CD38 inhibitor 78c   中文名称: CD38抑制剂 1

humanCD38 (IC50 = 7.3 nM; mouse CD38 (IC50 = 1.9 nM); WT hCD38 (Ki = 0.3 nM)[1]

78c通过抑制CD38 NADase活性增加NAD+水平。 78c是一种有效的、可逆的、非竞争性的CD38抑制剂。 78c是CD38的特异性抑制剂，不直接影响参与NAD+代谢的其他酶的活性或表达[2]。

口服 CD38 抑制剂（30 mg/kg；2 小时和 6 小时）可显着提高肝脏和肌肉中的 NAD 水平[1]。

pIC50测定人和小鼠CD38酶的生化分析细节[1]
CD38抑制剂通过比色法检测其抑制人CD38酶活性的能力。人CD38的细胞外结构域在毕赤酵母中表达并纯化至均匀性。酶活性测定在总容积为20 μL的384孔板上进行。将200 nL DMSO中一定浓度的试验化合物送入测定板孔。板的第6列和第18列含有不含化合物的DMSO，分别作为高信号和低信号对照（未添加CD38）。使用Multidrop Combi向板中添加所有检测试剂，每次添加后摇板3-5 s。反应开始前，CD38 （0.8 nM）与10 μL含100 mM HEPES、pH 7.4、4 mM EDTA、1 mM CHAPS的化合物孵育30 min。在10 μL的溶液中加入5 mM的乙酸钠、pH为4.5、1 mM的CHAPS、200 μM的NAD和500 μM的GW323424X，引发反应。两种添加物的溶液每天从单个成分的浓缩库存中新鲜制备。实验最终浓度为50 mM HEPES， 2 mM EDTA, 1 mM CHAPS， 2.5 mM乙酸钠，100 μM NAD, 250 μM GW323434X, 0.4 nM CD38。GW323434X是一种4-吡啶基化合物，作为亲核试剂参与NAD上烟酰胺的碱交换反应，形成一种新的二核苷酸，在405 nm处吸收。这种新型发色团的催化形成在Envision微孔板阅读器上通过读取两个时间点的吸光度来跟踪，通常在反应的前45分钟内相隔30分钟。这些时间点是根据经验建立的，以确保确定的速率在产品形成的线性范围内。使用ActivityBase XE （Abase XE）以以下方式执行数据分析。15和45分钟读取的数据通过对每个板孔进行45分钟读取值减去15分钟读取值的减法处理。使用公式100 × (（U - C1)/(C2 - C1)）将非控制井的结果值转换为抑制率%，其中U为测试井的值，C1为高信号（第6列）控制井的值的平均值，C2为低信号（第18列）控制井的值的平均值。绘制抑制百分比(y)与抑制剂浓度(x)的关系曲线，并使用以下四个参数方程进行曲线拟合：y = A + ((B - A)/(1 + (10x/10C)D))，其中A为最小响应，B为最大响应，C为log10IC50， D为Hill斜率。每种化合物的结果记录为pIC50值（上式中的−C）。对于本文中的数据，将pIC50值换算为摩尔IC50值，公式为IC50 = 10-pIC50。对IC50值进行统计[1]。
重组小鼠CD38胞外结构域在CHO - CGE细胞中表达并纯化至均匀性。利用酶在低体积384孔检测板上以10 μL的体积进行酶反应，用荧光法测定小鼠CD38抑制剂的pIC50值。该实验定量测定了CD38在45分钟的反应时间内催化NAD水解的速率是线性的。将100nl DMSO中一定浓度的试验化合物送入测定板孔。平板的第6列和第18列含有DMSO，分别作为低信号和高信号对照。柱18含有一种有效的小鼠CD38抑制剂来定义高信号（无酶活性）控制。使用Multidrop Combi向培养皿中添加化合物以外的其他成分，每次添加后摇晃培养皿3-5 s。反应开始前，CD38 （0.45 nM）与5 μL含20 mM HEPES、pH 7.2、1 mM EDTA和1 mM CHAPS的化合物孵育30 min。在5 μL的溶液中加入20 mM HEPES、pH 7.2、1 mM EDTA、1 mM CHAPS和60 μM NAD，引发反应。实验最终浓度为HEPES 20 mM， pH 7.2, EDTA 1 mM, CHAPS 1 mM, NAD 30 μM，小鼠CD38 0.225 nM。反应时间结束后，用乙醇脱氢酶（ADH）将NAD转化为NADH，定量测定NAD的残留量。ADH在5 μL中加入9U/mL ADH、90 mM焦磷酸钠、pH 8.8、90 mM乙醇、1 mM EDTA和1 mM CHAPS。加入5 μL 1 M HEPES， pH 7.0, 1.0 mM EDTA和1 mM含0.8 M二硫代糖醇（DTT）的CHAPS，阻断乙醇脱氢酶反应，在Envision平板读取仪（激发340 nm，发射460 nm）上测量NADH荧光。四种添加物的溶液都是每天从单个成分的浓缩原液中新鲜制备的，DTT是每天从新鲜原液中制备的。

