用激活 NAMPT 的小分子增强 NAD +


Boosting NAD+ with a small molecule that activates NAMPT



Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a “super catalyst” that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects.

促进细胞内 NAD + 的药理学策略因其治疗潜力而备受关注。一种方法是激活烟酰胺磷酸核糖基转移酶(NAMPT)以增加哺乳动物细胞中主要的 NAD + 前体——烟酰胺单核苷酸(NMN)的产生。高通量筛选的 NAMPT 激活剂和点击引导活动产生了 SBI-797812,一种结构类似于活性位点定向的 NAMPT 抑制剂的化合物,并阻断了这些抑制剂与 NAMPT 的结合。SBI-797812使 NAMPT 反应平衡向 NMN 生成转移,增加 NAMPT 对 ATP 的亲和力,稳定 His247磷酸化的 NAMPT,促进焦磷酸副产物的消耗,减弱 NAD + 的反馈抑制作用。SBI-797812的这些效应使 NAMPT 成为一种“超级催化剂” ,更有效地产生 NMN。SBI-797812处理培养细胞可增加细胞内 NMN 和 NAD + 。SBI-797812提高小鼠肝 NAD + 水平。小分子 NAMPT 激活剂如 SBI-797812是提高细胞内 NAD + 并实现其有益作用的开拓性方法。



NAD+ plays a vital role in diverse cellular processes that govern human health and disease1. The long-standing focus on NAD+ as a redox enzyme cofactor has been eclipsed by recent seminal discoveries establishing NAD+ as a co-substrate for sirtuins and poly-ADP-ribose polymerases (PARPs)2,3. These revelations have implicated NAD+ in additional cellular processes including cell signaling, DNA repair, cell division, and epigenetics. Elevated tissue levels of NAD+ were linked to salutary effects including healthy aging4. Thus, there is keen interest in pharmacological and nutraceutical strategies to boost intracellular NAD+ levels5,6.

NAD + 在控制人类健康和疾病的多种细胞过程中发挥着重要作用。长期以来人们对 NAD + 作为一种氧化还原酶辅助因子的关注已经被最近发现的 NAD + 作为去乙酰化酶和多聚 adp 核糖聚合酶(PARPs)2,3的共底物所取代。这些发现暗示 NAD + 参与了其他细胞过程,包括细胞信号传导、 DNA 修复、细胞分裂和表观遗传学。组织中 NAD + 水平升高与有益的影响有关,包括健康老化。因此,人们对提高细胞内 NAD + 水平的药理学和营养学策略非常感兴趣。

Enzymatic activities catalyzed by sirtuins and PARPs consume intracellular NAD+7. Hence, a cellular biosynthetic pathway to preserve the NAD+ level is imperative. In mammalian cells, the principle contributor to NAD+ synthesis is the nicotinamide (NAM) salvage pathway involving sequential actions of nicotinamide phosphoribosyltransferase (NAMPT) and NMN adenylyltransferases (NMNAT1-3)8. NAMPT forms NMN and pyrophosphate (PP) from NAM (generated by sirtuins and PARPs) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP). In turn, NMNAT1-3 produce NAD+ from NMN and ATP.

去乙酰化酶和 PARPs 催化的酶活力消耗细胞内 NAD + 7。因此,保持 NAD + 水平的细胞生物合成途径势在必行。在哺乳动物细胞中,NAD + 合成的主要促进因子是烟酰胺(NAM)补救通路,涉及烟酰胺磷酸核糖基转移酶(NAMPT)和 NMN 腺苷酰转移酶(NMNAT1-3)8的连续作用。NAMPT 由去乙酰化酶和 PARPs 产生的 NAM 和 α-d-5- 磷酸核糖 -1- 焦磷酸酯(PRPP)形成 NMN 和焦磷酸盐(PP)。反过来,NMNAT1-3从 NMN 和 ATP 生成 NAD + 。

NAMPT, a homodimeric type II phosphoribosyltransferase, is the putative rate-limiting step in the NAM salvage pathway9. The canonical NAMPT reaction scheme involves the following sequential steps: (1) ATP binding, NAMPT phosphorylation at His247 to form pHisNAMPT, followed by ADP release; (2) PRPP binding to pHisNAMPT, followed by NAM binding and (3) catalysis producing NMN and PP, followed by product release and regeneration of non-phosphorylated NAMPT10,11,12. The NAMPT protein structure with and without various ligands has been solved by X-ray crystallography10,13,14,15,16.

NAMPT,一个二聚体 II 型磷酸核糖转移酶,被认为是 NAM 挽救途径中的限速步骤。经典的 NAMPT 反应方案包括以下几个步骤: (1) ATP 结合,NAMPT 磷酸化在 His247形成 pHisNAMPT,其次是 ADP 释放; (2) PRPP 结合 pHisNAMPT,其次是 NAM 结合和(3)催化产生 NMN 和 PP,其次是非磷酸化 NAMPT10,11,12的产物释放和再生。用 x 射线晶体衍射方法分别求解了含有和不含配体的 NAMPT 蛋白的结构。

Our pursuit of a pharmacological approach to boost intracellular NAD+ levels focuses on discovering compounds that increase the activity of NAMPT. High-throughput screening (HTS) of a small molecule library using a protein thermal shift (PTS) assay17 yielded novel NAMPT ligands. Subsequent evaluation with a NAMPT activity assay identified a subset of HTS hits that increase NMN production. The ensuing medicinal chemistry campaign produced SBI-797812, our NAMPT activator prototype. Herein, we describe the mechanism of action (MOA) of SBI-797812 and test the ability of this small molecule to raise NMN and NAD+ in cultured cells and mice.

我们追求的药理学方法,以提高细胞内 NAD + 水平的重点是发现化合物,增加活性的 NAMPT。利用蛋白质热位移(PTS)分析技术,从一个小分子文库中分离得到了一种新型的 NAMPT 配体,这种新型的 NAMPT 配体可以用于分子生物学的研究。随后通过 NAMPT 活性分析进行评估,确定了提高 NMN 产量的 HTS 子集。随后的药物化学运动生产了 SBI-797812,我们的 NAMPT 催化剂原型。在此,我们阐述了 SBI-797812的作用机制,并在培养的细胞和小鼠中测试了该小分子提高 NMN 和 NAD + 的能力。



Discovery of a small molecule NAMPT activator

一种小分子 NAMPT 激活剂的发现

A chemical library (57,004 compounds) was screened for small molecules that bound to human NAMPT using a PTS assay (Fig. 1a). The negative control was DMSO-treated NAMPT. The positive control was NAMPT treated with 20 μM CHS-828 (Fig. 1b), a potent NAMPT inhibitor18. NAMPT ligands stabilized the enzyme against thermal denaturation (i.e., increased Tm) as detected by binding of Sypro orange fluorescent dye. Five hundred fifteen compounds (0.9%) were identified as NAMPT ligands. While the majority of the hits from the PTS assay were inhibitors or had no activity, 30 compounds (5.8%) were NAMPT activators as determined with NAMPT activity assays (NAD/NADH-Glo assay kit and NMN fluorometric assay19).

一个化学文库(57,004化合物)被筛选小分子结合到人的 NAMPT 使用 PTS 试验(图1a)。阴性对照采用 dmso 处理的 NAMPT。阳性对照采用20μmchs-828(图1b)处理的 NAMPT 作为有效的 NAMPT 抑制剂18。NAMPT 配体稳定酶的热变性(即增加 Tm)检测结合 Sypro 橙色荧光染料。鉴定了515个化合物(0.9%)为 NAMPT 配体。PTS 检测的大部分结果为抑制剂或无活性,其中30个化合物(5.8%)为 NAMPT 活性检测剂(NAD/NADH-Glo 检测试剂盒和 NMN 荧光检测19)。

Fig. 1 图一

The HTS hit, which was the focus of our hit-to-lead campaign, SBI-136892 (Fig. 1b), dose-dependently increased the NAMPT Tm (Supplementary Fig. 1a) and stimulated NAMPT-mediated NMN production (Supplementary Fig. 1b). Interestingly, SBI-136892 was structurally similar to active-site-directed NAMPT inhibitors (possessing a urea core and pyridyl group) such as compound 50 (GNI-50; IC50 = 7 nM), from Genentech20(Fig. 1b). We confirmed that GNI-50 was a potent NAMPT inhibitor (Fig. 1c). A close structural analog of GNI-50, GNI-5 (Fig. 1b), was shown to bind to the NAMPT active site similarly to FK-866 (Fig. 1b), the prototypical NAMPT inhibitor20. Surmising that the 4-pyridyl group was crucial for NAMPT activation, we synthesized the 4-pyridyl analog of GNI-50 to produce SBI-797812 (Fig. 1b). Remarkably, moving the pyridine nitrogen from the 3 position (GNI-50) to 4 position (SBI-797812) converted a potent NAMPT inhibitor to a NAMPT activator. SBI-797812 increased NAMPT-catalyzed NMN synthesis by 2.1-fold. (Fig. 1c). The SBI-797812 isomer with the 2-pyridyl group, SBI-796950 (Fig. 1b), slightly inhibited NMN production (Fig. 1c).

高温超导作用,也就是我们的主要作用,SBI-136892(图1b) ,剂量依赖性地增加了 NAMPT Tm (图1a) ,刺激了 NAMPT 介导的 NMN 产生(图1b)。有趣的是,SBI-136892的结构类似于活性位点定向的 NAMPT 抑制剂(具有尿素核心和吡啶基团) ,如化合物50(GNI-50; IC50 = 7nm) ,来自 Genentech20(图1b)。我们证实 GNI-50是一个有效的 NAMPT 抑制剂(图1c)。一个 GNI-50的近结构类似物,GNI-5(图1 b) ,被显示与 NAMPT 活性位点 FK-866结合类似(图1 b) ,这是典型的 NAMPT 抑制剂20。由此推测,4- 吡啶基团对 NAMPT 的活化起关键作用,我们合成了 GNI-50的4- 吡啶基类似物,生成了 SBI-797812(图1b)。显著地,将吡啶氮从3位(GNI-50)转移到4位(SBI-797812) ,使一个强有力的 NAMPT 抑制剂转变为一个 NAMPT 激活剂。SBI-797812使 nampt 催化的 NMN 合成增加了2.1倍。(图1c)。2- 吡啶基的 SBI-797812同分异构体 SBI-796950(图1b)对 NMN 的产生有轻微的抑制作用(图1c)。

SBI-797812 caused concentration-dependent activation of human NAMPT-mediated NMN production in the presence of NAM, PRPP and ATP (Fig. 1d). After adjusting for baseline NMN synthesis without NAMPT activator, the EC50 for SBI-797812 was 0.37 ± 0.06 μM and maximal NAMPT activity was 55 ± 3 U nmol−1. The maximal fold stimulation of NMN formation by SBI-797812 was 2.1-fold. The ability of SBI-797812 to activate NAMPT was abolished by NAMPT inhibitors including GNI-50, FK-866, and CHS-828 (Supplementary Fig. 2).

