NAMPT (IC50 = 0.023 μM)

Evaluation of ATTEC-Induced NAMPT Degradation [1]
Initially, the NAMPT inhibitory activities of all the designed NAMPT autophagy degraders were determined by the assays described previously. As shown in Table 1, all the target compounds showed good inhibitory activities with the IC50 values ranging from 10 to 34 nM, suggesting the good binding affinity between the target compounds and NAMPT. Then, we investigated whether target compounds could degrade NAMPT in human ovarian cancer A2780 cells, in which NAMPT is overexpressed. The results indicated that all the compounds were able to trigger NAMPT degradation after treatment for 48 h (Figure 3A). The degradation efficiency increased gradually along with the linker length. When the length was 5–7 atoms (compounds A6, A1 and A2), they showed relatively weak efficiency with the maximum degradation rates in the range of 1–9% at 0.1 μM. As the length was extended to 8-atom (NAMPT degrader-1 (compound A3)), it showed the best degradation activity. NAMPT degrader-1 (compound A3) induced the NAMPT degradation in a concentration- and time-dependent manner (Figure S3 in the Supporting Information) with a maximal degradation rate of 91% at 3 μM. When the linker length was further extended to 10-atoms (compound A5), the degradation activity was obviously decreased (maximum degradation rate: 33% at about 0.1 μM). In addition, surface plasmon resonance (SPR) experiments were performed to further verify the binding affinity between compound A3 and LC3 protein, with a KD value of 933 nM (Figure S4 in the Supporting Information). Ispinesib was a well-known kinesin spindle protein (KSP) inhibitor. Therefore, we further evaluated the selectivity of compound A3 by monitoring the degradation of KSP. As shown in Figure S5, compound A3 had no effect on KSP degradation, indicating the selective NAMPT degrading activity. To evaluate whether the NAMPT degradation could led to antitumor potency, the cellular antiproliferative activity of compound A3 was assayed for several cell lines (Figure 3B), including A2780, MBA-MB-231 (NAMPT overexpression), HCT116 (NAMPT in normal), and A549 (NAMPT low expression, Figure S6 in the Supporting Information). Interestingly, the excellent inhibitory activities were observed in cell lines with overexpressed NAMPT. Compound A3 also significantly inhibited the viability of HCT116 cells, which were highly dependent on the NAD salvage pathway for survival. In particular, the IC50 value was 46 nM for compound A3 against A2780 cells, which was significantly more potent than NAMPT inhibitor MS2 (IC50 = 490 nM). Moreover, compound A3 was stable under the experimental condition and did not degrade into free drugs (Figure S7 in the Supporting Information). Investigation of the Mechanism of Action of NAMPT Autophagic Degrader NAMPT degrader-1 (compound A3) [1]
The underlying mechanism for the NAMPT-degrading effect of NAMPT degrader-1 (compound A3) was further studied. First, direct target engagement was performed in the cell lysate by cellular thermal shift assay (CETSA) to investigate whether the designed compounds bound to NAMPT directly. As shown in Figure S8, treatment with NAMPT degrader-1 (compound A3) enhanced the stability of NAMPT proteins compared to the blank control, indicating that it was capable of stabilizing NAMPT in cells. In addition, the CETSA assay also revealed that compound A3 was able to bind with KSP, which might partly contribute to the cytotoxicity of compound A3. Thus, as an ATTEC warhead, ispinesib remained to be further optimized to reduce the binding activity to KSP. In order to investigate whether the target compounds caused the NAMPT degradation via lysosomal-mediated autophagy, different autophagy inhibitors (ammonium chloride, chloroquine, wortmannin, 3-methyladenine, and LY294002) were added to evaluate their effects on reversing the expression level of NAMPT. Among them, ammonium chloride (NH4Cl) and chloroquine can block the function of lysosomes by acidifying the environment of lysosomes and then impede the fusion process of autophagy and lysosome. Wortmannin, 3-methyladenine and LY294002 are able to block or interfere with the formation of autophagosome, thus inhibiting autophagy. (19) As shown in Figure 4A, the total protein level of NAMPT was reversed upon the addition of autophagy inhibitors, indicating that compound A3 induced NAMPT degradation through lysosomal-mediated autophagy. Meanwhile, the coculture of NAMPT inhibitor MS2 or FK866 also led to the recuperation of NAMPT expression, indicating that the binding mode of compound A3 with NAMPT was similar to that of FK866 and MS2. LC3 ligand ispinesib was added to compete with compound A3 for binding with LC3 protein. However, the A2780 cells were all dead after pretreating with ispinesib because of high toxicity. As an alternative, probe P1 was used in the competitive experiment and the result revealed that the degrading activity was reversed due to the blockage of LC3 binding (Figure S10 in the Supporting Information). Atg7 is essential to lysosome-mediated autophagy. (13) When Atg7 was knocked down through infection via lentivirus in A2780 cells, compound A3 was unable to induce NAMPT degradation (Figure 4B), indicating that the degradation relied on the pathway of lysosome-mediated autophagy.

