烈丝氨酸合成倾向的乳腺癌和黑色素瘤细胞系增殖。

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CBR-5884 抑制癌细胞中的丝氨酸从头合成。在Carney细胞中使用13C6-葡萄糖标记的急性（3小时）处理实验中，30 μM的 CBR-5884 将新合成（M+3）丝氨酸的比例降低了约30%。这种降低作用对丝氨酸及其前体3-PG具有特异性，对糖酵解终产物丙酮酸和乳酸无显著影响。 [1]
CBR-5884 选择性抑制具有高内在丝氨酸合成活性（高PHGDH表达）的乳腺癌和黑色素瘤细胞系的增殖。例如，在丝氨酸充足的培养基中，30 μM的 CBR-5884 可抑制高PHGDH细胞系（MDA-MB-468, MDA-MB-436, HCC70, Hs578T）35%至60%的生长，而对低PHGDH细胞系（MDA-MB-231, MCF10A）无影响。当细胞在丝氨酸缺乏的培养基中培养时，其生长抑制作用显著增强。 [1]
在作用机制上，CBR-5884 是PHGDH的非竞争性抑制剂（针对底物3-PG和NAD+）。它表现出时间依赖性抑制，与酶预孵育后效力增强（预孵育4小时后IC50降至约7 μM）。该化合物破坏了PHGDH的天然四聚体状态，使其平衡向二聚体移动。 [1]

体内研究显示，CBR-5884显著延缓肿瘤生长，组织学分析表明CBR-584的安全性。最后，利用患者来源的类器官（PDO）模型来探索CBR-5884对EOC细胞的临床前疗效，结果表明，CBR-584在EOC PDO模型中阻碍增殖并下调ITGB4的表达。[2]

PHGDH小分子抑制剂的筛选。[1]
一种适用于高通量筛选（HTS）的PHGDH活性的体外酶法测定是通过将3-PG氧化后NADH的产生与使用黄递酶作为偶联酶将雷沙祖林还原为间苯二酚偶联而开发的。因此，间苯二酚荧光作为PHGDH活性的替代物。该测定被小型化为1536孔格式，Z因子>0.75，表明该测定具有高质量。在13μM下以单点形式筛选了80万个小分子的文库。将阈值Z分数设置为−3，对应于至少50%的PHGDH抑制，可获得0.5%的命中率，产生3906次命中。假定的命中率以一式三份的形式重新确认，并对黄递酶进行反筛选，以排除针对黄递酶的假阳性。计数器筛选消除了3498种化合物，得到408种PHGDH抑制剂。
设计了一种基于命中效力和选择性的试验策略。我们推断，与打击多种脱氢酶的化合物相比，PHGDH特异性抑制剂将最大限度地降低一般细胞毒性。因此，测定了一组NAD（P）+依赖性脱氢酶的半数最大抑制浓度（IC50），这些脱氢酶包括PHGDH、异柠檬酸脱氢酶（IDH1）、苹果酸脱氢酶（MDH1）和3α-羟基类固醇脱氢酶（3α-HSD）。化合物对PHGDH的选择性提高了至少四倍以供进一步分析。基于这一试验，选择了七种最有效的PHGDH抑制剂作为先导化合物，用于在基于细胞的测定中进行评估；选定的结构如图1E所示。许多这些化合物可能靶向巯基，因此可能与PHGDH半胱氨酸残基反应。例如，CBR-587和CBR-6936都含有巯基反应性二硫中心。有趣的是，CBR-5807（二硫仑）是一种已批准用于治疗酒精中毒的药物，已知通过与巯基反应抑制乙醛脱氢酶。

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建立了一个高通量筛选（HTS）实验来鉴定PHGDH抑制剂。该实验通过双酶偶联，将PHGDH催化3-磷酸甘油酸（3-PG）氧化产生的NADH，用于将刃天青还原为荧光产物试卤灵。试卤灵的荧光强度作为PHGDH活性的读数。反应中包含磷酸丝氨酸氨基转移酶（PSAT1）以防止产物反馈抑制。该实验已微型化至1,536孔板格式。 [1]
为确定抑制模式和抑制常数（Ki），进行了机制研究。在几种固定浓度的 CBR-5884 存在下，测量了一个底物（3-PG或NAD+）浓度变化而另一个底物浓度恒定时，PHGDH的初始反应速率（V）。数据拟合到非竞争性抑制模型以得出Vmax、Km和Ki值。 [1]
通过在不同时间（0.5、1、4小时）将 CBR-5884 与PHGDH预孵育，然后再加入底物启动酶反应，来评估时间依赖性抑制。针对每个预孵育时间测定IC50值。 [1]
通过交联实验评估对PHGDH寡聚化的影响。将PHGDH蛋白与 CBR-5884 或对照溶剂孵育，然后用交联剂处理。通过SDS-PAGE分析反应混合物，以观察PHGDH的寡聚状态（单体、二聚体、四聚体）。 [1]