HEK293T分别用0.5 μM 78c、5 μM olaparib （LC Laboratories）、5 μM EX-527 （Tocris）或5 μM烟酰胺处理24小时。MEFs用0.2 μM 78c处理24小时。A549细胞分别用DMSO、78c （0.2 ~ 0.5 μM）或5 μM奥拉帕尼处理24小时。A549和293T细胞在含0.5% FBS的培养基中用药物处理，mef在含10% FBS的培养基中处理。为了检测CD38i的可逆性，A549细胞用载药或0.5 μM 78c处理16小时。然后将细胞洗净，加（78c）或不加（78c+release） 78c再孵育8小时。对照细胞在整个处理期间放置在载具中。处理后，制备细胞裂解液用于测量CD38活性。共培养实验中，AML12细胞下腔镀，HEK293T或Jurkat T细胞上腔镀。转染CD38质粒的方法如上所述。在加入100 μM NMN前4小时向上腔中加入0.5-1 μM 78c。4小时后，两个腔室一起再孵育20小时。然后收集AML12细胞进行NAD+检测。在AML12细胞与重组CD38在细胞培养基中孵育的实验中，第一步是将重组蛋白（100ng/mL）与1 μM 78c在含1% FBS的细胞培养基中，37°C孵育30分钟。30分钟后，将重组蛋白-78c混合物加入细胞中，加入100μM NMN。18小时后，收集AML12细胞进行NAD+检测或Western blot。[2]

Animal/Disease Models: Diet-induced obesity (DIO) C57Bl6 mice [1]
Doses: 30 mg/kg
Route of Administration: Orally once.
Experimental Results: NAD levels in liver and muscle were Dramatically increased at both the 2-hour and 6-hour time points.

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CD38 inhibitor (78c) 78c was administered to C57BL/6 (3, 12, and 22 to 26 months old) and ICR (1-year-old) mice by intraperitoneal injection (i.p., 10 mg/kg/dose) twice daily over a period of 4 to 14 weeks. 3-month-old, 1-year-old and 2-year-old mice were treated with 78c for up to 14 weeks. Combination of 78c and FK866 was performed for 10 weeks. P44+/+ progeroid mice due to their accelerated aging were treated with 78c for 4 weeks. For the short treatment, mice received a 15 mg/kg/dose twice daily for 8 days. Control mice received vehicle (5% DMSO, 15% PEG400, 80% of 15% hydroxypropyl-γ-cyclodextrin (in citrate buffer pH 6.0)) injections. We also measured the concentration of 78c in multiple tissues and plasma. Samples and standards were extracted by protein precipitation with acetonitrile containing internal standards. The supernatant was diluted with 0.1% formic acid in water before injection into an HPLC-MS/MS system for separation and quantitation. The analytes were separated from matrix components using reverse phase chromatography on a 30x2.1 mm 5 μm Fortis Pace C18 using gradient elution at a flow rate of 0.8 mL/min. The tandem mass spectrometry analysis was carried out on SCIEX™ triple quadrupole mass spectrometer with an electrospray ionization interface, in positive ion mode. Data acquisition and evaluation were performed using Analyst® software (SCIEX™). The results of the measurements were: plasma (0.007 μg/mL); brain (0.000 μg/g); heart (0.003 μg/g); kidney (0.005 μg/g); liver (0.024 μg/g); pancreas (0.002 μg/g) and, spleen (0.0048 μg/g). We also observed that 78c could be detected in cellular extracts of cultured cells treated with different concentration of 78c.[2]