SBI-797812在 NAM、 PRPP 和 ATP 的存在下引起人 nampt 介导的 NMN 产生的浓度依赖性激活(图1d)。调整无 NAMPT 活化剂的 NMN 基线合成后,SBI-797812的 EC50为0.37 ± 0.06 μm,最大 NAMPT 活性为55 ± 3ummol-1。SBI-797812对 NMN 形成的最大倍数刺激为2.1倍。NAMPT 抑制剂包括 GNI-50、 FK-866和 CHS-828均能阻断 SBI-797812激活 NAMPT 的能力(附图2)。

The close structural similarity between SBI-797812 and NAMPT competitive inhibitors strongly suggested that SBI-797812 also bound to the NAMPT active site. This inference was supported by two independent experimental findings. Firstly, the NAMPT(G217R) mutant that was resistant to inhibition by CHS-82818 or FK-866 (Fig. 1e) was also refractory to the stimulatory effect of SBI-797812 (Fig. 1e). Modeling showed that the R217 side chain sterically obstructed the pyridinium binding pocket thus precluding inhibitor binding at the NAMPT active site18. We inferred that the R217 side chain also blocked SBI-797812 binding to the NAMPT active site. Secondly, FK-866 and CHS-828 prevented direct binding of SBI-797812 to NAMPT (Fig. 1f). This approach used a spin (desalting) column to separate free SBI-797812 from the NAMPT•SBI-797812 complex, and subsequent detection of NAMPT-bound SBI-797812 in the column eluent by mass spectrometry. When SBI-797812 was applied to the spin column, it did not appear in the column eluent. When NAMPT and SBI-797812 were mixed and applied to the column, SBI-797812 was detected in the column eluent along with NAMPT. When the NAMPT and SBI-797812 mixture contained an active site-directed NAMPT inhibitor (FK-866 or CHS-866), SBI-797812 was not found in the eluent.

SBI-797812和 NAMPT 竞争性抑制剂之间的结构相似性结合强烈表明 SBI-797812也与 NAMPT 活性位点结合。这一推论得到了两个独立实验结果的支持。首先,耐 CHS-82818或 FK-866抑制的 NAMPT (G217R)突变体也对 SBI-797812的刺激作用不敏感(图1e)。模拟结果表明,R217侧链立体地阻塞了吡啶盐结合囊,从而阻止了抑制剂在 NAMPT 活性位点的结合。我们推断,R217侧链也阻止了 SBI-797812结合到 NAMPT 活性位点。其次,FK-866和 CHS-828阻止了 SBI-797812与 NAMPT 的直接结合(图1f)。这种方法使用旋转(脱盐)柱从 NAMPT · SBI-797812络合物中分离出游离的 SBI-797812,然后用质谱法在洗脱液中检测到 NAMPT 结合的 SBI-797812。当 SBI-797812应用于旋转柱时,在柱洗脱液中没有出现。当 NAMPT 和 SBI-797812混合并应用于柱上时,在洗脱液和 NAMPT 中检测到了 SBI-797812。当 NAMPT 和 SBI-797812混合物含有活性定点 NAMPT 抑制剂(FK-866或 CHS-866)时,洗脱液中未发现 SBI-797812。

An earlier compound, P7C3, was claimed to be a direct NAMPT activator21. Our investigation of P7C3 using the assays described herein failed to confirm that this compound bound to NAMPT or directly stimulated its activity (Supplementary Fig. 3).

早期的一种化合物 P7C3被称为 NAMPT 直接活化剂21。我们对 P7C3的研究使用了本文描述的化验方法,未能证实该化合物与 NAMPT 结合或直接刺激其活性(附图3)。

Mechanistic studies


ATP promotes NAMPT-mediated NMN formation, but ATP is not obligatory for this reaction11 (result replicated in Fig. 2a). Notably, stimulation of NMN production by SBI-797812 required ATP (Fig. 2a). SBI-797812 slightly inhibited NMN formation in the absence of ATP. We next examined the impact of SBI-797812 on the affinity of NAMPT for ATP in the presence of NAM and PRPP (Fig. 2b). The Km values of NAMPT for ATP without and with SBI-797812 were 1.73 ± 0.32 and 0.29 ± 0.03 mM, respectively. The approximate six-fold lower Km value of NAMPT for ATP in the presence of SBI-797812 will enable saturation of the enzyme at lower ATP concentrations. The corresponding Vmax values in the absence and presence of SBI-797812 were 271 ± 28 and 316 ± 9 U nmol−1, respectively.

ATP 促进 nampt 介导的 NMN 的形成,但 ATP 并不是这种反应的必需物(结果复制在图2a 中)。值得注意的是,SBI-797812刺激 NMN 生产需要 ATP (图2a)。在缺乏 ATP 的情况下,SBI-797812对 NMN 的形成有轻微的抑制作用。接下来我们研究了在 NAM 和 PRPP 存在下,SBI-797812对 NAMPT 与 ATP 的亲和力的影响(图2b)。不加 SBI-797812和加入 SBI-797812后,ATP 的 NAMPT Km 值分别为1.73 ± 0.32和0.29 ± 0.03 mM。在 SBI-797812存在下,ATP 的 NAMPT 的 Km 值大约降低了6倍,这使得在较低的 ATP 浓度下该酶能够饱和。SBI-797812缺失和存在时相应的 Vmax 分别为271 ± 28和316 ± 9umol-1。

Fig. 2 图二

Crucial insight into the NAMPT activator MOA was gleaned from a reaction equilibrium study (Fig. 2c). NAMPT was incubated with substrates for both the forward (NAM, PRPP) and reverse (NMN, PP) reactions. A time-dependent increase of NAM and concomitant decrease of NMN was observed during the first 3 h. The dominance of the reverse reaction leading to nearly complete conversion of NMN to NAM was shown previously11. Under these same assay conditions, SBI-797812 had no discernible impact on the reaction. After 3 h, ATP was added, and the reactions proceeded for an additional 2 h. Without SBI-797812, a time-dependent NMN increase and concomitant NAM depletion was recorded. Hence, ATP caused the directionality of NAMPT activity to pivot towards the forward reaction. This ATP effect was also consistent with published data11. At equilibrium, the NAM:NMN ratio was approximately 6.5:3.5. Notably, SBI-797812 elicited a further dramatic shift of the reaction equilibrium towards NMN synthesis. NAMPT converted nearly all of the NAM to NMN within 1 h after ATP addition in the presence of SBI-797812.

对 NAMPT 活化剂 MOA 的重要认识来自反应平衡研究(图2c)。NAMPT 与底物共同孵育前(NAM,PRPP)和反(NMN,PP)反应。结果表明,在头3小时内,不结核分枝杆菌含量呈时间依赖性增加,而 NMN 含量随时间增加而减少。以前的研究表明,导致 NMN 几乎完全转化为 NAM 的逆反应占主导地位。在相同的测定条件下,SBI-797812对反应没有明显的影响。3小时后,加入 ATP,在没有 SBI-797812的情况下,反应继续进行2小时,记录到 NMN 的时间依赖性增加和伴随的 NAM 损耗。因此,ATP 使 NAMPT 活动的方向性转向前向反应。这种 ATP 效应也与已发表的数据一致。在平衡状态下,NAM 与 NMN 的比值约为6.5:3.5。值得注意的是,SBI-797812引起了反应平衡向 NMN 合成的进一步戏剧性的转变。在 SBI-797812存在下,加入 ATP 后1小时内,NAMPT 将几乎所有 NAM 转化为 NMN。

The NAMPT forward reaction (NAM + PRRP + ATP ± SBI-797812) was performed and the levels of NAM, NMN, PP, ADP, and Pi were measured (Fig. 2d). NAM consumption and NMN production were directly correlated, and both reactions were markedly stimulated by SBI-797812 (thus corroborating the results shown in Fig. 2c). The synchronous ATPase activity of NAMPT produced equimolar amounts of ADP and Pi in a reaction that was also stimulated by SBI-797812. The ratio of ADP to NMN production in the absence and presence of SBI-797812 were comparable (1.9- and 1.8-fold, respectively) (Supplementary Fig. 4). Surprisingly, PP accumulation was not stoichiometric with NMN accumulation. This PP shortfall was more prominent in the presence of SBI-797812 (Fig. 2d).

采用 NAMPT 前向反应(NAM + PRRP + ATP ± SBI-797812) ,测定 NAM、 NMN、 PP、 ADP、 Pi 水平(图2d)。NAM 消耗量与 NMN 产量直接相关,SBI-797812对两种反应均有明显的促进作用(从而证实了图2c 所示的结果)。同步 atp 酶活性的 NAMPT 产生等摩尔数量的 ADP 和 Pi 的反应,也刺激 SBI-797812。在没有 SBI-797812和存在 SBI-797812的情况下,ADP 与 NMN 的产量比值可比(分别为1.9倍和1.8倍)(补充图4)。令人惊讶的是,PP 的积累与 NMN 的积累不存在化学计量关系。在 SBI-797812的存在下,这种 PP 缺口更加突出(图2d)。

To shed light on the anomalous PP production, NAMPT was incubated with PP or (ATP + PP) and assayed for residual PP. NAMPT did not deplete PP in the absence of ATP (Fig. 3a). In contrast, incubation of NAMPT with PP and ATP resulted in a diminished PP level. Such PP consumption was markedly enhanced by SBI-797812 (Fig. 3a) and abolished by the NAMPT inhibitor, CHS-828 (Supplementary Fig. 5). The fate of the PP was predicted by modeling using density functional theory to solve the unrestricted transition state structure of the NAMPT-catalyzed reaction. Our in silico model (i.e., residues Asp313, Asp279, and pHis247, with PP, magnesium atoms and water molecules) generated a transition structure matching a late SN2 attack by an activated PP on the pHis247 (dN–P = 2.38 Å and dP–O = 1.96 Å) to produce triphosphate (P3) (Fig. 3b).

为了揭示异常的聚丙烯生产,NAMPT 与聚丙烯或(ATP + 聚丙烯)孵育,并测定残留聚丙烯。在缺乏 ATP 的情况下,NAMPT 没有消耗 PP (图3a)。与此相反,NAMPT 与 PP 和 ATP 的孵育使 PP 水平下降。SBI-797812(图3a)显著增加了这种 PP 消耗,而 NAMPT 抑制剂 CHS-828(图5)则消除了这种消耗。采用密度泛函理论建模,解决了 nampt 催化反应过渡态结构不受限制的问题,预测了聚丙烯的归宿。我们的硅树脂模型(即残基 Asp313、 Asp279和 pHis247,包括 PP、镁原子和水分子)产生了一个与活化 PP 对 pHis247(dN-p = 2.38 å 和 dP-o = 1.96 å)产生三磷酸盐(P3)的晚期 SN2攻击相匹配的过渡结构。

Fig. 3 图3

An LC-MS/MS method to assay P3 was established to test this prediction. Incubation of NAMPT with ATP and PP produced P3 (Fig. 3c). P3 production was abolished in the presence of CHS-828. SBI-797812 stimulated P3 production by 3.8-fold (Fig. 3c). Notably, P3 was not produced when the NAMPT complete reaction (NAM, PRPP, NMN, PP) was performed. The explanation for this finding likely stems from the ability of NMN to blunt P3 production (Fig. 3c). While SBI-797812 reduced the levels of PP during the complete reaction (Fig. 2d), the actual fate of PP coincident with concomitant NMN production requires further investigation.

