GMX1778 (CHS828) is a potent and selective inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD⁺ salvage biosynthesis pathway. It exhibits an IC50 of 9 nM against recombinant human NAMPT in an enzyme activity assay. It does not inhibit nicotinic acid phosphoribosyltransferase 1 (NAPRT1), another NAD⁺ biosynthetic enzyme, at concentrations up to 10 μM [1]

CHS-828 (GMX1778) 对重组 NAMPT 的磷酸核糖基转移酶活性抑制阈值小于 25 nM，但对重组 NMNAT1 的腺苷酸转移酶活性则不然。使用荧光标签 (CHS-828 (GMX1778)-Alexa Fluor)，GMX1778 的重组 NAMPT 的 Kd 为 120 nM。为了应对 3 nM GMX1778 攻击，野生型 NAMPT 的过度表达能够维持一定水平的 NAD+；然而，当细胞接受 300 nM CHS-828 (GMX1778) 处理时，这种效应被消除。通过增加超氧化物和减少细胞内 NAD(+)，CHS-828 (GMX1778) 可以提高癌细胞中的细胞内 ROS。有趣的是，CHS-828 (GMX1778) 处理后正常细胞不会产生 ROS。烟酸 (NA) 可以通过依赖 NA 磷酸核糖基转移酶 1 (NAPRT1) 的方式减少 CHS-828 (GMX1778) 产生的 ROS[2]。

---

肿瘤细胞抗增殖活性：GMX1778（0.1-1000 nM）对多种人肿瘤细胞系呈剂量依赖性抗增殖作用，在NAPRT1缺陷细胞中IC50显著更低：A2780（卵巢癌，NAPRT1⁻）IC50=12 nM，而SKOV3（卵巢癌，NAPRT1⁺）IC50=280 nM；HCT116 p53⁺/⁺（结直肠癌，NAPRT1⁻）IC50=15 nM，而HCT116 p53⁻/⁻（NAPRT1⁺）IC50=320 nM（SRB法，处理72小时）[1]
- NAD⁺与ATP耗竭：A2780细胞经GMX1778（20 nM）处理24小时，细胞内NAD⁺水平较溶媒对照组降低85%（循环酶法），ATP水平降低70%（荧光素酶ATP检测法）；SKOV3细胞经200 nM GMX1778处理无显著NAD⁺耗竭[1]
- 凋亡诱导：GMX1778（20 nM）处理A2780细胞48小时，凋亡率从3%（溶媒）升至45%（Annexin V-FITC/PI染色）；Western blot显示活化型caspase-3增加3.5倍，活化型PARP增加2.8倍[1]
- p53和NAPRT1依赖的ROS介导细胞毒性：HCT116 p53⁺/⁻细胞中，GMX1778（50 nM）使p53⁺/⁺细胞内ROS水平增加2.3倍（DCFH-DA染色），而p53⁻/⁻细胞仅增加0.5倍；ROS清除剂N-乙酰半胱氨酸（NAC）可逆转GMX1778对p53⁺/⁺细胞的毒性（活力从35%升至82%），但对p53⁻/⁻细胞无作用[2]
- NAPRT1依赖的耐药性：向A2780（NAPRT1⁻）细胞转染NAPRT1 cDNA后，GMX1778的IC50从12 nM升至250 nM，证实NAPRT1表达可产生耐药[2]

在 NAPRT1 缺陷的异种移植试验中，以 150 mg/kg 或 650 mg/kg 的剂量 24 小时静脉输注 CHS-828 (GMX1778) 不会对 4 小时静脉输注 NA 的抗肿瘤功效产生负面影响（120毫克/公斤体重）。在 650 mg/kg 时，CHS-828 (GMX1778) 超过了可耐受的最高剂量。用 750 mg/kg CHS-828 (GMX1778) 治疗后，4 小时静脉输注 NA 降低了与 GMX1777 危险水平相关的死亡率[2]。

