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

78c increases NAD+ levels through inhibition of CD38 NADase activity. 78c is a potent, reversible, and uncompetitive inhibitor of CD38. 78c is a specific inhibitor of CD38 and does not directly affect the activity or expression of other enzymes involved in NAD+ metabolism.[2]

NAD levels in liver and muscle are markedly elevated by oral CD38 inhibitors (30 mg/kg; 2 hours and 6 hours) [1].

Biochemical Assay Details for the pIC50 Determinations against the Human and Mouse CD38 Enzymes[1]
CD38 inhibitors were tested for their capacity to inhibit human CD38 enzyme activity in a colorimetric-based assay. The extracellular domain of human CD38 was expressed in Pichia pastoris and purified to homogeneity. The enzyme activity assay was performed in a low-volume 384-well plate in a total volume of 20 μL. A range of concentrations of test compound in 200 nL of DMSO was delivered into the assay plate wells. Columns 6 and 18 of the plate contained DMSO with no compound and served as the high signal and low signal controls (no CD38 added), respectively. All additions of assay reagents to the plate were done using a Multidrop Combi, and the plate was shaken 3–5 s after each addition. CD38 (0.8 nM) was incubated with test compound in 10 μL containing 100 mM HEPES, pH 7.4, 4 mM EDTA, and 1 mM CHAPS for 30 min prior to initiation of the reaction. The reaction was initiated by a 10 μL addition containing 5 mM sodium acetate, pH 4.5, 1 mM CHAPS, 200 μM NAD, and 500 μM GW323424X. The solutions for each of the two additions were prepared fresh each day from concentrated stocks of the individual components. The final concentrations in the assay were 50 mM HEPES, 2 mM EDTA, 1 mM CHAPS, and 2.5 mM sodium acetate, 100 μM NAD, 250 μM GW323434X, and 0.4 nM CD38. GW323434X is a 4-pyridynal compound that acts as a nucleophile that participates in the base exchange reaction with the nicotinamide on NAD to form a novel dinucleotide that absorbs at 405 nm. Catalytic formation of this novel chromophore was followed in an Envision microplate reader by reading absorbance at two time points, typically 30 min apart within the first 45 min of the reaction. These time points were established empirically to ensure the rates determined were in a linear range of product formation. Data analysis was performed in the following way using ActivityBase XE (Abase XE). The data from the 15 and 45 min reads was processed by performing a subtraction function of 45 min read value minus 15 min read value for each plate well. The resulting values for noncontrol wells were converted to % inhibition using the formula 100 × ((U – C1)/(C2 – C1)), where U is the value of the test well, C1 is the average of the values of the high signal (column 6) control wells, and C2 is the average of the values of the low signal (column 18) control wells. Percent inhibition (y) was plotted versus inhibitor concentration (x), and curve fitting was performed with the following four parameter equation: y = A + ((B – A)/(1 + (10x/10C)D)), where A is the minimum response, B is the maximum response, C is the log10IC50, and D is the Hill slope. The results for each compound were recorded as pIC50 values (−C in the above equation). For the data presented in this manuscript, the pIC50 values were converted to molar IC50 values according to the equation IC50 = 10–pIC50. Statistics were performed on the IC50 values.[1]
The recombinant extracellular domain of mouse CD38 was expressed in CHO CGE cells and purified to homogeneity. The pIC50 values for the inhibitors against mouse CD38 were generated using the enzyme in a fluorescence-based assay in which the enzyme reaction occurred in a 10 μL volume in a low-volume 384-well assay plate. The assay quantitated CD38 catalyzed NAD hydrolysis over 45 min of reaction time in which the rate was linear. A range of concentrations of test compound in 100 nL of DMSO was delivered into the assay plate wells. Columns 6 and 18 of the plate contained DMSO and served as the low signal and high signal controls, respectively. Column 18 contained a potent mouse CD38 inhibitor to define the high signal (no enzyme activity) control. Additions to the plate other than compound were done using a Multidrop Combi, and the plate was shaken 3–5 s after each addition. CD38 (0.45 nM) was incubated with test compound in 5 μL containing 20 mM HEPES, pH 7.2, 1 mM EDTA, and 1 mM CHAPS for 30 min prior to initiation of the reaction. The reaction was initiated by a 5 μL addition containing 20 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM CHAPS, and 60 μM NAD. The final concentrations in the assay were 20 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM CHAPS, 30 μM NAD, and 0.225 nM mouse CD38. After the reaction time, the amount of NAD remaining was quatitated by converting it to NADH using alcohol dehydrogenase (ADH). The ADH was added in 5 μL containing 9U/mL ADH, 90 mM sodium pyrophosphate, pH 8.8, 90 mM ethanol, 1 mM EDTA, and 1 mM CHAPS. The alcohol dehydrogenase reaction was stopped by the addition of 5 μL of 1 M HEPES, pH 7.0, 1.0 mM EDTA, and 1 mM CHAPS containing 0.8 M dithiothreitol (DTT), and the NADH fluorescence was measured in an Envision plate reader (340 nm excitation, 460 nm emission). The solutions for each of the four additions were prepared fresh each day from concentrated stocks of the individual components, except the DTT which was prepared fresh daily from solid. In this assay, an increase in enzyme activity results in a decreased measured fluorescent signal. Each compound plate was run in duplicate with (plate A) and without (plate B) ADH. Data were acquired by reading plates in pairs and subtracting the values for plate B from plate A to obtain “corrected” data (accounts for intrinsic fluorescence from test compound). Using Abase XE, “corrected” fluorescence signals for noncontrol wells were converted to percent inhibition values using the formula 100 – 100 × ((U – C2)/(C1 – C2)), where U is the “corrected” fluorescence signal value of the test well, C1 is the average of the “corrected” fluorescence values of the low signal (column 6; full CD38 enzyme activity) control wells, and C2 is the average of the “corrected” fluorescence values of the high signal (column 18; 100% inhibited CD38 enzyme activity) control wells. Percent inhibition data were fit using the four-parameter curve fit equation described above. For the data presented in this manuscript, the pIC50 values were converted to molar IC50 values according to the equation IC50 = 10–pIC50. Statistics were performed on the IC50 values.