NAMPT Enzymatic Inhibition Assay [1]
All of the enzymatic reactions were conducted at 30 °C for 90 min in a 50 μL mixture containing 50 mM Tris HCl (pH 8.0), 12.5 mM MgCl2, 2 mM ATP, 1 mM DTT, 0.02% bovine serum albumin (BSA), 0.4 mM phosphoribosyl pyrophosphate, 20 μM nicotinamide, 30 μg/mL of alcohol dehydrogenase, 1.5% alcohol, 0.01% Tween 20, 10 μg/mL NAMPT, and the test compounds. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm, and then, the fluorescent intensity data were analyzed using Graphpad Prism.

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Fluorescence Polarization Measurements [1]
KD values were measured using the fluorescence anisotropy assay with fluorescein-labeled ispinesib as the ligand in black flat-bottom half-area 96-well plates. The fluorescein-labeled ispinesib was diluted to 50 nM with N-(2-hydroxyethyl)piperazine-N’-ethanesulfonic acid (HEPES) buffer. A twofold serial dilution of LC3 protein in HEPES buffer (100 μL) was added to a 200 μL of solution of fluorescent probe (final concentration at 25 nM). The fluorescence anisotropy was measured on BioTek Synergy H2 with the 485 nm excitation and 535 nm emission filters after 30 min incubation at room temperature. The KD values were determined by fitting the displacement curves to the following equation using Mathamatica 9

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Isothermal Titration Calorimetry [1]
The thermodynamic parameters of the binding of ispinesib to recombinant LC3B were determined by ITC using VP-ITC or ITC200 calorimetry. Since the solubility of ispinesib in the ITC buffer is relatively low, we performed the titration by injecting a 12 μl of aliquot of LC3B protein sample (1500 μM) into the cell containing 100 μM ispinesib (the ITC buffer includes 20 mM HEPES-HCl, pH 7.3, 1% DMSO, and 100 mM NaCl) at a time interval of 120 s to ensure that the titration peak returned to baseline. Altogether, 13 aliquots were titrated in each individual experiment. The dissociation constant KD, stoichiometry of binding (n), and the binding enthalpy ΔH were calculated using MicroCal Origin 7.0 software with a one-site binding model.

Western Blot Analysis [1]
A2780 cells were seeded in six-well plates at a density of 1 × 106/well. After incubation at 37 °C with 5% CO2 overnight, compounds at different concentrations were added. After 48 h, the medium was discarded. Cells were washed with phosphate-buffered saline (PBS) three times and then lysed on ice with RIPA cell lysis buffer for 30 min. Cell lysates were collected and centrifuged at 1.2 × 105 g at 4 °C for 15 min. The supernatants were quantified via the bicinconinic acid assay kit and then degenerated at 100 °C. Then, they were analyzed at an equal amount of protein via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Subsequently, they were transferred to the polyvinylidene fluoride membrane and then blocked with 5% BSA in Tris-buffered saline and Tween 20 at room temperature for 2 h. Then, they were incubated with primary antibodies at 4 °C overnight separately. After being washed with PBS three times every 10 min, they were incubated with secondary antibodies at room temperature for 1 h. After being washed with PBS another three times every 10 min, the membranes were read by Odyssey Infrared Imaging.

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Cell Proliferation Assay [1]
Cells were seeded at a density of 5 × 103 cells per well. After being incubated overnight, they were treated with DMSO, MS2, or A3 as indicated for 72 h. The culture medium was discarded, and 10% CCK8 containing culture medium was added. After being incubated at 37 °C, the OD values at a wavelength of 450 nm were read. The cell proliferation rates versus concentration of compounds was analyzed using Graphpad Prism.

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Cell Thermal Shift Assay [1]
A2780 cells were seeded at a density of 1 × 107/well and then incubated at 37 °C with 5% CO2 overnight. Then, compounds at the indicated concentration were added. After 4 h, culture medium was discarded and the cells were collected and lysed under treatment in liquid nitrogen for 5 min (three times). After being centrifuged at 1.2 × 105 g at 4 °C for 15 min, the supernatants were divided into four parts and heated at 48 , 52, 55, and 58 °C, respectively. Subsequently, they were centrifuged at 1.2 × 105 g at 4 °C for 15 min. The supernatants were degenerated at 100 °C and then analyzed via SDS-PAGE to detect the expression level of NAMPT protein.

