Discovery of a Novel Nicotinamide Phosphoribosyl Transferase (NAMPT) Inhibitor via in Silico Screening

Abstract

Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC₅₀ of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC₅₀ of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT.

Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes a rate-limiting step in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis. Recently, intracellular NAD has received substantial attention due to the recent discovery that several enzymes, including poly(ADP-ribose) polymerase (PARP) and the Sirtuin-family proteins, use NAD as a substrate,^1–3) suggesting that intracellular NAD level may regulate cytokine production,⁴⁾ circadian rhythm,^5,6) metabolism, and aging^3,7) through these enzymes.

FK866, an NAMPT inhibitor, has been shown to induce apoptosis of cancer cells⁸⁾ and immune cells,⁹⁾ suggesting the possibility of NAMPT inhibitors as a novel drug class. As such, FK866 and CHS-828, another NAMPT inhibitor, are currently being investigated in clinical trials for cancer treatment.^10,11) However, these compounds were discovered by chance using cell-based assay,^12,13) and discovery of new NAMPT inhibitors with better structures has been hindered by the difficulty of conducting high-throughput screening (HTS) with good sensitivity. HTS presents difficulties because a radioisotope (RI)-labeled substrate is needed to retain good assay sensitivity, but a complicated procedure including separation of product from substrate prevents evaluation of large numbers of compounds.

Here we report our finding of novel compounds with potent NAMPT inhibitory activity as determined using in silico screening and a 96-well-based sensitive RI-based assay.

MATERIALS AND METHODS

Reagents

FK866 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). AS1604498, AS2292427 and AS2334990 were in our chemical library, and the structure was confirmed via electrospray mass spectrometry, and ¹H nuclear magnetic resonance (¹H-NMR) or UV spectrometry. Cell Counting Kit-8 kits were purchased from Dojindo (Kumamoto, Japan). Caspase-Glo™ 3/7 Assay kits were purchased from Promega (Madison, WI, U.S.A.). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), phenazine ethosulfate, βNAD, and alcohol dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [¹⁴C]-Nicotinamide was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, U.S.A.).

In Silico Screening

Human NAMPT and FK866 complex structure data (PDB code: 2GVJ) were used as a template for the docking simulation. To prepare the protein for docking, hydrogens were added to the complex structure, and energy was minimized with all heavy atoms restrained by the Protein Preparation module (Schrodinger, LLC, Portland, OR, U.S.A.). Virtual screening experiments were performed using the docking program GOLD 4.1 (CCDC, Cambridge, U.K.) for our proprietary chemical inventory. The docking simulation reproduced the conformation of co-crystallized FK866. Top-scoring compounds were subjected to the in-house post-docking procedure¹⁴⁾ to be re-ranked for the final selection.

Cell Culture

THP-1 (JCRB Cell Bank, Osaka, Japan) and K562 cells (RIKEN Bioresource Center, Ibaraki, Japan) were grown as suspension cultures in RPMI1640 (Invitrogen, Grand Island, NY, U.S.A.). Media were supplemented with 10% fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS, U.S.A.), 100 units/mL penicillin, and 100 µg/mL streptomycin. All cell lines were incubated in a humidified atmosphere containing 5% CO₂ at 37°C.

Cloning and Expression of NAMPT cDNA

Full-length mouse NAMPT (residues 1–491) was cloned from Mouse Liver Marathon-Ready cDNA (Clontech, Mountain View, CA, U.S.A.) into pET21a vector (NOVAGEN, WI, U.S.A.) with an His₆ tag at the N-terminal, and expressed in Escherichia coli BL21 strain at 15°C for 24 h. The protein was purified using nickel-agarose affinity chromatography (QIAGEN, Venlo, the Netherlands). Full length human NAMPT (GenBank Accession Number: U02020) was cloned into a pET26b vector (NOVAGEN) with an His₆ tag at the C-terminal as described in a previous report¹⁵⁾ and purified as described above. The purity was found to be >90% using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by Coomassie Brilliant Blue staining.