建立了液相色谱-串联质谱(LC-MS/MS)分析 P3蛋白的方法。NAMPT 与 ATP 和 PP 的孵育产生 P3(图3c)。由于 CHS-828的存在,P3的生产被取消。SBI-797812刺激 P3产生3.8倍(图3c)。显著地,当 NAMPT 完全反应(NAM,PRPP,NMN,PP)进行时,不产生 P3。这一发现的解释可能源于 NMN 抑制 P3生成的能力(图3c)。虽然 SBI-797812在完全反应过程中降低了 PP 的水平(图2d) ,但 PP 的实际命运与 NMN 的同时产生还需要进一步的研究。

Assaying the facultative ATPase activity of NAMPT (enzyme incubated with ATP only) yielded another clue regarding the impact of SBI-797812 on NAMPT catalytic activity. SBI-797812 increased the ATPase activity of NAMPT as judged by ADP production (Supplementary Fig. 6a). Interestingly, this was not matched by equimolar Pi production (Supplementary Fig. 6b) but instead gave rise to adenosine tetraphosphate (Ap4) (Supplementary Fig. 6c). Ap4 was recently shown to be a NAMPT product formed during ATP hydrolysis22. The ability of SBI-797812 to augment NAMPT-mediated production of either P3 (when incubated with ATP and PP) or Ap4 (when incubated with ATP) suggested that the reactivity of pHis247 in NAMPT was modulated by the NAMPT activator.

测定 NAMPT (仅与 ATP 孵育的酶)的兼性 ATP 酶活性,提供了有关 SBI-797812对 NAMPT 催化活性影响的另一线索。SBI-797812通过 ADP 产量判断,增加了 NAMPT 的 atp 酶活性(补充图6a)。有趣的是,这并不匹配等摩尔 Pi 产生(补充图6b) ,而是产生四磷酸腺苷(补充图6c)。Ap4最近被证明是在 ATP 水解过程中形成的一种 NAMPT 产物。SBI-797812与 ATP 和 PP 共同孵育时增加 NAMPT 介导的 P3和 Ap4的产生,提示 NAMPT 激活剂可调节 pHis247在 NAMPT 中的反应性。

Our hypothesis that SBI-797812 altered the reactivity of pHis247 was supported by a western blotting approach that detected pHisNAMPT. pHisNAMPT was widely considered to be a highly-labile phosphoenzyme intermediate11. Autophosphorylated NAMPT was previously detected but only as a weak signal by autoradiography after incubation of the enzyme with [γ-32P]-ATP followed by SDS-PAGE13. We showed that NAMPT treated with ATP was visualized by western blotting using anti-1-pHis(δ1N) antibody (1pHisAb)23(Fig. 3d, Supplementary Fig. 7). NAMPT not exposed to ATP failed to react with 1pHisAb (Supplementary Fig. 7). Antibody raised against 3-pHis (ε3N) did not detect pHisNAMPT. The existence of 1-pHis (δ1N) but not 3-pHis (ε3N) in NAMPT agreed with the assignment from the crystal structure of NAMPT and beryllium fluoride (PDB: 3DHF), a putative pHisNAMPT mimic10. Consistent with the known chemical lability of 1-pHis(δ1N)24, the pHisNAMPT band was abolished by heating the sample at 95 °C (Supplementary Fig. 7). While the pHisNAMPT band was suppressed by NMN or PP, NAM or PRPP had no effect (Fig. 3d, Supplementary Fig. 7). Incubation of NAMPT with ATP in the presence of SBI-797812 had little impact on pHisNAMPT accumulation (Fig. 3d). However, SBI-797812 stabilized pHisNAMPT in the presence of NMN or PP (Fig. 3d). We thus hypothesized that SBI-797812 exerted a water shield effect whereby the NAMPT activator promoted the ability of nucleophiles other than water (e.g., PP) to abstract phosphate from the pHis247 residue.

我们的假设是 SBI-797812改变了 pHis247的反应性,这一假设得到了检测 pHisNAMPT 的西方墨点法方法的支持。pHisNAMPT 被广泛认为是一种高活性磷酸酶中间体11。自磷酸化 NAMPT 以前被检测到,但只是作为一个微弱的信号,放射自显影后,与[ γ-32p ]-ATP 孵育,其次是 SDS-PAGE13。我们用抗1-φ (δ1-n)抗体(1pHisAb)23(图3 d,补充图7)显示了经 ATP 处理过的 NAMPT 西方墨点法。未暴露于 ATP 的 NAMPT 未能与1pHisAb 产生反应(图7)。抗3-φ (ε3-n)抗体未检测到 pHisNAMPT。在 NAMPT 中存在1-φ (δ1-n)但不存在3-φ (ε3-n) ,这与 NAMPT 和氟化铍(PDB: 3DHF)晶体结构的结论一致。与已知的1- 具有1- 雌激素受体(δ1-n)24的化学不稳定性一致,pHisNAMPT 带被加热到95 ° c 时消失(附图7)。NMN 或 PP 抑制 pHisNAMPT 带时,NAM 或 PRPP 无效(图3d,补充图7)。在 SBI-797812存在下,NAMPT 与 ATP 的孵育对 pHisNAMPT 的积累影响不大(图3d)。然而,SBI-797812在 NMN 或 PP 存在下稳定了 pHisNAMPT (图3d)。因此,我们假设 SBI-797812发挥了水屏蔽作用,即 NAMPT 活化剂促进亲核试剂(如 PP)从 pHis247残留物中提取磷酸盐的能力。

Given the dramatic rightward shift of the NAMPT reaction equilibrium by SBI-797812, we explored if intracellular enzymes that might be “metabolic sinks” for NMN and PP (NMNAT1 and cytosolic inorganic pyrophosphatase PPA1, respectively) would compromise the SBI-797812 effect. NMNAT1 curtailed NMN accumulation in the assay because NMN was channeled towards NAD+ synthesis (Supplementary Fig. 8). Importantly, SBI-797812 exerted comparable effects on (i) NMN production in the absence of NMNAT1 and (ii) combined NMN and NAD+ production in the presence of NMNAT1. We next examined the impact of PPA1 in the absence and presence of SBI-797812 (Supplementary Fig. 9). The amount of PPA1 used in the assay was sufficient to completely degrade 20 μM PP in 10 min. PPA1 slightly “pulled” the NAMPT reaction towards NMN production (1.2-fold increase) in the absence of SBI-797812. Importantly, stimulation of NAMPT activity by SBI-797812 was not diminished in the presence of PPA1.

鉴于 SBI-797812的 NAMPT 反应平衡发生了戏剧性的向右转移,我们探讨了可能成为 NMN 和 PP“代谢汇”的胞内酶(NMNAT1和胞质无机焦磷酸酶 PPA1,分别)是否会影响 SBI-797812效应。NMNAT1减少了 NMN 的积累,因为 NMN 被导向 NAD + 的合成(补充图8)。更重要的是,SBI-797812在 NMNAT1缺失和 NMNAT1缺失的情况下对 NMN 的生产产生了类似的影响,在 NMNAT1缺失的情况下对 NMN 的生产产生了类似的影响。我们接下来研究了在 SBI-797812缺失和存在的情况下 PPA1的影响(补充图9)。结果表明,PPA1的用量足以使20μM PP 在10min 内完全降解。在 SBI-797812缺失的情况下,PPA1使 NAMPT 反应对 NMN 的产生有轻微的“拉动”作用(增加1.2倍)。重要的是,在 PPA1存在时,SBI-797812对 NAMPT 活性的刺激作用没有减弱。

We also probed the effect of SBI-797812 on feedback inhibition of NAMPT activity by NAD+11. Such suppression of NAMPT activity by NAD+ which is formed in tandem with intracellular NMNAT activity would stymie NAD+ booster strategies. Interestingly, SBI-797812 relieved NAMPT inhibition mediated by NAD+ (Fig. 2e). This unanticipated impact of the small molecule NAMPT activator should further promote intracellular production of NMN and NAD+.

探讨了抑制剂 SBI-797812对 NAD + 11反馈抑制 NAMPT 活性的影响。这种由 NAD + 与细胞内 NMNAT 活性同时形成的对 NAMPT 活性的抑制会阻碍 NAD + 增强策略。有趣的是,SBI-797812减轻了 NAD + 介导的 NAMPT 抑制(图2e)。小分子 NAMPT 激活剂的这种意想不到的影响应该进一步促进细胞内 NMN 和 NAD + 的产生。

Cellular effects of the NAMPT activator

NAMPT 激活剂的细胞效应

We used A549 human lung carcinoma cells for routine testing of NAMPT activators. Exposure of A549 cells to SBI-797812 for 4 h produced dose-dependent elevations of intracellular NMN (Fig. 4a) and NAD+ (Fig. 4b). The NMN level in A549 cells was 30 ± 7 pmol mg−1 protein. The fold elevations of NMN were 2.7, 6.1, and 16.7 in the presence of 0.4, 2, and 10 μM SBI-797812, respectively. The level of NAD+ in A549 cells was 7.4 ± 0.8 nmol mg−1 protein. The fold elevations of NAD+ were 1.5, 1.7, and 2.2 in the presence of 0.4, 2, and 10 μM SBI-797812, respectively. SBI-7979812 had no significant impact on the levels of NADP (Fig. 4c), NADH (Fig. 4d) or NADPH (Fig. 4e) in A549 cells. The apparent decreased potency of SBI-797812 in the cellular assays likely reflects binding by intracellular proteins and serum-containing cell culture media.

我们使用 A549人肺癌细胞进行 NAMPT 激活剂的常规检测。将 A549细胞暴露于 sbi-7978124小时后,细胞内 NMN (图4a)和 NAD + (图4b)出现剂量依赖性升高。A549细胞 NMN 水平为30 ± 7pmol mg-1蛋白。在0.4、2和10μM SBI-797812存在下,NMN 的褶皱高度分别为2.7、6.1和16.7。A549细胞 NAD + 水平为7.4 ± 0.8 nmol mg-1蛋白。在0.4、2和10μM SBI-797812存在下,NAD + 的褶皱高度分别为1.5、1.7和2.2。SBI-7979812对 A549细胞的 NADP (图4c)、 NADH (图4d)和 NADPH (图4e)水平无明显影响。SBI-797812在细胞分析中的明显降低可能反映了细胞内蛋白质和含血清的细胞培养基的结合。

Fig. 4 图4

To further explore the impact of SBI-797812 on NAD+ synthesis, A549 cells were treated with 13C/15N-labelled NAM [NAM(M + 4)] and intracellular levels of NAM(M + 4), 1-methylnicotinamide (1-MeNAM)(M + 4), NMN(M + 4), NAD+(M + 4) and NADP(M + 4) were measured (Fig. 4f). The study was performed in the absence or presence of SBI-797812 or FK-866 for 4 h. Cellular NAM(M + 4) did not accumulate, except for a small but significant increase when cells were also treated with FK-866. No differences in the levels of 1-MeNAM(M + 4) were observed. The meager appearance of intracellular NADP(M + 4) revealed that conversion of NAD+ to NADP by NAD+ kinase was not a dominant pathway in A549 cells. Intracellular NMN(M + 4) was slightly increased in the presence of SBI-797812 and decreased in the presence of FK-866. By far, the 13C/15N-labelled NAM-containing species that displayed the largest intracellular accumulation was NAD+(M + 4). SBI-797812 dramatically increased the level of NAD+(M + 4) by 5-fold as compared to control whereas NAD+(M + 4) production was abolished by FK-866. Importantly, this experiment unveiled a more robust effect of SBI-797812 on NAMPT activity than inferred by assaying the total intracellular NAD+ pool. This finding undoubtedly reflects cellular homeostatic mechanisms that exert a ceiling effect on the intracellular NAD+levels.