细胞增殖测定[1]
细胞类型：乳腺癌和黑色素瘤细胞系
测试浓度：15 或 30 μM
孵育时间：3-5天
实验结果：抑制黑色素瘤和乳腺癌细胞系的增殖。

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采用急性抑制剂处理结合稳定同位素示踪技术来测量对丝氨酸从头合成的影响。细胞先用 CBR-5884 或对照预处理1小时，然后更换为含有13C6-葡萄糖（示踪剂）和药物的培养基继续培养2小时。细胞经快速洗涤后速冻。提取极性代谢物，并通过气相色谱-质谱联用（GCMS）分析，以确定丝氨酸和其他代谢物的同位素富集度（M+3比例）。 [1]
进行增殖实验以评估生长抑制作用。将细胞接种在含丝氨酸的培养基中。第二天，更换为含有 CBR-5884 （通常为15和30 μM）或对照的新鲜丝氨酸充足或丝氨酸缺乏的培养基。细胞培养3-5天，每天更换培养基/药物，最后量化相对细胞数量。 [1]
进行急性毒性实验，以排除短期代谢示踪处理中存在的普遍细胞毒性。用 CBR-5884 （1至40 μM）处理细胞3小时。然后更换为不含药物的培养基，并使用基于发光或荧光的活力测定法评估细胞活力。 [1]

The study utilized 12 female BALB/c nude mice aged 4–6 weeks, procured from the Experimental Animal Center of Southern Medical University. These mice were housed in autoclaved, ventilated cages and provided with autoclaved water. All mice used were bred in the specific pathogen free laboratory. The mice received a subcutaneous injection of 1 × 106 ID8 cells in the right armpit. When the tumors reached an average volume of 65 mm3, the mice were randomly divided into two groups using the random number method: (1) an experimental group, wherein the mice received intragastric administration of CBR-5884 (70 mg/kg, qd; n = 6); and (2) a control group, wherein the mice received intragastric administration of an equal volume of vehicle (corn oil) (n = 6). CBR-5884 treatment lasted for 12 consecutive days, with continuous drug administration. Tumors were resected when the average tumor volume in the control group reached 400 mm3. Tumor growth was tracked using caliper measurements, and tumor volume was calculated using the formula: length × width2/2. Subsequently, images of mice and tumors were captured, and tumors were weighed. Liver, spleen, kidney, and tumors were subjected to histological analysis after hematoxylin–eosin (H&E) staining. The maximal tumor size/burden permitted by our institutional review board is 10% of body weight and mean tumor diameter = or > 15 mm in adult mice (~ 25 g). The maximal tumor size/burden permitted by our institutional review board was not exceeded.[2]

CBR-5884 is an ethyl ester prodrug. Its corresponding carboxylic acid derivative retains similar in vitro enzymatic inhibitory activity and selectivity against PHGDH, suggesting intracellular hydrolysis by esterases may not be required for activity, but the acid derivative showed reduced cellular activity likely due to poor membrane permeability. [1]
CBR-5884 was found to be unstable in mouse plasma. [1]

In acute (3-hour) treatment assays, CBR-5884 at concentrations up to 40 μM did not cause significant cytotoxicity in Carney cells as measured by two independent viability assays. [1]
In longer-term proliferation assays (3-5 days), CBR-5884 at 30 μM significantly inhibited the growth of PHGDH-dependent cancer cell lines, indicating a cytostatic or cytotoxic effect under conditions of serine synthesis dependence. [1]

[1]. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers Proc Natl Acad Sci U S A, 2016 Feb 16, 113(7):1778-83.

Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial–mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC[2].

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CBR-5884 is a tool compound identified from a high-throughput screen of 800,000 compounds. It represents a proof-of-concept for pharmacological inhibition of PHGDH. [1]
The compound is proposed to be more of a starting point for medicinal chemistry optimization rather than a direct drug candidate, due to its plasma instability and need for improved cellular potency. [1]
The mechanism of action involves noncompetitive, time-dependent inhibition of PHGDH and disruption of its active tetrameric form. [1]
PHGDH is a metabolic enzyme overexpressed in certain cancers (e.g., breast cancer, melanoma, non-small cell lung cancer) and is essential for de novo serine synthesis. Inhibiting PHGDH is a proposed strategy to target cancers addicted to this pathway. [1]
The selectivity of CBR-5884 was assessed against a panel of other NAD(P)+-dependent dehydrogenases (e.g., LDH, MDH1, IDH1, 3α-HSD), where it showed at least 4-fold selectivity for PHGDH. [1]

C14H12N2O4S2

336.386

336.023

C, 49.99; H, 3.60; N, 8.33; O, 19.02; S, 19.06

681159-27-3

4674993

Typically exists as light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

378.1±42.0 °C at 760 mmHg

182.5±27.9 °C

0.0±0.9 mmHg at 25°C

1.628

4.15

146Ų

1

7

6

22

479

0

O=C(C1=C(C)C(SC#N)=C(NC(C2=CC=CO2)=O)S1)OCC

QBVIRPJBDIZKBC-UHFFFAOYSA-N

InChI=1S/C14H12N2O4S2/c1-3-19-14(18)11-8(2)10(21-7-15)13(22-11)16-12(17)9-5-4-6-20-9/h4-6H,3H2,1-2H3,(H,16,17)

ethyl 5-(furan-2-carboxamido)-3-methyl-4-thiocyanatothiophene-2-carboxylate

CBR-5884; CBR 5884; CBR5884.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~148.64 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.18 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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