Measurement of NAMPT Activity

NAMPT activity was determined by miniaturization of the methods described by Eliott et al.¹⁶⁾ Briefly, the enzyme activity was determined in 70 µL of reaction solution consisting of 50 mM Tris–HCl (pH 8.8), 2 mM ATP, 5 mM MgCl₂, 0.5 mM 5′-phosphorylribose-1-pyrophosphate (PRPP), 1 µM [¹⁴C]-nicotinamide (1.5×10⁵ cpm/nmol), 5 µg/mL of recombinant NAMPT, and compound in 1% dimethyl sulfoxide (DMSO) for 1 h at room temperature. After the reaction, 50 µL of reaction solution was added to deep-welled plates filled with 1 mL acetone and absorbed through GF/B plates. The plates were then dried at 50°C for 1 h and measured using a Topcount NXT (Packard, Meriden, CT, U.S.A.).

Cell Culture Assays

The cells were plated at 1.5–2×10⁴/100 µL in 96-well plates and incubated with compounds for the time indicated in Figs. 3 and 4. The WST-8 assay was performed in accordance with the manufacturer’s protocol (Dojindo, Kumamoto, Japan) and measured at an optical density of 450 nm. In some experiments, nicotinamide was added to examine whether nicotinamide reversed the growth inhibition caused by test compounds. To determine Caspase activation, cells were plated at 1×10⁴/100 µL, and Caspase-Glo™ 3/7 Assay was conducted in accordance with the manufacturer’s protocol.

NAD Measurement

NAD was quantified via an enzymatic cycling procedure described by Pittelli et al.¹⁷⁾ with slight modification. Briefly, K562 cells were plated at 1×10⁵/200 µL in 96-well plates and incubated with compounds for 5 h. After washing out with PBS chilled to 4°C, cells were treated with 50 µL of 1 N HClO₄ for 15 min on ice and then neutralized with an equal volume of 1 N KOH and 100 µL of Bicine (200 mM, pH 8). After 25 µL of the cell extract was diluted with the same volume of 100 mM Bicine buffer with a new plate, the dilution was mixed with an equal volume of the Bicine buffer containing 23 µL/mL of ethanol, 0.17 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.57 mg/mL of phenazine ethosulfate, and 2.5 µg of alcohol dehydrogenase. The mixture was kept at room temperature for 30 min, and then absorbance at 570 nm was measured using an Infinite M1000 microplate reader (Tecan, Männedorf, Switzerland). A standard curve allowed quantification of NAD.

Statistical Analysis

Significance was assessed using one-way analysis of variance followed by Dunnett’s multiple comparison test. Values of p<0.05 were considered significant.

RESULTS

Rational Approach to Identifying NAMPT Inhibitors

To identify NAMPT inhibitors with novel structures, we first evaluated all compounds in our in-house chemical library using computer modeling using the data of co-crystal structures of NAMPT and FK866, selecting 468 compounds for further examination. These compounds were then evaluated with a mouse NAMPT assay, and 14 compounds showed over 50% inhibition at 5 µM, our first criteria. Although these 14 did not show potent activity in the cell culture assay, we performed a second cycle with 274 analogs of these compounds using mouse NAMPT assay, and 25 showed over 50% inhibition at 1 µM. Three compounds showed over 50% inhibition in the cell culture assay as well and were selected for validation. These compounds showed no activity at 5–10 µM with other protein-based HTS, indicating that the activity of these compounds is highly specific to NAMPT. These compounds’ structures are shown in Fig. 1. AS2292427 and AS2334990 had similar characteristics, but since AS1604498 showed the greatest NAMPT inhibition and was therefore the most promising candidate, this compound became the focus of our study.

Fig. 1. Structure and Molecular Basis of Hit Compounds and FK866

(A) Chemical structures of hit compounds and FK866. (B) Docking mode of AS1604498 (orange) and the crystal structure of FK866 (green) with NAMPT.

In the docking simulation, AS1604498 exhibited a binding mode similar to that of FK866 in the complex structure determined by X-ray crystallography.¹⁵⁾ All residues interacting with AS1604498 were involved in hydrophobic contacts. The imidazole ring of AS1604498 showed good overlap with the pyridine ring of FK866, located between the side chains of Phe193 and Tyr18′. Note that FK866’s binding pocket is involved in the dimer interface, with the prime indicating the second monomer. The distal benzene ring of AS1604498 occupied the same region as that of FK866.