为了进一步探讨 SBI-797812对 NAD + 合成的影响,用13c/15n 标记的 NAM [ m + 4]处理 A549细胞,测定细胞内 NAM (m + 4)、1- 甲基烟酰胺(1- 甲基烟酰胺)(m + 4)、 NMN (m + 4)、 NAD + (m + 4)和 NADP (m + 4)的含量(图4f)。本研究在 SBI-797812或 FK-866不存在或存在的情况下进行4小时,细胞内 NAM (m + 4)没有积累,除了 FK-866处理后细胞内 NAM (m + 4)有少量但显著的增加。1-MeNAM (m + 4)水平无显著性差异。细胞内 NADP (m + 4)的缺失表明 NAD + 通过 NAD + 激酶转化为 NADP 的途径在 A549细胞中不占主导地位。细胞内 NMN (m + 4)在 SBI-797812存在时略有升高,在 FK-866存在时略有降低。到目前为止,13c/15n 标记的含 nam- 的物种显示最大的细胞内积累是 NAD + (m + 4)。与对照相比,SBI-797812显著提高 NAD + (m + 4)水平5倍,而 NAD + (m + 4)产量则被 FK-866消除。重要的是,这个实验揭示了一个更强大的影响,SBI-797812对 NAMPT 活性比推断的总细胞内 NAD + 池。这一发现无疑反映了细胞内稳态机制,对细胞内 NAD + 水平产生天花板效应。

We also examined the impact of SBI-797812 on intracellular levels of NMN (Fig. 4g) and NAD+ (Fig. 4h) in human primary myotubes. Treatment of these cells for 4 h with SBI-797812 elicited dose-dependent increases in the intracellular levels of NMN and NAD+. SBI-797812 at 10 μM elicited 2.5- and 1.25-fold increases of intracellular NMN and NAD+, respectively. Exposure of mouse primary myotubes to SBI-797812 (10 μM) for 4 h also elicited significant increases of NMN and NAD+ (Supplementary Fig. 10).

我们还检测了 SBI-797812对人初级肌管内 NMN (图4g)和 NAD + (图4h)水平的影响。用 SBI-797812处理这些细胞4小时后,细胞内 NMN 和 NAD + 水平呈剂量依赖性增加。SBI-797812在10μM 处诱导细胞内 NMN 和 NAD + 分别增加2.5倍和1.25倍。小鼠初级肌管暴露于 SBI-797812(10μM)4h 后,NMN 和 NAD + 也显著增加(补充图10)。

Recent assessment of NAD+ biosynthetic flux in cultured cells concluded that PARP1/2 and SIRT1/2 are major NAD+ consumers and their cellular activities are governed by the intracellular NAD+ concentration7. Hence, elevated intracellular NAD+ mediated by SBI-797812 might elicit concomitant activation of sirtuins and PARPs. To probe a possible impact of SBI-797812 on sirtuin activity, we examined acetylation of histone H4, a known SIRT1 target25. Addition of SBI-797812 to A549 cells for 4 h decreased the H4-AcK16/H4 ratio (Fig. 5a), an effect that is consistent with SIRT1 activation. We next examined a possible impact of SBI-797812 on PARP-1 activity. PARP-1 activation elicits conspicuous auto-PARylation as detected by western blotting26. Addition of SBI-797812 to A549 cells for 4 h did not increase auto-PARylated PARP-1 whereas treating A549 cells with H2O2, a PARP-1 activation trigger, increased auto-PARylated PARP-1 (Fig. 5b). SBI-797812 did not alter the auto-PARylated PARP-1 level in the presence of H2O2. However, when cell lysates were prepared in the absence of a PARP-1 inhibitor (used to block post-cell lysate PARylation artifacts), lysates from cells treated with SBI-797812 displayed markedly elevated auto-PARylated PARP-1 (Fig. 5c). This result established that increased intracellular NAD+ levels in A549 cells treated with SBI-797812 promoted PARP-1 activity in the cellular lysate. The lack of an SBI-797812 effect on PARP-1 activation in intact cells might reflect subcellular compartmentalization constraints or the dissimilar substrate (NAD+) concentrations in intact cells versus cellular lysates (this latter explanation relates to the Km value of PARP-1 for NAD+)27.

近年来对培养细胞中 NAD + 生物合成通量的研究表明,PARP1/2和 SIRT1/2是 NAD + 的主要消耗者,其细胞活性受细胞内 NAD + 浓度的控制。因此,SBI-797812介导的细胞内 NAD + 升高可能引起去乙酰化酶和 PARPs 的同时激活。为了探讨 SBI-797812可能对 sirtuin 活性的影响,我们检测了组蛋白 H4的乙酰化,一个已知的 SIRT1靶标25。SBI-797812加入 A549细胞4h 后 H4-AcK16/H4比值降低(图5a) ,与 SIRT1激活一致。我们接下来研究了 SBI-797812对 PARP-1活性的可能影响。用 western blotting26法检测 PARP-1激活诱导明显的自动 parp 化反应。将 SBI-797812添加到 A549细胞中4小时不会增加 PARP-1的自身分泌,而用 H2O2(PARP-1激活触发器)处理 A549细胞可增加 PARP-1的自身分泌(图5b)。SBI-797812在 H2O2存在时不改变自身 PARP-1水平。然而,当在没有 PARP-1抑制剂的情况下制备细胞裂解物时(用于阻断细胞后裂解物 PARylation 伪迹) ,用 SBI-797812处理的细胞裂解物显示自 PARP-1明显升高(图5c)。这一结果表明,SBI-797812处理后 A549细胞内 NAD + 水平升高,细胞裂解液中 PARP-1活性增强。缺乏 SBI-797812对完整细胞 PARP-1激活的影响可能反映了亚细胞防火分区的约束或完整细胞与细胞裂解物中不同底物(NAD +)的浓度(后者的解释与 NAD + 的 PARP-1的 Km 值有关)27。

Fig. 5 图5

In vivo effects of NAMPT activators in mice

NAMPT 激活剂在小鼠体内的作用

SBI-797812 was administered to mice (10 mg compound kg−1 body weight) by oral or intraperitoneal (i.p.) dosing, blood was drawn at increasing times and plasma levels of SBI-797812 were measured by LC-MS/MS. Plasma concentrations of SBI-797812 after oral administration were low (Supplementary Fig. 11a). Higher plasma levels of SBI-797812 were seen after i.p. dosing (Cmax value: 3297 ng ml−1, 8.2 μM) (Supplementary Fig. 11b). The transient plasma exposure of SBI-797812 was a key consideration when evaluating the pharmacodynamic effects of SBI-797812 in mice.

采用口服或腹腔注射 SBI-797812方法给小鼠(体重10mg 复方 kg-1) ,血液循环次数增加,液相色谱-串联质谱法(LC-MS/MS)测定血浆 SBI-797812浓度。口服给药后血浆 SBI-797812浓度较低(补充图11a)。静脉注射后血浆 SBI-797812水平升高(Cmax 值: 3297ng ml-1,8.2 μm)(补充图11b)。在评价 SBI-797812对小鼠的药效学作用时,SBI-797812的瞬时血浆暴露是一个关键因素。

The potency of SBI-797812 against mouse NAMPT was also investigated. The specific activity (U nmol−1 NAMPT) of mouse NAMPT using the in vitro NMN production assay was approximately eight-fold higher than human NAMPT. Moreover, the apparent affinity (EC50) of SBI-797812 for mouse NAMPT was approximately 8-fold less than for human NAMPT, whereas maximal fold activation by SBI-797812 was comparable between the mouse and human NAMPT. These differences between human and mouse NAMPT are another important factor when evaluating the in vivo efficacy of SBI-797812 in murine preclinical models.

还研究了 SBI-797812对小鼠 NAMPT 的抑制作用。体外 NMN 生成试验表明,小鼠 NAMPT 的比活性(u nmol-1 NAMPT)约为人 NAMPT 的8倍。此外,SBI-797812对小鼠 NAMPT 的表观亲和力(EC50)约为人 NAMPT 的8倍,而 SBI-797812对小鼠和人 NAMPT 的最大褶皱活化能力相当。这些人类和小鼠 NAMPT 之间的差异是另一个重要因素,当评价 SBI-797812在小鼠临床前模型的体内疗效时。

For the tissue biomarker study, mice were dosed with SBI-797812 (20 mg kg−1 i.p.) and liver, heart, gastrocnemius muscle and quadriceps muscle were harvested after 4 h. Despite the transient plasma exposure of SBI-797812 and lower apparent affinity of SBI-797812 for mouse NAMPT, a significant 1.3-fold increase of NAD+ was detected in liver (Fig. 6a). There was also a trend towards increased NAD+ levels in cardiac tissue (Fig. 6b). Skeletal muscle, either gastrocnemius (Fig. 6c) or quadriceps (Fig. 6d), did not exhibit increased NAD+ levels after dosing with SBI-797812. The mean tissue levels of SBI-797812 (2 h post-dose) as measured by LC-MS/MS were 0.311, 0.144, 0.078, and 0.078 μg/mg dry powder in liver, heart, gastrocnemius and quadriceps, respectively (Supplementary Fig. 12). Hence, liver which displayed a statistically significant increase of NAD+ after SBI-797812 dosing exhibited the highest level of the compound.

在组织标志物研究中,给小鼠注射 SBI-797812(20mg kg-1 i.p.) ,4小时后取肝脏、心脏、腓肠肌和股四头肌。心肌组织中 NAD + 水平也呈上升趋势(图6b)。骨骼肌,无论是腓肠肌(图6c)还是股四头肌(图6d) ,在服用 SBI-797812后都没有表现出 NAD + 水平的增加。LC-MS/MS 法测定 SBI-797812(2h 后)在肝、心、腓肠肌和股四头肌干粉中的平均含量分别为0.311、0.144、0.078和0.078 μg/mg (图12)。因此,在服用 SBI-797812后,肝脏中 NAD + 的含量显著升高,表现为最高水平。

Fig. 6 图6



Our search for small molecules that stimulated NAMPT-mediated NMN formation yielded SBI-797812 which activated purified NAMPT, raised NMN and NAD+ levels in cultured cells, and boosted hepatic NAD+ in mice. The structural similarity between SBI-797812 and active-site targeted NAMPT inhibitors was striking. Remarkably, moving the pyridyl nitrogen from the 4-position in SBI-797812 to the 2 or 3-positions changed the compound from a NAMPT activator to an inhibitor. Binding of SBI-797812 to the NAMPT active site was supported by two independent experimental approaches. Firstly, the NAMPT (G217R) mutant was refractory to activation by SBI-797812, just as it was insensitive to inhibition by CHS-82818 and FK-866. Secondly, FK-866 and CHS-828 blocked binding of SBI-797812 to NAMPT as determined with a “spin column” method that separated free ligand from NAMPT-bound ligand. The fact that SBI-797812, an active-site targeted ligand, was a NAMPT activator rather than an inhibitor posed a vexing yet fascinating conundrum.

