CHEMOTHERAPY, Aug. 1990, p. 1473-1479

0066-4804/90/081473-07$02.00/0 Copyright C 1990, American Society for Microbiology

Vol. 34, No. 8

In Vitro Evaluation of Nicotinamide Riboside Analogs against Haemophilus influenzae CHRISTOPHER P. GODEK AND MICHAEL H. CYNAMON*

Department of Medicine, Veterans Administration Medical Center, and State University of New York Health Science Center, Syracuse, New York 13210 Received 24 January 1990/Accepted 7 May 1990

Exogenous NAD, nicotinamide mononucleotide, or nicotinamide riboside is required for the growth of Haemophilus influenzae. These compounds have been defined as the V-factor growth requirement. We have previously shown that the internalization of nicotinamide riboside is energy dependent and carrier mediated with saturation kinetics. Thionicotinamide riboside, 3-pyridinealdehyde riboside, 3-acetylpyridine riboside, and 3-aminopyridine riboside were prepared from their corresponding NAD analogs. These compounds and several other nicotinamide riboside analogs were evaluated for their ability to support the growth of H. influenzae and for their ability to block the uptake of [carbonyl-'4C]nicotinamide riboside by H. influenzae. 3Aminopyridine riboside blocked the uptake of [carbonyl-14CJnicotinamide riboside and inhibited the growth of H. influenzae when NA), nicotnamide mononucleotide, or nicotinamide riboside served as the V factor. The antibacterial activity of 3-aminopyridine riboside was found to be specific for H. influenzae but had no effect on the growth of Staphylococcus aureus or Escherichia coli. In additional experiments by reversed-phase highperformance liquid chromatography, it was determined that whole cells of H. influenzae degrade 3aminopyridine adenine dinucleotide to 3-aminopyridine riboside, which is then internalized. Inside the cell, 3aminopyridine riboside has the ability to interfere with the growth of H. influenzae by an undetermined mechanism.

Early studies regarding Haemophilus species determined that Haemophilus parainfluenzae requires NAD (V factor) for growth (21-23). Similar studies with Haemophilus influenzae found that both NAD and hemin (X factor) are required for growth (7, 8). The inability of H. influenzae to grow in the presence of quinolinic acid or nicotinic acid indicated the lack of a de novo biosynthetic pathway or a Preiss-Handler pathway (11). The inability of nicotinamide (Nam) to serve as a V factor indicated that H. influenzae was also incapable of synthesizing NAD through any of the previously investigated pyridine nucleotide cycles (10, 14). The ability of nicqtinamide riboside (NR) and nicotinamide mononucleotide (NMN) to support the growth of H. influenzae suggested the existence of a synthetic NAD pathway in which NMN or NR is used as a precursor (3, 12, 21, 25). Subsequent studies with H. influenzae determined that NAD is initially cleaved to NMN and AMP by a periplasmic nucleotide pyrophosphatase (15). NMN is then dephosphorylated to NR and internalized (5). Inside the cell, NR is either metabolized to NAD or is degraded to Nam and ribose-5-phosphate. Intracellularly produced NAD can be degraded to Nam and ADP-ribose by an NAD glycohydrolase, as shown in Fig. 1 (3). The utilization of exogenous NAD and the synthetic pathway of H. influenzae are unusual among pathogenic bacteria. This pathway offers several specific targets for the development of selective antimetabolic agents. Here we report the effects that NAD and NR analogs have on the growth of H. influenzae and the internalization of NR.