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Assay Details for Ki Determinations with the Human Wild-Type CD38 Enzyme[1]
The CD38-catalyzed hydrolysis of NAD resulted in a decrease in absorbance at 280 nm (using Δε280 = −1.2 mM–1 cm–1 for NAD). The fully glycosylated human recombinant enzyme used for Ki determination was purchased from R&D Systems. The enzyme was diluted 1:500 into standard buffer (Hepes (K+) pH 7.0), and the reaction was initiated using 100 μM NAD. Progress curves were fitted using a mixed inhibition model to determine Kis for individual compounds.

HEK293T were treated with 0.5 μM 78c, 5 μM olaparib (LC Laboratories), 5 μM EX-527 (Tocris), or 5 μM nicotinamide for 24 hours. MEFs were treated with 0.2 μM 78c for 24 hours. A549 cells were treated for 24 hours with vehicle (DMSO), 78c (0.2–0.5 μM) or 5 μM olaparib. A549 and 293T cells were treated with drugs in media containing 0.5% FBS and MEFs were treated in media containing 10% FBS. To test the reversibility of the CD38i, A549 cells were treated with vehicle or 0.5 μM 78c for 16 hours. Then, cells were washed and incubated with (78c) or without (78c+release) 78c for another 8 hours. Control cells were left in vehicle for the whole treatment period. After treatment, cell lysates were prepared for measurements of CD38 activity. For the co-culture experiment, AML12 cells were plated in the lower chamber and HEK293T or Jurkat T cells were plated in the upper chamber. Transfections with CD38 plasmid were done as described above. 0.5–1 μM 78c was added to the upper chamber 4 hours before addition of 100 μM NMN. 4 hours later, both chambers were incubated together for an additional 20 hours. Then AML12 cells were collected for NAD+ measurements. In experiments where AML12 cells were incubated with recombinant CD38 in the cell culture media, the first step involved incubation of the recombinant protein (100ng/mL) with 1 μM 78c for 30 minutes at 37 °C in cell culture media containing 1% FBS. After 30 minutes, the recombinant protein-78c mixture was added to the cells and 100μM NMN was added. After 18 hours, AML12 cells were collected for NAD+ measurements or 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (24.18 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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Solubility in Formulation 5: 10 mg/mL (24.18 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)