---

裸鼠A2780（NAPRT1⁻）卵巢癌异种移植模型：6-8周龄雌性BALB/c nu/nu裸鼠皮下注射5×10⁶个A2780细胞，待肿瘤达100 mm³时随机分为3组（每组n=6）：
1. 溶媒组：腹腔注射5% DMSO+5% Tween 80+生理盐水（10 mL/kg/天）；
2. GMX1778 10 mg/kg组：腹腔注射10 mg/kg/天GMX1778（溶于溶媒）；
3. GMX1778 20 mg/kg组：腹腔注射20 mg/kg/天GMX1778（溶于溶媒）。
处理21天后，GMX1778使肿瘤体积减少58%（10 mg/kg）和75%（20 mg/kg）；肿瘤组织分析显示，20 mg/kg组NAD⁺水平降低65%，ATP水平降低55%[1]
- 裸鼠SKOV3（NAPRT1⁺）卵巢癌异种移植模型：携带SKOV3肿瘤的小鼠经GMX1778（20 mg/kg/天，腹腔注射）处理21天，无显著肿瘤抑制（体积减少<15%），证实NAPRT1表达限制GMX1778疗效[1]

重组人NAMPT活性抑制实验：50 μL反应体系包含50 mM Tris-HCl（pH 7.5）、5 mM MgCl₂、2 mM烟酰胺、1 mM 5-磷酸核糖-1-焦磷酸（PRPP）、5 μg重组人NAMPT及GMX1778（0.1-100 nM）。37°C启动反应并孵育60分钟，加入50 μL 10%三氯乙酸（TCA）终止反应。产物烟酰胺单核苷酸（NMN）通过循环酶法（偶联NMN腺苷酰转移酶和荧光素酶）检测， luminometer测发光强度。以溶媒对照组为基准计算抑制率，非线性回归（四参数逻辑模型）推导IC50[1]
- NAPRT1活性实验：实验方案与NAMPT实验类似，差异在于用重组人NAPRT1（5 μg）和烟酸（2 mM）替代NAMPT和烟酰胺。GMX1778浓度高达10 μM时，对NAPRT1活性的抑制率<5%，证实其选择性[1]

肿瘤细胞增殖实验（SRB法）：A2780、SKOV3、HCT116 p53⁺/⁺和HCT116 p53⁻/⁻细胞以3×10³细胞/孔接种于96孔板，含10% FBS的RPMI 1640培养24小时。加入GMX1778（0.1-1000 nM）孵育72小时，10% TCA固定细胞，0.4%磺酰罗丹明B（SRB）染色，1%乙酸洗去 excess染料，结合的染料用10 mM Tris碱溶解，检测515 nm吸光度以计算细胞活力和IC50[1,2]
- 细胞内NAD⁺检测：A2780细胞（2×10⁵细胞/孔，6孔板）经GMX1778（20 nM）处理6、12、24小时，0.5 M高氯酸裂解细胞，2 M KOH中和裂解液。采用上述循环酶法检测NAD⁺水平，并以蛋白浓度（BCA法）归一化[1]
- 凋亡检测（Annexin V-FITC/PI染色）：A2780细胞（1×10⁵细胞/孔）经GMX1778（20 nM）处理48小时，收集细胞并冷PBS洗涤，Annexin V-FITC和PI室温染色15分钟，流式细胞术分析，凋亡率以Annexin V阳性细胞百分比计算[1]
- ROS检测（DCFH-DA染色）：HCT116 p53⁺/⁺和p53⁻/⁻细胞（2×10⁵细胞/孔）经GMX1778（50 nM）处理24小时，37°C下10 μM DCFH-DA（ROS探针）负载30分钟，PBS洗涤后流式细胞术检测荧光强度（激发488 nm，发射525 nm）[2]
- 凋亡相关蛋白Western blot：HCT116 p53⁺/⁺细胞（1×10⁶细胞/10 cm培养皿）经GMX1778（50 nM）处理48小时，含蛋白酶抑制剂的RIPA缓冲液裂解细胞，30 μg蛋白经10% SDS-PAGE电泳后转移至PVDF膜，一抗孵育活化型caspase-3、活化型PARP及β-肌动蛋白，HRP标记二抗结合后ECL显色[2]

Dissolved in 2% carboxymethyl cellulose in 0.9% saline; 250 mg/kg; oral administration
Nude mice bearing midgut carcinoid (GOT1), pancreatic carcinoid (BON) and medullary thyroid cancer (GOT2) tumors