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NAMPT inhibitor: Aged C57BL/6 mice received FK866 (25 mg/kg/dose, i.p., once daily), 78c (10 mg/kg/dose, i.p., twice daily), or a combination of FK866 and 78c (same doses) for 10 weeks. Control mice received equivalent injections of vehicle for FK866 (1% Hydroxypropyl-β-cyclodextrin, 12% Propylene glycol) and vehicle for 78c (5% DMSO, 15% PEG400, 80% of 15% hydroxypropyl-γ-cyclodextrin (in citrate buffer pH 6.0)), a group was treated with 78c, and one group was treated with a combination of 78c (10 mg/kg/dose, i.p twice daily) and FK866 (25 mg/kg/dose, i.p once daily).

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NAD+ precursors: For the treatment with nicotinamide mononucleotide (NMN), C57BL/6 mice received a single dose of NMN (500 mg/kg) or vehicle (PBS) by gavage. Mice were sacrificed after 2 hours, and tissues harvested. For the study with nicotinamide riboside (NR), aged C57BL/6 mice were pretreated with a single dose of 78c (10 mg/kg, i.p). Sixteen hours later, they received NR (100 mg/kg) by gavage and a second injection of 78c (10 mg/kg). Blood was collected just prior to administration of NR, and 30 min, 1 hour, 2 hours, and 6 hours later. Control mice received NR alone (200 mg/kg), 78c alone (10 mg/kg/dose, 2 doses), or vehicle (NR=PBS; 78c=5% DMSO, 15% PEG400, 80% hydroxypropyl-γ-cyclodextrin). The mice were sacrificed 6 hours after administration of 78c and NR, and tissues were collected.[2]

[1]. Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors. J Med Chem. 2015 Apr 23;58(8):3548-71.

[2]. A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline. Cell Metab. 2018;27(5):1081-1095.e10.

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.[1]

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Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.[2]

C22H27N3O3S

413.5331

413.18

C, 63.90; H, 6.58; N, 10.16; O, 11.61; S, 7.75

1700637-55-3

1700637-55-3;MDK-7553 HCl;

118736856

Light yellow to brown solid powder

3

91.9

1

6

7

29

594

0

VJQALSOBHVEJQM-QAQDUYKDSA-N

InChI=1S/C22H27N3O3S/c1-25-20-8-3-15(21-13-23-14-29-21)11-18(20)19(12-22(25)26)24-16-4-6-17(7-5-16)28-10-9-27-2/h3,8,11-14,16-17,24H,4-7,9-10H2,1-2H3/t16-,17-

4-(((1r,4r)-4-(2-methoxyethoxy)cyclohexyl)amino)-1-methyl-6-(thiazol-5-yl)quinolin-2(1H)-one

CD38 inhibitor 78c; Compound-78c; CD38 inhibitor 1; 1700637-55-3; CD38-IN-78c; CHEMBL3426034; 4-((trans-4-(2-Methoxyethoxy)cyclohexyl)amino)-1-methyl-6-(thiazol-5-yl)quinolin-2(1H)-one; 4-[[trans-4-(2-Methoxyethoxy)cyclohexyl]amino]-1-methyl-6-(5-thiazolyl)-2(1H)-quinolinone; 4-(((1r,4r)-4-(2-Methoxyethoxy)cyclohexyl)amino)-1-methyl-6-(thiazol-5-yl)quinolin-2(1H)-one; compound 78c; CD38i_78c; 78c

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 : ~25 mg/mL (~60.46 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.05 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (6.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
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 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.05 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 10 mg/mL (24.18 mM) in Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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配方 5 中的溶解度: 10 mg/mL (24.18 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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。