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Knockdown of Atg7 in A2780 Cells [1]
Cells were seeded into 24-well plates at a density of 1.5×105/well. After being incubated at 37 °C with 5% CO2 overnight, 250 μL of medium containing shRNA and 2 μg/mL polybrene was replaced for culture medium. After 4 h, another 250 μL of culture medium was added. After 24 h, the medium was discarded and replaced by fresh culture medium. Cells were collected after another 48 h for analysis of the expression level of Atg7.

[1]. Ispinesib as an Effective Warhead for the Design of Autophagosome-Tethering Chimeras: Discovery of Potent Degraders of Nicotinamide Phosphoribosyltransferase (NAMPT). J Med Chem. 2022 Jun 9;65(11):7619-7628.

In summary, we discovered that ispinesib is an effective targeting ligand for designing autophagosome-anchored chimeras. Using ispinesib as the LC3 ligand, we designed and synthesized a series of novel NAMPT autophagy degraders. NAMPT degrader-1 (compound A3) effectively degrades NAMPT, exhibiting excellent antitumor activity. Mechanistic studies confirmed that NAMPT degrader-1 (compound A3) degrades NAMPT via a lysosome-mediated autophagy pathway. PROTAC technology has become a promising method in drug discovery and development. Recently, we used PROTAC technology to design and screen several effective NAMPT PROTAC degraders. Although the degradation activity of compound A3 was not superior to NAMPT PROTAC, this work demonstrates that NAMPT can also be effectively degraded by the ALP system, providing a new strategy for targeted NAMPT degradation. Furthermore, we discovered that ispinesib is an effective degradation tag and provides a useful tool for ALP-based targeted protein degradation. However, it should be noted that ispinesib is a potent KSP inhibitor and needs further optimization to improve its affinity and selectivity for LC3 protein. Currently, we are investigating the application of the LC3-binding ispinesib to the degradation of more drug targets and optimizing the structure of the reactive group of ispinesib. [1]

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Autophagosome anchored compounds (ATTECs) are an emerging technology in the field of targeted protein degradation. However, effective tools and successful cases for autophagosome anchored chimeras are still quite limited. This paper is the first to discover that ATTEC ispinesib is an effective reactive group for designing autophagosome anchored chimeras. As a proof-of-concept study, we developed a first-generation nicotinamide phosphoribosyltransferase (NAMPT) autophagy degrader by linking a NAMPT inhibitor and the LC3-binding ispinesib with a flexible linker. In particular, compound A3 significantly induced NAMPT degradation through the autophagy-lysosome pathway, thereby exhibiting excellent cellular antitumor activity. Ispinesib may have broad application prospects in the design of highly efficient autophagosome anchored chimeras. [1]

C56H68CLN9O5S2

1046.78

1045.44733

3011778-39-2

163409181

Typically exists as solid at room temperature

8.9

3

10

24

73

1880

1

CC1=CC=C(C=C1)C(=O)N(CCCNC(=O)CCCCCCCCN2CCN(CC2)S(=O)(=O)C3=CC=C(C=C3)NC(=S)NCC4=CN=CC=C4)[C@@H](C5=NC6=C(C=CC(=C6)Cl)C(=O)N5CC7=CC=CC=C7)C(C)C

LZCAADBODOWVEZ-OIVUAWODSA-N

InChI=1S/C56H68ClN9O5S2/c1-41(2)52(53-62-50-37-46(57)23-28-49(50)55(69)66(53)40-43-15-9-8-10-16-43)65(54(68)45-21-19-42(3)20-22-45)32-14-30-59-51(67)18-11-6-4-5-7-12-31-63-33-35-64(36-34-63)73(70,71)48-26-24-47(25-27-48)61-56(72)60-39-44-17-13-29-58-38-44/h8-10,13,15-17,19-29,37-38,41,52H,4-7,11-12,14,18,30-36,39-40H2,1-3H3,(H,59,67)(H2,60,61,72)/t52-/m1/s1

N-[(1R)-1-(3-benzyl-7-chloro-4-oxoquinazolin-2-yl)-2-methylpropyl]-4-methyl-N-[3-[9-[4-[4-(pyridin-3-ylmethylcarbamothioylamino)phenyl]sulfonylpiperazin-1-yl]nonanoylamino]propyl]benzamide

NAMPT degrader-1; CHEMBL5170949; BDBM50605982;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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