AS1604498 Showed the Most Potent Inhibitory Activity in the Human NAMPT Enzyme Assay

We further evaluated these 3 compounds with human NAMPT activity. As shown in Fig. 2, these compounds dose-dependently inhibited the production of nicotinamide mononucleotide, with AS1604498 showing the highest potency. IC₅₀ values were 44 nM for AS1604498, 81 nM for AS2292427, and 556 nM for AS2334990. The IC₅₀ value of FK866 for this assay was 51 nM, suggesting that AS1604498 has similar potency to FK866 in the human NAMPT enzyme assay.

Fig. 2. Inhibition of Human NAMPT Activity by Hit Compounds

The inhibition of human NAMPT activity by (A) AS1604498 (B) AS2292427 (C) AS2334990 (D) FK866. The values are expressed as mean±S.D. (n=2) and are from representative examples of three independent experiments.

AS1604498 Inhibited Cell Growth

We next evaluated the effects of these compounds in cell-based assay with respect to THP-1 viability, as depletion of NAD in cells is known to cause apoptosis.⁸⁾ As shown in Fig. 3A, AS1604498 inhibited THP-1 proliferation, and addition of 10 mM Nam reversed the inhibitory effect of AS1604498, suggesting that the inhibitory effect of AS1604498 was based on inhibition of NAMPT with AS1604498 working as a competitive inhibitor like FK866. FK866 showed a similar effect to AS1604498, but with higher potency (Fig. 3B). AS2292427 and AS2334990 also showed similar effects to AS1604498, but with lower potency (Table 1).

In addition, we also assessed the effects of these compounds on K562 chronic myelogenous leukemia cells, with results showing that all three hit compounds as well as FK866 exerted inhibitory activity against the cells (Figs. 3C, D, Table 1).

Fig. 3. Dose-Dependent Inhibition of Cell Growth and Reversal by Nicotinamide

Cells were seeded with compounds, and the growth was measured after 48 h with WST-8. AS1604498 inhibited A) THP-1 cell and B) K562 cell growth, and 10 mM Nicotinamide reversed the effect. FK866 inhibited C) THP-1 cell and D) K562 cell growth as well. Each column shown indicates mean±S.D. (n=3), and data are from a representative example of two independent experiments.

Table 1. Inhibitory Activity of Hit Compounds

	Human NAMPT IC50 (nM)	THP-1 growth IC50 (nM)	THP-1 growth (+Nam) IC50 (μM)	K562 growth IC50 (nM)	Caspase 3/7 activation	NAD inhibition
FK866	51.0±8.1	0.295±0.024	>10	1.46±3.7	+ (100 nM)	+
AS1604498	44.4±4.8	198±31	>10	673±190	+ (10 µM)	+
AS2334990	556±2	730±100	>10	3608±44	+ (10 µM)	+
AS2292427	80.8 ±6.4	894±306	>10	3071±1070	+ (10 µM)	+

Results are presented as the mean±standard error of at least two independent experiments.

AS1604498 Caused Cell Apoptosis

Since FK866 is known to induce apoptosis in cells, including cancer cell lines, we evaluated caspase activation in THP-1 cells. As shown in Fig. 4, apoptosis inducers Etoposide and Actinomycin D induced activation of caspase 3/7 after 5 h, while AS1604498 showed delayed activation, similar to findings with FK866. Further, unlike Etoposide or Actinomycin D, the caspase activation induced by AS1604498 was inhibited by Nicotinamide, strongly suggesting that AS1604498 causes apoptosis through NAD depletion. AS2292427 and AS2334990 also showed similar effect (Table 1).

Fig. 4. Delayed Apoptosis against THP-1 Cells Caused by NAMPT Inhibitors

THP-1 cells were treated with compounds for (A) 5 h and (B) 48 h with or without 10 mM nicotinamide (Nam), respectively. Cells were lysed and measured for caspase 3/7 activity. Each column shown indicates mean±S.D. (n=2) and are from a representative example of two independent experiments. Compounds were abbreviated as follows: AS, AS1604498; FK, FK866; ETO, Etoposide; ACTD, Actynomicin D, with concentrations of 10 µM, 100 nM, 10 µM, and 5 µg/mL, respectively. * p<0.05; ** p<0.001 vs. respective controls.