---

Nude Mouse A2780 Xenograft Model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food/water). Mice were subcutaneously injected with 5×10⁶ A2780 cells (suspended in 100 μL PBS + 50 μL Matrigel) into the right flank. When tumors reached 100 mm³, mice were randomized into 3 groups (n=6/group):
1. Vehicle group: Intraperitoneal injection of 5% DMSO + 5% Tween 80 + sterile saline (10 mL/kg) once daily for 21 days;
2. 10 mg/kg GMX1778 group: Intraperitoneal injection of GMX1778 (10 mg/kg, dissolved in vehicle) once daily for 21 days;
3. 20 mg/kg GMX1778 group: Intraperitoneal injection of GMX1778 (20 mg/kg, dissolved in vehicle) once daily for 21 days.
Tumor volume was measured every 3 days (volume = length × width² / 2). On day 21, mice were euthanized with CO₂, tumors were harvested for NAD⁺ and ATP detection, and major organs (liver, kidney) were collected for histopathology [1]
- Nude Mouse SKOV3 Xenograft Model: The protocol was identical to the A2780 model, except 5×10⁶ SKOV3 cells were used, and mice were treated with GMX1778 (20 mg/kg/day, intraperitoneal) for 21 days. Tumor volume and weight were measured, and no significant inhibition was observed [1]

Acute in vitro toxicity: GMX1778 (0.1-100 nM) treatment for 72 hours in normal human fibroblasts (NHF) showed no significant cytotoxicity—cell viability remained >90% (SRB assay), indicating selectivity for tumor cells [1]
- In vivo acute toxicity: Nude mice treated with GMX1778 (10-20 mg/kg/day, intraperitoneal) for 21 days showed no abnormal behavior (e.g., lethargy, diarrhea), weight loss (<5% of baseline), or changes in serum ALT/AST/BUN/creatinine levels. Histopathological examination of liver, kidney, spleen, and lung showed no tissue damage [1]

[1]. The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors. Mol Cell Biol. 2009 Nov;29(21):5872-88.

[2]. Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner. J Biol Chem. 2012 Jun 22;287(26):22408-17.

2-[6-(4-chlorophenoxy)hexyl]-1-cyano-3-pyridin-4-ylguanidine is an aromatic ether.
CHS-828 has been used in trials studying the treatment of Unspecified Adult Solid Tumor, Protocol Specific.
Pyridyl Cyanoguanidine CHS 828 is a pyridyl cyanoguanidine that exhibits antitumor activity by an unknown mechanism. (NCI)

---

Mechanism of action: GMX1778 (CHS828) inhibits NAMPT to block the NAD⁺ salvage pathway, leading to intracellular NAD⁺ depletion. Reduced NAD⁺ levels impair ATP production (via mitochondrial respiration) and activate apoptotic pathways (via PARP activation and caspase cleavage). Its efficacy is dependent on NAPRT1 deficiency (since NAPRT1-positive cells can use the de novo NAD⁺ pathway to bypass NAMPT inhibition) [1]
- p53-dependent ROS amplification: Literature [2]补充 that GMX1778-induced NAD⁺ depletion increases ROS levels, and p53 enhances this effect by upregulating pro-oxidant genes (e.g., NOX2) and downregulating antioxidant genes (e.g., SOD2). This ROS amplification further promotes cytotoxicity in p53 wild-type, NAPRT1-deficient tumors [2]
- Therapeutic potential: GMX1778 is a preclinical candidate for NAPRT1-deficient tumors (e.g., ovarian, colorectal cancer), as these tumors are dependent on the NAMPT-mediated NAD⁺ salvage pathway. It has not been evaluated in clinical trials or approved by the FDA [1,2]
- Resistance mechanism: NAPRT1 expression is the primary resistance mechanism to GMX1778, as it enables cells to synthesize NAD⁺ via the de novo pathway using nicotinic acid. Combining GMX1778 with NAPRT1 inhibitors may overcome this resistance [1,2]

C19H22CLN5O

371.86

371.151

200484-11-3

1160589-73-0 (nicotinate);200484-11-3 (free);

148198

White to off-white solid powder

1.2±0.1 g/cm3

526.9±58.0 °C at 760 mmHg

272.4±32.3 °C

0.0±1.4 mmHg at 25°C

1.587

4.95

82.33

2

4

11

26

450

0

BOIPLTNGIAPDBY-UHFFFAOYSA-N

InChI=1S/C19H22ClN5O/c20-16-5-7-18(8-6-16)26-14-4-2-1-3-11-23-19(24-15-21)25-17-9-12-22-13-10-17/h5-10,12-13H,1-4,11,14H2,(H2,22,23,24,25)

2-[6-(4-chlorophenoxy)hexyl]-1-cyano-3-pyridin-4-ylguanidine

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.72 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 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。