我们寻找能够刺激 NAMPT 介导的 NMN 形成的小分子,得到了能够激活纯化的 NAMPT 的 SBI-797812,提高了培养细胞中 NMN 和 NAD + 的水平,并促进了小鼠肝脏中 NAD + 的形成。在 SBI-797812和活性位点靶向 NAMPT 抑制剂之间的结构相似性是惊人的。值得注意的是,将吡啶基氮从 SBI-797812的4位移动到2位或3位,使该化合物从 NAMPT 活化剂转变为抑制剂。SBI-797812与 NAMPT 活性位点的结合得到两种独立实验方法的支持。首先,NAMPT (G217R)突变体对 SBI-797812的激活不敏感,对 CHS-82818和 FK-866的抑制也不敏感;。其次,FK-866和 CHS-828阻断了 SBI-797812与 NAMPT 的结合。SBI-797812是一种活性位点靶向配体,它是一种 NAMPT 激活剂而非抑制剂,这一事实提出了一个令人烦恼而又迷人的难题。

SBI-797812 had a dramatic effect on the equilibrium position of the reversible NAMPT reaction eliciting a marked elevation of NMN and concomitant depletion of NAM. Several clues to the SBI-797812 MOA have been gleaned from our investigation (Fig. 7). First, ATP was obligatory for the activating effect of SBI-797812. SBI-797812 increased the apparent affinity of NAMPT for ATP. Second, SBI-797812 stabilized pHisNAMPT in the presence of NAMPT products (NMN or PP) as shown by western blotting using an 1pHisAb. Stabilization of pHisNAMPT should manifest itself as increased affinity for NAMPT substrates11. The water shield effect of SBI-797812 on pHis247 was likely responsible for the remarkable ability of the NAMPT activator to promote formation of P3 and Ap4 from the nucleophiles PP and ATP, respectively. Our observations that NMN both abolished P3 production and blocked the appearance of pHisNAMPT (when NAMPT was incubated with ATP) are probably inextricably linked. Third, NAMPT consumed the PP by-product in a reaction that was stimulated by SBI-797812. The fate of PP in the presence of NMN remains to be determined but PP consumption would certainly contribute to the “rightward shift” of the NAMPT reaction.

SBI-797812对可逆 NAMPT 反应的平衡位置有显著影响,引起 NMN 明显升高和 NAM 的伴随性损耗。从我们的调查中收集到了几条有关 SBI-797812 MOA 的线索(图7)。首先,三磷酸腺苷对 SBI-797812的激活作用是必需的。SBI-797812增加了 NAMPT 对 ATP 的亲和力。第二,SBI-797812在 NAMPT 产品(NMN 或 PP)存在下稳定了 pHisNAMPT,如西方墨点法使用1pHisAb 所示。pHisNAMPT 的稳定应该表现为与 NAMPT 子层的亲和力增强。SBI-797812对 pHis247的水屏蔽效应可能是 NAMPT 活化剂促进亲核试剂 PP 和 ATP 合成 P3和 Ap4的显著能力的原因。我们的观察表明,NMN 既阻断了 P3的产生,又阻断了 pHisNAMPT (当时 NAMPT 与 ATP 孵化)的出现,这两者之间可能有着千丝万缕的联系。第三,NAMPT 在 SBI-797812刺激的反应中消耗了 PP 副产物。聚丙烯在 NMN 存在下的命运仍有待确定,但聚丙烯的消费肯定会促进 NAMPT 反应的“向右转移”。

Fig. 7 图7

We established that the ability of SBI-797812 to activate purified NAMPT was recapitulated in cultured cells. While the fold-increase of NMN in SBI-797812-treated A549 cells (17.4-fold) was much larger than the increase of NAD+ (2.2-fold), the total NAD+ rise greatly exceeded the total NMN rise by approximately 17.5-fold. Hence, NMNAT did not emerge as the rate-limiting step when NAMPT was activated with SBI-787812.

我们在培养细胞中证实了 SBI-797812激活纯化的 NAMPT 的能力。结果表明,在 sbi-797812处理的 A549细胞中,NMN 的增加倍数(17.4倍)远大于 NAD + 的增加倍数(2.2倍) ,而 NAD + 的增加总量大于 NMN 的增加总量约17.5倍。因此,当 NAMPT 被 SBI-787812激活时,NMNAT 并没有出现作为限速步骤。

In an intracellular milieu where NAMPT works in tandem with NMNAT1-3 to produce NAD+, the interaction of SBI-797812 with NAMPT mediated two other effects that should also promote NAD+ accumulation. First, SBI-797812 blunted feedback inhibition of NAMPT activity by NAD+, thus opposing product inhibition of NAD+ synthesis. Suppression of feedback inhibition by NAD+ with a small molecule NAMPT activator might be a key advantage for raising intracellular NAD+ as compared to nicotinamide riboside (NR), an NMN precursor5,28. Second, colocalization of NAMPT and NMNAT in various subcellular compartments29 suggests that PP produced by the NMNAT reaction might be also consumed by NAMPT in a process stimulated by SBI-797812. As the equilibria of the NMNAT reactions favor NAD+ catabolism30, such PP depletion by NAMPT would act to further displace the NAM salvage pathway equilibrium towards NAD+-end product formation.

在 NAMPT 与 NMNAT1-3协同作用产生 NAD + 的细胞内环境中,SBI-797812与 NAMPT 的相互作用介导了另外两种促进 NAD + 积累的效应。首先,SBI-797812减弱了 NAD + 对 NAMPT 活性的反馈抑制作用,从而对抗了 NAD + 合成的产物抑制。用小分子 NAMPT 激活剂抑制 NAD + 的反馈抑制可能是提高细胞内 NAD + 的关键优势。其次,NAMPT 和 NMNAT 在不同亚细胞间隔区的共定位表明,NAMPT 也可能在 SBI-797812的刺激下消耗 NMNAT 反应产生的 PP。由于 NMNAT 反应的平衡有利于 NAD + 分解代谢 m30,NAMPT 的这种 PP 耗尽将进一步取代 NAM 补救路径平衡,促进 NAD +-终产物的形成。

The vast arsenal of intracellular enzymes that consume NAD+ constitutes a powerful homeostatic mechanism that exerts a ceiling effect on cellular NAD+ levels. The activities of PARPs and sirtuins in T47D cultured cells were shown to be regulated by the intracellular NAD+ concentration (i.e., NAD+ elevation is opposed by increased NAD+ consumption)7. To better recognize the ability of SBI-797812 to activate NAMPT in cells, we co-treated cells with 13C/15N-NAM and the ensuing NAM-containing metabolites were monitored for the presence of this stable isotope tracer. The dominant fate of the NAM tracer in A549 cells was the NAD+ pool. Importantly, SBI-797812 dramatically elevated the intracellular NAD+(M + 4) level. The NAM tracer was not enriched in the 1-MeNAM pool which is formed by nicotinamide N-methyltransferase (NNMT), a key regulatory enzyme in numerous cell types31. The rise of NMN(M + 4) was very slight compared to NAD+(M + 4) which suggested that NMNAT activity was not rate determining even when NAMPT activity was increased by SBI-797812. There was very modest appearance of NADP(M + 4). This result was concordant with the recent NAD+ flux study showing that NADP was slowly labeled in T47D cells7.

消耗 NAD + 的巨大细胞内酶库构成了一个强大的内稳态机制,对细胞 NAD + 水平施加了天花板效应。结果表明,细胞内 NAD + 浓度(即 NAD + 浓度增加与 NAD + 浓度增加相反)可调节 T47D 培养细胞 PARPs 和 sirtuins 的活性。为了更好地认识 SBI-797812激活细胞内 NAMPT 的能力,我们与13C/15N-NAM 共处理细胞及随后产生的含 nam- 的代谢物监测这种稳定的同位素示踪剂的存在。NAM 示踪剂在 A549细胞中的主要归宿是 NAD + 池。重要的是,SBI-797812显著升高细胞内 NAD + (m + 4)水平。NAM 示踪剂在多种细胞类型31的关键调节酶——烟酰胺 n- 甲基转移酶(NNMT)所形成的1-MeNAM 池中没有富集。与 NAD + (m + 4)相比,NMN (m + 4)的升高很小,说明即使在 NAMPT 活性增加的情况下,NMNAT 活性也不是速率决定因素。NADP (m + 4)的表达非常温和。这一结果与近年来 NAD + 通量研究结果一致,即 NADP 在 T47D 细胞中被缓慢标记。

Achieving the desired pharmacological effect when dosing mice with a NAMPT activator is contingent on several prerequisites. Firstly, compound dosing must yield adequate plasma exposure. The pharmacokinetic profile of SBI-797812 was mediocre but appreciable plasma exposure was seen up to 4 h after i.p. dosing. Secondly, the NAMPT activator must cross-react with murine NAMPT. This was the case even though SBI-797812 was a weaker activator of mouse NAMPT as compared to human NAMPT. Thirdly, NAD+ biosynthetic flux in target tissues must be rapid relative to the study duration. This prerequisite appeared to be met given the ability of SBI-797812 to elevate NMN and NAD+ in A549 cells at 4 h post-dosing. However, the smaller increases of NMN and NAD+ in primary mouse myotubes portended that responsiveness to SBI-797812 was not uniform across different cell types. This latter concern was heightened by recent data showing that mammalian NAD+ metabolism involves extensive tissue-specific pathway regulation, which is not replicated in standard cell lines7.

达到预期的药理作用时,给予小鼠的 NAMPT 激活剂是依赖于几个先决条件。首先,复合剂量必须产生足够的血浆暴露。SBI-797812的药代动力学曲线平平,但可观的血浆暴露直至静脉注射后4小时。其次,NAMPT 激活剂必须与小鼠的 NAMPT 发生交叉反应。即使 SBI-797812与人类的 NAMPT 相比是一个较弱的小鼠 NAMPT 激活剂,情况也是如此。第三,NAD + 生物合成通量在靶组织中必须随着研究时间的延长而迅速增加。SBI-797812在给药后4小时能提高 A549细胞中的 NMN 和 NAD + ,这个前提条件似乎得到了满足。然而,小鼠初级肌管中 NMN 和 NAD + 的增加幅度较小,表明不同类型的细胞对 SBI-797812的反应不一致。最近的数据显示,哺乳动物 NAD + 代谢涉及广泛的组织特异性途径调节,而这种调节在标准细胞系中没有复制,这加剧了后者的担忧。

Despite these shortcomings of SBI-797812, administration of our prototypical NAMPT activator to mice increased hepatic NAD+ levels. Future studies with superior compounds will allow us to decipher if the relatively modest in vivo NAD+ elevating effects of SBI-797812 can be improved with analogs that exhibit more favorable pharmacokinetics and/or potency versus murine NAMPT. In addition, changes in the study design such as longer duration of dosing and interrogation of other tissues may also unveil more robust effects of a NAMPT activator. It is also possible that the NAD+ levels are not an ideal biomarker for NAMPT activation due to cellular mechanisms that divert the newly- generated NAD+ to other metabolic by-products as strongly suggested by our cell study using the stable-isotope NAM tracer.

尽管 SBI-797812有这些缺点,给予我们的原型 NAMPT 激活剂增加小鼠肝脏 NAD + 水平。未来对高级化合物的研究将允许我们破译相对较小的体内 NAD + 升高效应的 SBI-797812是否可以通过相对于小鼠 NAMPT 而表现出更有利的药代动力学和/或效力的类似物得到改善。此外,研究设计的变化,如更长的剂量和其他组织的讯问时间,也可能揭示更强大的作用的 NAMPT 激活剂。也有可能 NAD + 水平不是一个理想的 NAMPT 激活的生物标志物,因为细胞机制转移新产生的 NAD + 到其他代谢副产物,正如我们使用稳定同位素 NAM 示踪剂的细胞研究强烈建议的那样。

Comparing the therapeutic utility of small molecule NAMPT activators to NAD+ boosters such as NR or NMN will be essential. The fact that SBI-797812 acts catalytically to promote NAD+ synthesis along with its ability to suppress feedback inhibition of NAMPT activity by NAD+ (Fig. 2e) are two discriminating attributes that are likely to be advantageous. In any event, a small molecule NAMPT activator exemplified by SBI-797812 represents a pioneering pharmacological approach to raise intracellular NAD+ and realize diverse and potentially impactful therapeutic benefits.