AS1604498 Caused NAD Reduction in Cell-Based Assay

To confirm the effect of the novel NAMPT inhibitors, we evaluated the direct effect of these drugs on the NAD concentration in the cells. K562 cells were plated in a 96-well plate with the compounds, and after 5 h, NAD was quantified using an enzymatic cycling procedure.¹⁷⁾ AS1604498 inhibited the concentration of NAD in the cells (Fig. 5A), similar to the effect of FK866 (Fig. 5B), but these inhibitory effects were completely reversed by the addition of 10 mM Nam (data not shown). AS2292427 and AS2334990 showed results similar to those achieved with AS16044498 (Table 1).

Fig. 5. Effect on Intracellular NAD Level by NAMPT Inhibitors

K562 Cells were treated with compounds for 5 h and the contents of NAD were measured. Each column represents the mean±S.D. (n=2) of representative examples of at least two experiments.

DISCUSSION

While progression in technology has changed how various stages of drug development research are conducted, some parts of enzyme assay, such as RI-based assay, remain problematic due to complicated procedures and necessary rate-limiting steps. Here, we combined an in silico screening strategy with a traditional RI-based enzyme assay and identified novel NAMPT inhibitors easily in our large-scale library.

Our hit compounds inhibited NAMPT with high potency, showed cell growth inhibition, delayed apoptosis induction, and caused NAD reduction in the cells. These effects were reversed by addition of nicotinamide, strongly suggesting that these effects are based on NAMPT inhibition.⁸⁾

The reason why these compounds showed moderate potency against cell-based assay despite the potent activity in the enzyme assay remains unclear. We evaluated the protein binding, stability in human serum, log D and Parallel Artificial Membrane Permeability Assay for both AS1604498 and FK866, and no major differences were noted in these assays (data not shown). Therefore, differences in potency between cell-based assay and enzyme assay are likely to be induced via excretion from the cell, protein binding in the cell, or degradation of compounds. Further investigation is needed to clarify the mechanism leading to reduced potency in cell-based assay. Chemical modification may be able to improve this deficiency.

Conflicting results have been reported for the effects of FK866 on K562 leukemia cells. While Nahimana et al. reported an EC₅₀ value of 7.2 nM using the MTT assay,¹⁸⁾ Thakur et al. found no inhibition at 100 nM using the trypan blue exclusion assay.¹⁹⁾ Here, we showed that FK866 inhibits K562 cell growth with high potency using the WST-8 assay, which is similar to the MTT assay. This discrepancy in findings may be due to differences in the mode of measurement. In our observation, FK866 inhibited K562 cell growth immediately but required time to kill this cell lineage, possibly because of the P53 mutation.²⁰⁾ In addition, K562 cells did not grow well when cell confluency was low. These findings suggest that Thakur et al. may not have adequately evaluated the effect of FK866 on K562 cells. We confirmed that FK866 did indeed inhibit K562 cell growth using a DNA content-based assay (data not shown).¹⁹⁾

We demonstrated that our hit compounds reduced NAD content in the cytoplasm to about 60% of baseline levels at 5 h after addition of hit compounds and FK866. Previous studies showed time-dependent decreases in NAD induced by FK866 in THP-1 and other cells,^12,17) and our data showed that caspase 3/7 activation in THP-1 occurred after 24 h (data not shown), suggesting that apoptosis was caused by NAD depletion.

In conclusion, our result showed that the combination of in silico screening and a low-throughput screening method is effective at identifying potential chemicals for drug development, and that NAMPT is a suitable target for in silico screening. Our hit compounds—including AS1604498, which we found to be the most highly potent inhibitor—are promising candidates to be modified for new therapies as well as for use as research tools.

Acknowledgment

We thank Dr. Katsuyuki Maki and Dr. Masato Watanabe for their invaluable suggestions and support.

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