比较小分子 NAMPT 激活剂与 NAD + 助推剂如 NR 或 NMN 的治疗效果是必要的。SBI-797812对 NAD + 的合成具有催化作用,并能抑制 NAD + 对 NAMPT 活性的反馈抑制(图2e)。无论如何,以 SBI-797812为代表的一种小分子 NAMPT 激活剂代表了提高细胞内 NAD + 和实现多样化和潜在的有效治疗效果的开创性药理学方法。



Materials and reagents


Commercially-available materials and reagents are listed in Supplementary Table 1.


Custom produced recombinant proteins


Human NAMPT (N-terminal His-tagged) was expressed in E.coli BL21 (DE3) pLysS containing expression plasmid (pBAD-DEST 49) with DNA encoding human NAMPT11. Cells were harvested and lysed with a pneumatic high shear fluid homogenizer (Microfluidizer LM10, Microfluidics) in lysis buffer (PBS containing 10 mM imidazole, pH 7.4). The lysed sample was centrifuged (30 min at 13,000 rpm) and the supernatant was applied to Complete His-tag purification resin (Roche Diagnostics). The column was washed with PBS buffer (pH 7.4) containing 20 mM imidazole and His-tagged NAMPT was eluted with 0.5 M imidazole in PBS. The penultimate purification step used a HiTrap Q column (GE Healthcare) equilibrated with 20 mM Tris buffer (pH 8.0). NAMPT was eluted with a 0–500 mM NaCl gradient. NAMPT-containing fractions were concentrated and treated with thrombin (5 IU thrombin for each 1 mg NAMPT at room temperature for 2 h) to remove the N-terminal His tag extension. Final NAMPT purification involved re-chromatography with the HiLoad 16/60 column (100 mM HEPES pH 7.5, 100 mM NaCl and 10 mM 2-mercaptoethanol). Purified human NAMPT was stored at −80 °C. Protein concentration was determined by amino acid composition analysis performed by the Proteomics and Mass Spectrometry Facility at the Donald Danforth Plant Science Center (St. Louis, MO). Mouse NAMPT was expressed in E.coli and purified similarly. The human NAMPT(G217R) mutant was cloned from the NAMPT WT cDNA and expressed/purified essentially as above.

在含有编码人 NAMPT11的 DNA 表达质粒 pbd-dest 49的大肠杆菌 BL21(DE3) pLysS 中表达了人 NAMPT (n 端 his 标记)。用气动高剪切流体匀浆器(微流控器 LM10,微流控器)在含10mm 咪唑的 PBS 溶液中进行细胞裂解。将裂解后的样品进行离心分离(13,000 rpm,30 min) ,上清液用于完成 his 标签纯化树脂(罗氏诊断)。用含20mm 咪唑的 PBS 缓冲液(pH 7.4)洗涤柱,用0.5 m 咪唑洗脱标记的 NAMPT。倒数第二个纯化步骤使用 HiTrap q 色谱柱(GE Healthcare)与20mm Tris 缓冲液(pH 8.0)平衡。采用0-500mm NaCl 梯度洗脱 NAMPT。含 NAMPT 组分经浓缩后用凝血酶(室温下每1mg NAMPT 5 IU 凝血酶处理2h)去除 n- 末端 His 标签。最终的 NAMPT 纯化采用 hiload16/60色谱柱(100mmhepes ph7.5,100mmnacl 和10mm2- 巯基乙醇)。纯化的人 NAMPT 保存在 -80 °c。蛋白质的浓度是由 Donald Danforth 植物科学中心(密苏里州圣路易斯)的蛋白质组学和质谱法实验室进行的氨基酸组成分析测定的。小鼠 NAMPT 在大肠杆菌中表达并纯化。从 NAMPT WT cDNA 中克隆了人 NAMPT (G217R)突变体,并对其进行了表达和纯化。

Human NMNAT1 was synthesized in E.coli BL21(DE3) containing expression plasmid pET20b (+) with cDNA encoding NMNAT1. The custom-synthesized oligonucleotide primers used for PCR-mediated amplification of the NMNAT1 cDNA (35 cycles; 56 °C annealing temperature) are shown in Supplementary Table 2. Cells were lysed and His-tagged NMNAT1 was isolated using the Complete His-tag purification resin (Roche Diagnostics) as described above. The NMNAT1-containing fractions were pooled, dialyzed against 20 mM Tris-HCl, 500 mM NaCl, 3 mM DTT, 10% glycerol (pH 7.4) and stored at −80 °C.

以含有 NMNAT1基因的表达质粒 pET20b (+)为载体,在大肠杆菌 BL21(DE3)中合成人 NMNAT1。用于 pcr 介导 NMNAT1 cDNA 扩增的自制寡核苷酸引物(35个周期; 56 ° c 退火温度)见补充表2。细胞裂解和 his 标记 NMNAT1分离使用完整的 his 标记纯化树脂(罗氏诊断)如上所述。将 nmnat1组分混合,对20mm Tris-HCl、500mm NaCl、3mm DTT、10% 甘油(ph7.4)透析,保存在 -80 ° c。

Protein thermal shift assay


The Conrad Prebys Center for Chemical Genomics (CPCCG) at SBP-La Jolla screened 57,004 compounds (ChemBridge Premium Set; 10 mM compound stocks in DMSO) to identify chemical structures that caused a thermal shift of purified human NAMPT. The protein thermal shift (PTS) assay was performed in 384 well plates (10 μl well volume). The PTS assay used 2 μM NAMPT, 5x Sypro Orange dye (Molecular Probes), 2.5 mM ATP in 50 mM HEPES, pH 7.5 buffer containing 50 mM NaCl, 5 mM MgCl2 and 1 mM TCEP. Compounds (25 μM) were added using an Echo acoustic dispenser and incubated for 15 min prior to the assay. The final DSMSO concentration was 0.25%. The assay was performed with a ViiA7 real time PCR system (Thermo Fisher Scientific) at 0.15° C sec−1 ramping speed. The temperature ranged from 25 to 95 °C. The primary screen hit criterion was Z-score ≥ 5 or ∆Tm D ≥ 1 °C. Hit confirmation was performed with fresh powders and used the following PTS criterion: Z-score ≥ 7 or delta Tm D ≥ 1 °C (triplicate wells at a compound concentration of 25 μM). Further details of the PTS assay are presented in Supplementary Table 3.

位于 SBP-La Jolla 的康拉德 · 普雷比化学基因组学中心筛选了57004种化合物(ChemBridge Premium Set; DMSO 中的10mm 化合物库存) ,以确定导致纯化的人类 NAMPT 发生热位移的化学结构。在384个(10μl 阱容积)孔板上进行蛋白热位移(PTS)测定。PTS 法采用2 μm NAMPT、5 x Sypro 橙色染料(分子探针公司)、2.5 mM ATP、 pH 7.5、 NaCl、5 mM MgCl2和1 mM TCEP 缓冲液。化合物(25微米)加入使用回声分配器和孵化15分钟前的分析。最终的 DSMSO 浓度为0.25% 。采用 ViiA7实时荧光定量 PCR (Thermo Fisher Scientific)系统,以0.15 ° c sec-1的速度进行检测。气温由摄氏25度至95度不等。主要的屏幕命中准则是 z 评分≥5或者 Tm d ≥1 ° c。用新鲜粉末进行命中验证,采用以下 PTS 准则: z 评分≥7或 δTm d ≥1 ° c (复合浓度为25μM 的三叉井)。进一步的 PTS 分析的细节见补充表3。

Detection of NAMPT activators in PTS hit set

PTS 命中集中 NAMPT 激活子的检测

PTS hits were tested for their abilities to stimulate NAMPT activity with a 2-step sequential assay. Firstly, NAMPT (16 nM), NAM (5 μM), PRPP (6.25 μM), ATP (0.12 mM), yeast inorganic pyrophosphatase (0.04 U ml−1) and NMNAT3 (5 μg ml−1) with or without PTS hits (25 μM final concentration; performed in triplicate) were incubated for 2 h at room temperature in 50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.005% Tween 20 (1536 well format). Next, NAD+ was quantified with NAD-Glo assay kits (Promega Corp). The NAD-Glo master reagent was added and luminescence was detected after 30 min at room temperature.

用两步序贯分析法测试 PTS 中药刺激 NAMPT 活性的能力。首先将 NAMPT (16nm)、 NAM (5μM)、 PRPP (6.25 μm)、 ATP (0.12 mM)、酵母无机焦磷酸酶(0.04 u ml-1)和 NMNAT3(5 μg-1)加入 PTS (25 μm 终浓度,一式三份) ,室温培养2h,ph7.5,50 mM NaCl,5 mM MgCl2,1 mM TCEP,0.005% Tween 20(1536 well 格式)。接下来,使用 NAD-glo 检测试剂盒(Promega 公司)对 NAD + 进行定量。在室温下加入 NAD-Glo 母体试剂,30min 后检测其发光性能。

NAMPT enzymatic reactions


The NMN production assay involved incubation of NAMPT at 37oC with NAM (25 μM), PRPP (50 μM) and ATP (2 mM) at 37 °C in TMD buffer (50 mM Tris-HCl, 10 mM MgCl2, 2 mM DTT, pH 7.5). Where indicated (see Fig. 2c), NMN and PP were also included. Also, where specified (see Fig. 2b), the ATP concentration was varied from 0 to 2 mM. The values for Vmax and Km (ATP hydrolysis) were deduced using the on-line Michaelis-Menten kinetics tool at http://www.graphpad.com/quickcalcs/ttest1/?Format=SEM. The reactions were performed in the absence or presence of NAMPT activators, inhibitors or other agents as specified. The final DMSO concentration was 1%. NAM, NMN and ADP were assayed by LC-MS/MS (see Supplementary Information) after quenching samples with equal volume of 1 M perchloric acid (PCA). NMN was also assayed using a chemical method which converts NMN into a fluorescent derivative19. For the latter assay, an aliquot (37.5 μl) of the NMN-containing sample was sequentially mixed with 15 μl of 20% acetophenone (in DMSO) and 15 μl of 2 M KOH. The mixture was placed on ice for 10 min. Next, 67.5 μl of 100% formic acid was added to each sample, vortexed, and then incubated at 37 °C for 20 min. Samples (100 μl) were transferred to a 96-well opaque bottom plate and fluorescence (Ex/Em = 382/445 nm) was measured using a SpectraMax M5 plate reader (Molecular Devices). Pi was assayed using the PiColorLock Gold Phosphate detection system (Innova Biosciences). PP concentration was calculated from the difference in the amount of Pi without and with treatment with yeast inorganic pyrophosphatase (40 ng ml−1 for 10 min at 37 °C).

NMN 的产生方法包括在37oC 时,在 TMD 缓冲液(50mm Tris-HCl,10mm MgCl2,2mm DTT,pH 7.5)中,用 NAM (25μM)、 PRPP (50μM)和 ATP (2mm)孵育 NAMPT。如果表明(见图2c) ,NMN 和 PP 也包括在内。此外,在指定的地方(见图2b) ,ATP 浓度从0到2毫米不等。使用米氏方程在线工具在 http://www.graphpad.com/quickcalcs/ttest1/?format=sem 计算了 Vmax 和 Km 值。反应是在没有或存在 NAMPT 激活剂,抑制剂或其他指定的代理人进行。最终二甲基亚砜浓度为1% 。用等体积的1m 高氯酸(PCA)淬灭样品后,用 LC-MS/MS (见补充信息)测定 NAM、 NMN 和 ADP。此外,还采用化学方法将 NMN 转化为荧光衍生物19。对于后一种分析方法,将含有 nmn 的样品分别与20% 苯乙酮(DMSO)的15 μl 和2m KOH 的15 μl 混合。混合物在冰上放置10分钟。其次,每个样品加入67.5 μl 100% 甲酸,旋转,然后在37 ° c 下培养20分钟。样品(100μl)转移到96孔不透明底板上,用 SpectraMax M5平板阅读器(Molecular Devices)测量荧光(Ex/Em = 382/445nm)。Pi 的测定使用皮科洛克金磷酸盐检测系统(Innova Biosciences)。聚丙烯浓度计算的差异,Pi 的数量没有和处理与酵母无机焦磷酸酶(40ng ml-110min 在37 ° c)。

The PP consumption assay involved incubation of NAMPT at 37 °C with PP, ATP and other agents as indicated. PP was measured using the PiColorLock Gold Phosphate Detection System as described above. P3 was measured by LC-MS/MS (see Supplementary Information).

PP 消耗试验包括在37 ° c 下,用 PP、 ATP 和其它试剂进行 NAMPT 的培养。聚丙烯测量使用皮科洛克金磷酸盐检测系统如上所述。采用 LC-MS/MS 法测定 P3(见补充资料)。

The ATPase assay involved incubation of NAMPT at 37oC with 2 mM ATP in TMD buffer and other agents as indicated. ADP was measured by LC-MS/MS (see Supplementary Information). Pi was measured using the PiColorLock Gold Phosphate Detection system.

ATP 酶试验包括 NAMPT 在37oC 培养,2 mM ATP 在 TMD 缓冲液和其他药物中培养。采用 LC-MS/MS 法测定 ADP (见补充资料)。采用皮金磷酸盐检测系统进行 Pi 的测定。

SBI-797812 binding to NAMPT

与 NAMPT 绑定的 SBI-797812

Human NAMPT (2 μM) and SBI-797812 (5 μM) were combined in T8MD-Tw buffer (50 mM Tris pH 8.0, 10 mM MgCl2, 2 mM DTT, 0.01% Tween 80). Where indicated, NAMPT was first treated with FK-866 (5 μM) or CHS-828 (5 μM). Samples were incubated at 37 °C for 10 min and then placed on ice. Samples (60 μl) and T8MD-Tw buffer (15 μl) were sequentially added to Zeba Spin Desalting Columns (7 K MWCO; 89883, Thermo Fisher Scientific) equilibrated with T8MD-Tw buffer. Columns were centrifuged at 1500 × g at 4 °C for 2 min. Eluents were collected and frozen at −80 °C. SBI-797812 was assayed as follows. Samples (25 μl) were extracted with 100 μl acetonitrile (ACN) containing 1 μg ml−1 indomethacin as the internal standard (IS). Samples were vortexed 5 min, centrifuged 3700 x rpm at 4 °C for 10 min, and 100 μl aliquots of each supernatant were transferred to a 96-well plate. Ten microliter aliquots of the extracts were injected onto a Thermo HPLC system equipped with PAL CTC plate sampler (96-well plate), Dionex Ultimate 3000 binary pump (flow rate at 0.25 ml min−1), Dionex Ultimate 3000 thermostatted column compartment (temperature held at 40 °C), Thermo Endura Mass Spectrometer (ESI source), and Thermo Scientific Accucore C18 (2.6 μm, 2.1 × 50 mm, 100 Å) column. The HPLC solvents were ACN / 0.1% formic acid (A) and 0.1% formic acid (B). The column gradient was: 5 to 95% A from 0 to 5.0 min, 95% A until 5.5 min and step reduction to 5% A for 1 min for column reequilibration. SBI-797812 peak areas were measured, and analyte amounts were calculated from calibration curves after adjusting for IS concentrations. SBI-797812 calibration curves were constructed with 8 concentrations (1, 5, 10, 50, 100, 500, 1000, and 5000 nM) by spiking 10 μl of 50x concentration DMSO stocks into 490 μl buffer, extracting 25 μl of the resulting sample and analyzing as described above.

在 T8MD-Tw 缓冲液(50mm Tris ph8.0,10mm MgCl2,2mm DTT,0.01% Tween 80)中,将人 NAMPT (2μM)和 SBI-797812(5μM)结合。如有指示,首先用 FK-866(5μM)或 CHS-828(5μM)处理 NAMPT。样品在37 ° c 温度下培养10分钟,然后放在冰上。样品(60μl)和 T8MD-Tw 缓冲液(15μl)依次加入 Zeba Spin Desalting 柱(7k MWCO; 89883,Thermo Fisher Scientific)与 T8MD-Tw 缓冲液相平衡。离心柱在4 ° c 下1500 × g,离心时间2min。收集洗脱液,在 -80 °c 下冷冻。SBI-797812测定结果如下。以含1μg/ml 吲哚美辛的100μl 乙腈(ACN)为内标物,提取样品(25μl)。样品旋转5min,在4 ° c 下离心3700xrpm 10min,将每个上清液的100μl 溶液转移到96孔板上。将10微升提取物分别注入装有 PAL CTC 板采样器(96孔板)、 Dionex Ultimate 3000二元泵(流量0.25 ml min-1)、 Dionex Ultimate 3000恒温柱(温度40 ° c)、 Thermo Endura 质谱仪(源)和 Thermo acc18(2.6 μm,2.1 × 50 mm,100 å)柱的热高效液相色谱系统中。高效液相色谱溶剂为 ACN/0.1% 甲酸(a)和0.1% 甲酸(b)。柱梯度为: 5ー95% a,从0ー5.0 min,95% a 至5.5 min,步进还原至5% a,再平衡1min。测定了 SBI-797812的峰面积,并根据校正后的 IS 浓度曲线计算分析物含量。在490μl 缓冲液中加入50x 浓度的 DMSO 原料10μl,提取25μl 样品,分别以1,5,10,50,100,500,1000,5000nm 为8个浓度,构建了 SBI-797812的校正曲线。

Western blotting for pHisNAMPT

为 pHisNAMPT 设计的西方墨点法

pHisNAMPT was produced by treating NAMPT (10 μg ml−1) with 2 mM ATP in TMD buffer. Where indicated, other agents (SBI-797812, NAM, PRPP, NMN, PP) were also included. Samples were incubated at 37 °C for 10 min and combined with SDS-containing sample preparation buffer. Samples were kept on ice and not heated prior to loading on the gel. Two hundred ng of protein was run on a 4–20% Criterion TGX gel (Bio-Rad) at 150 V for 1.5 h and transferred to a polyvinylidene fluoride (PVDF) membrane (Roche) at 100 V for 30 min. Both the gel running and PVDF transfer steps were performed at 4 °C. PVDF membranes were treated with Blocking Buffer (LI-COR Bioscience) for 1 h at 4 °C, and then exposed to rabbit monoclonal anti-N1-phosphohistidine (1-pHis) (Millipore Sigma, cat no. MABS1330, clone SC1-1) 1:500-dilution for 16 h at 4 °C. PVDF membranes were washed and treated with IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences) for 1 h at 4 °C. PVDF membranes were washed and bands were visualized with an Odyssey Digital Infrared Imaging System (LI-COR Biosciences).

在 TMD 缓冲液中加入2mm ATP 处理 NAMPT (10μg ml-1) ,产生 pHisNAMPT。如有需要,还包括其他药物(SBI-797812、 NAM、 PRPP、 NMN、 PP)。样品在37 ° c 下培养10min,并与含有 sds 的样品制备缓冲液相结合。样品保存在冰上,在加载凝胶之前不加热。200ng 的蛋白质在4-20% 标准 TGX 凝胶(Bio-Rad)上在150v 下运行1.5 h,然后在100v 的聚偏二氟乙烯(PVDF)膜上转移30min。在4 ° c 条件下分别进行了凝胶运转和 PVDF 转移步骤。采用阻断缓冲液(LI-COR Bioscience)对 PVDF 膜进行4 ° c 处理1h,然后与兔抗 n1-磷酸组氨酸单克隆抗体1- 表达载体(Millipore Sigma,cat no。马布斯1330,克隆 SC1-1)1:500稀释4 °c 16h。用 IRDye 800CW 山羊抗兔 IgG (LI-COR Biosciences)对 PVDF 膜进行清洗和处理,在4 ° c 条件下处理1h。利用奥德赛数字红外成像系统(LI-COR 生物科学公司)对 PVDF 膜进行清洗和显示条带。

Cellular studies


Human A549 lung carcinoma cells (American Type Culture Collection; Manassas, VA, USA) were grown in DMEM, 4.5 g l−1 D-(+)-glucose, 10% fetal bovine serum, penicillin/streptomycin mixture; 10 cm dishes) and treated with DMSO (vehicle control) or SBI-797812 for 4 h. Cells were washed with cold PBS and immersed in liquid nitrogen. After decanting the liquid nitrogen, the cells were scraped, collected and stored at −80 °C. In a related protocol, A549 cells were exposed to media also containing 20 μM NAM (13C315N) (Cambridge Isotope Laboratories) along with vehicle or SBI-797812 for 4 h. Cells were harvested and processed as described above. Intracellular NMN, NAD+, NADP, NADH and NADPH were quantitated by LC-MS/MS (see Supplementary Information). LC-MS/MS was also used to measure the levels of NAM-containing metabolites that possessed the NAM(M + 4) moiety. Different cell extraction protocols were used for oxidized and reduced pyridine nucleotides. For oxidized pyridine nucleotides, 10 μl of the thawed cells was quickly removed for the protein assay, and 200 μl 1 M PCA was added to the remainder. Samples were vortexed, centrifuged and volumes were recorded. Samples were transferred to microfuge tubes and volumes were normalized to 400 μl with distilled water. A 100 μl aliquot was removed and used for targeted metabolite profiling. Total cellular protein was measured using the Pierce BCA Protein assay kit (ThermoFisher Scientific) with BSA as the standard. Reduced pyridine nucleotides were assayed similarly except that that 500 μl 50:50 0.1 M NaOH/MeOH was used to lyse the cells.

采用 DMEM 培养基、4.5 g l-1 d-(+)-葡萄糖、10% 胎牛血清、青霉素/链霉素混合液、10cm 培养皿培养人肺癌 A549细胞,并用 DMSO (载体对照)或 SBI-797812处理4h。液氮倒出后,刮取细胞,收集细胞,保存在 -80 °c。在一个相关的方案中,A549细胞暴露在同样含有20μM NAM (13C3,15N)的培养基中,连同载体或 SBI-797812一起培养4小时。细胞内 NMN、 NAD + 、 NADP、 NADH 和 NADPH 用 LC-MS/MS 定量(见补充信息)。同时采用液相色谱-串联质谱(LC-MS/MS)法测定了含 NAM-4的 NAM (m + 4)结构代谢产物的含量。采用不同的细胞提取方案对氧化还原吡啶核苷酸进行提取。对于氧化吡啶核苷酸,快速去除10 μl 融合细胞中的蛋白质,剩余部分加入200 μl 1 m PCA。对样品进行旋转、离心和体积记录。将样品转移到微量管中,用蒸馏水将体积归一化为400μl。取出100微升的碘片,用于靶向代谢产物分析。总细胞蛋白测定采用 Pierce BCA 蛋白测定试剂盒(ThermoFisher Scientific) ,BSA 为标准。除500 μl 50:500.1 mnaoh/meoh 溶解细胞外,其余还原吡啶核苷酸的测定结果基本一致。

Western blotting for sirtuin and PARP-1 activities

用于 sirtuin 和 PARP-1活动的西方墨点法

A549 cells (10 cm dishes) were treated with vehicle or SBI-797812 (10 μM) for 4 h as described above. Where indicated, 0.5 mM H2O2 was also added to the cells for 30 min (from 3.5 to 4 h). Cell lysis buffer contained RIPA, NAM (10 mM), trichostatin-A (10 μg ml−1; Sigma-Aldrich), protease inhibitor cocktail (1×, Roche), olaparib (5 μM; Cayman Chemical), ADP-HPD (250 nM; Millipore Sigma) and benzonase nuclease (0.2 U μl−1; Sigma-Aldrich). Where indicated (Fig. 5c), olaparib and ADP-HPD were excluded during the cell lysis step. Samples were probed by western blotting with the following antibodies: rabbit monoclonal anti-PARP1 (Cell Signaling, cat no. 9532, clone 46D11) 1:1000 dilution, rabbit polyclonal (affinity-purified) anti-PAR (Trevigen 4336-APC-050) 1:1000-dilution, rabbit monoclonal anti-histone H4 (Cell Signaling, cat no. 13919, clone D2X4V) 1:1000-dilution, and rabbit polyclonal (affinity-purified) anti-acetyl-histone H4(Lys16) (Millipore Sigma, cat no. 07-329) 1:5000-dilution. Protein bands were visualized with IRDye-labeled secondary antibodies (LI-COR Biosciences) followed by scanning with the Odyssey Digital Infrared Imaging System (LI-COR Biosciences).

A549细胞(10厘米培养皿)用载体或 SBI-797812(10微米)处理4小时。如果有,0.5 mM H2O2也加入细胞30分钟(从3.5到4小时)。细胞裂解缓冲液包括 RIPA、 NAM (10mm)、曲古抑菌素 -a (10μg ml-1; Sigma-Aldrich)、蛋白酶抑制剂鸡尾酒(1 × ,Roche)、 olaparib (5μM; Cayman Chemical)、 ADP-HPD (250nm; Millipore Sigma)和苯并酶核酸酶(0.2 μl-1;-aldrich Sigma)。如图5c 所示,奥拉帕尼和 ADP-HPD 在细胞裂解过程中被排除。用免疫组织化学方法检测兔抗 parp1(Cell signal,cat no. 9532,clone 46D11)1:1000稀释液,兔多克隆抗 par (Trevigen 4336-APC-050)1:1000稀释液,兔单克隆抗组蛋白 H4(Cell signal,cat no. 13919,clone D2X4V)1:1000稀释液,免疫组织化学方法检测兔抗 parp1(Cell signal,cat no. 13919,clone D2X4V)兔抗乙酰组蛋白 H4(Lys16)多克隆(亲和纯化)(密理波 Sigma,cat 号07-329)1:5000稀释。用 irdye 标记的二级抗体(LI-COR 生物科学)观察蛋白质带,然后用奥德赛数字红外成像系统(LI-COR 生物科学)进行扫描。

In vivo testing of SBI-797812


Eight-week-old male C57BL/6 J mice (The Jackson Laboratory) were fed a standard chow diet (Product #2016, Harlan Teklad). All animal studies and procedures were approved by the SBP-Orlando Institutional Animal Care and Use Committee (protocol # 2016-0136). After 1 h of fasting, mice were dosed by i.p. injection of vehicle (10% DMSO, 10% Tween 80 in sterile saline solution) or 20 mg kg−1 SBI-797812 (solubilized in vehicle). After 4 h, mice were administered Buthanasia-D (165 mg kg−1 body weight; i.p.). Tissues were harvested, flash frozen in liquid nitrogen, lyophilized to dryness and powdered using a Precellys Homogenizer (Bertin Instruments). Tissue powders (15 mg) were subjected to targeted metabolite profiling.

8周大的雄性 C57BL/6 j 小鼠(杰克逊纪念实验室)被喂以标准的食物(产品 # 2016 Harlan Teklad)。所有的动物研究和程序都得到了 SBP-Orlando 机构动物护理和使用委员会的批准(议定书 # 2016-0136)。空腹1h 后,静脉注射10% DMSO、10% Tween 80无菌盐水溶液或20mg · kg-1 SBI-797812(车载增溶剂)。4h 后,给予补他纳西 d (165mg/kg-1体重; i.p.)。组织被收集,在液氮中快速冷冻,冻干和粉末使用 Precellys 匀浆机(Bertin Instruments)。组织粉末(15毫克)受到靶向代谢物谱。

Quantitation of SBI-797812 in tissue homogenates

组织匀浆中 SBI-797812的定量分析

Mouse tissues were harvested 2 h after i.p. dosing of SBI-797812 (20 mg kg−1). Tissues were lyophilized and powdered using a Precellys bead-based homogenizer attached to a Crylolys cooling system. Tissue powders (2 mg) were homogenized in 200 µl of 50 mM Tris, pH 7.4 using the Precellys system. Homogenates (25 µl) were extracted with 100 µl ACN. Samples were vortexed and centrifuged at 18,000 × g for 5 min at 10 °C. Supernatants (100 µl) were passed through an AcroPrep Advance 3 K Omega Filter Plate by centrifugation at 3500 × g for 60 min. SBI-797812 in the deproteinized tissue extracts were fractionated using a Dionex Ultimate 3000 UHPLC outfitted with a 2.1 mm × 100 mm, 1.8 µm Cortecs C18 column (Waters Corp.) and run at 55 °C. Samples were injected (2 µl) by an autosampler maintained at 10 °C during the entire run. The mobile phase gradient was 95% A (0.1% formic acid in water) and 5% B (0.1% formic acid in ACN) to 50% A and 50% B over 5.2 min. The gradient began at 5% B (0.7 ml min−1flow rate), was increased from 5 to 10% B (0.7 ml min−1 flow rate) from 0.0–5.1 min, and was increased from 10 to 50% B (0.7 ml min−1 flow rate) from 5.1–5.2 min. The retention time for SBI-797812 was 4.78 min. The stock solution of SBI-797812 (50 mM) was prepared in DMSO. The working calibration solutions of SBI-797812 (0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 µM) were prepared by spiking the DMSO stock solution in 50 mM Tris buffer, pH 7.4. SBI-797812 was quantified with an Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer (Waters Corp.) operated in positive ion mode using electrospray ionization with an ESI capillary voltage of 3500 V. The electron multiplier voltage was set to 100 V. The ion transfer tube temperature was 325 °C and vaporizer temperature was 325 °C. The ESI source sheath gas flow was set at 10 l min−1. The mass spectrometer was operated with a mass resolution of 0.7 Da, cycle time of 1.9 cycles s−1, and nitrogen collision gas pressure was 45 psi for the generation and detection of product ion of SBI-797812. The SRM transition was 403 → 290 and the collision energy to produce the product ion was 25 V for SBI-797812. SBI-797812 raw data was processed using Mass hunter quantitative analysis software (Agilent). The SBI-797812 calibration curve was plotted using the raw area counts from known working calibration solutions.

静脉注射 SBI-797812(20mg kg-1)2h 后取小鼠组织。组织用 Precellys 珠状匀浆机连接 Crylolys 冷却系统进行冻干粉碎。采用 Precellys 系统,在 ph7.4,50mm Tris,200μl 的温度下均匀化组织粉末(2mg)。用100μl 活性炭纤维素(ACN)提取匀浆(25μl)。样品在10 °c,18,000 × g 温度下旋转离心5min。上清液(100μl)通过 AcroPrep Advance 3k Omega 过滤板,3500 × g 离心60min。采用 Dionex Ultimate 3000uhplc,配以2.1 mm × 100 mm,1.8 μm Cortecs C18柱(Waters corp.) ,在55 ° c 条件下分离得到组织脱蛋白提取物 SBI-797812。在整个运行过程中,样品由维持在10 ° c 的自动采样器注入(2μl)。流动相梯度为95% a (水中0.1% 甲酸)和5% b (ACN 中0.1% 甲酸)至50% a 和50% b,分别为5.2 min。梯度始于5% b (0.7 ml min-1流量) ,从0.0-5.1 min 由5% b (0.7 ml min-1流量)增加到10% b (0.7 ml min-1流量) ,从5.1-5.2 min 由10% b (0.7 ml min-1流量)增加到50% b (0.7 ml min-1流量)。SBI-797812的保留时间为4.78 min。在二甲基亚砜溶液中制备了 SBI-797812(50mm)原液。在50mm Tris 缓冲液中加入 DMSO 原液,ph7.4,制备了 SBI-797812(0.01,0.05,0.1,0.5,1,5,10,50μM)的工作标准溶液。用 Agilent 1290 HPLC/6490三重四极质谱仪(Waters corp.)在正离子模式下使用电喷雾毛细管电压为3500 v 的电喷雾离子法进行定量。电子倍增管电压设置为100v。离子转移管温度为325 ° c,蒸发器温度为325 ° c。电喷雾源鞘气体流量设定为10l/min-1。该质谱仪的质谱分辨率为0.7 Da,循环时间为1.9个循环 s-1,氮气碰撞压力为45psi,产生和检测产物离子。SBI-797812的 SRM 跃迁为403→290,产生产物离子的碰撞能为25v。利用安捷伦质量猎人定量分析软件对 SBI-797812原始数据进行处理。利用已知工作校准溶液的原始面积计数绘制了 SBI-797812校准曲线。

For details of the Medicinal Chemistry, Mass Spectrometry and Transition Structure Modeling see the Supplementary Methods.


Statistical analysis


Statistical analyses (ANOVA or t-test) were performed using GraphPad Prism software. For ANOVA, Dunnett’s multiple comparison test or Tukey’s multiple comparison tests were performed as indicated in the figure legends. Data was expressed as means ± s.d. P < 0.05 was considered statistically significant.

统计学分析(方差分析或 t 检验)使用 GraphPad Prism 软件进行。方差分析方面,Dunnett 的多重比较测验或 Tukey 的多重比较测验如图例所示进行。资料以平均值表示,p < 0.05具有统计学意义。

Reporting summary


Further information on research design is available in the Nature Research Reporting Summary linked to this article.


Data availability


The source data underlying Figs. 1c–f2a–e3a3c3d4a–h5a–c6a–d and Supplementary Figs. 112 are provided as a Source Data file. Any other relevant data is available upon reasonable request from the corresponding authors.

图1c-f、2a-e、3a、3c、3d、4a-h、5a-c、6a-d 和补充图1-12所示的源数据作为源数据文件提供。任何其他相关数据可在通讯作者的合理要求下获得。


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