obtained from the Clinical Microbiology Laboratory, Health Science Center, State University of New York at Syracuse, Syracuse. Chocolate agar plates (Remel, Lenexa, Kans.) or brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) supplemented with 3 ,uM NAD and 15.8 ,uM hematin was used to culture the H. influenzae (6). The V-factor requirement for optimal growth could be provided either by NADH or NADP at 3 p.M or by NMN or NR at 10 ,uM in BHI broth (37 g of BHI per liter of distilled H20). Viable cell counts were determined by dilution on chocolate agar plates by using 0.9% NaCl with 0.05% Tween 80 for dilutions. Chemicals and reagents. NAD, NADH, NADP, NMN, nicotinic acid adenine dinucleotide (NAAD), nicotinamide hypoxanthine dinucleotide (NHD), nicotinamide guanine dinucleotide (NGD), nicotinamide 1,N6-ethenoadenine dinucleotide (NED), Nam, 3-aminopyridine adenine dinucleotide (3-AmPAD), 3-acetylpyridine adenine dinucleotide (3AcPAD), thionicotinamide adenine dinucleotide (Thio NAD),2,4-dinitrophenol (DNP), hematin, and ATP were obtained from Sigma Chemical Co. (St. Louis, Mo.). 5-(P-DRibofuranosyl)-nicotinamide (RFN), 1-(P-D-ribofuranosyl)-2 (1H)-pyridone-3-carboxamide (SK32023), 1-(P-D-ribofuranosyl)-6(1H)-pyridone-3-carboxamide (SK32024), 1-(P-D-arabinofuranosyl)-2-(lH)-pyridone-3-carboxamide (SK32025),2, 2'-anhydro-2-hydroxy-1- -D-arabinofuranosylnicotinamide (SK32027), and 6,2 -anhydro-6-hydroxy-1-,3-D-arabinofuranosyl nicotinamide (SK32028) were graciously supplied by Krzystztof W. Pankiewicz (Memorial Sloan-Kettering Cancer Center, Rye, N.Y.). 2-p-D-Ribofuranosylpyrimidine-4carboxamide (SW-17) and ribavirin were obtained from Roland K. Robins (Nucleic Acid Research Institute, Costa Mesa, Calif.). Nicotinamide xyloside was supplied by John Welch (State University of New York at Albany, Albany). Tiazofuran was supplied by Nancita R. Lomax (National


Organisms and culture media. The H. influenzae strain our studies was clinical isolate T4681, which was

used in *







Nicotinamide Adenine Dinucleotide

Nicotinamide mononucleotide




Nicotinamide riboside


cel meb Nicotinamide

Nicotinamide riboside


-_o mononucleotide 5


6+ Nicotinamide Adenine Dinucleotide

FIG. 1. NAD degradative and synthetic pathway for H. influEnzyme activities were identified as follows: 1, nucleotide pyrophosphatase; 2, phosphatase; 3, nucleoside phosphorylase (end product includes ribose 5'-phosphate from an inorganic phosphate); 4, nicotinamide ribonucleoside kinase (requires both ATP and Mg2+); 5, nicotinamide mononucleotide adenyltransferase (requires both ATP and Mg2+); 6, nicotinamide adenine dinucleotide glycohydrolase. enzae.

Cancer Institute, Bethesda, Md.). 3-Pyridinealdehyde adenine dinucleotide (3-PAAD) was obtained from P-L Biochemicals, Inc. (Milwaukee, Wis.). Deuterium oxide was obtained from Aldrich Chemical Co., Inc. (Milwaukee, Wis.). The [carbonyl-14C]NAD (specific activity, 44 mCi/ mmol) was obtained from Amersham Corp. (Arlington Heights, Ill.). NR, [carbonyl-14C]NR, 3-aminopyridine riboside (3AmPR), 3-acetylpyridine riboside (3-AcPR), thionicotinamide riboside (ThioNR), and 3-pyridinealdehyde riboside (3-PAR) were prepared through a two-step enzymatic degradation process starting with the respective dinucleotide compound. The dinucleotide was dissolved in 50 mM Tris hydrochloride buffer (pH 7.4, 37°C) that was supplemented with 10 mM MgC12 to a final concentration of 1 mg/ml and was then treated with a snake venom nucleotide pyrophosphatase isolated from Crotalus atrox venom (Sigma) (17, 19). The nucleotide pyrophosphatase was added at a concentration of 0.7 mg/ml. Incubation at 37°C for 8 h resulted in almost complete conversion of the dinucleotide compounds to the respective mononucleotides, as indicated by thin-layer chromatography (TLC), which was performed as described below. The mononucleotide reaction mixture was adjusted to pH 4.5 with concentrated HCI. Prostatic acid phosphatase (Calbiochem-Behring, La Jolla, Calif.) was added to a final concentration of 0.1 mg/ml. Incubation for 12 h resulted in almost complete dephosphorylation of each mononucleotide, as indicated by TLC (18). Approximately 1 ml of the nucleoside mixture, at a concentration of 1 mg/ml, was then loaded onto a column (1 by 10 cm) packed with Dowex 1-X8, 200-400 mesh anionexchange resin in the chloride form (4, 19). The column was eluted with deionized H20 by gravity, and 1-ml fractions were collected over 20 min. The pyridine ribosides were found in fractions 4 through 7 and adenosine was eluted in fractions 11 through 16, as determined by TLC. Trace amounts of the dinucleotide, mononucleotide, and AMP

were also separated from the pyridine ribosides during this procedure. Purity and structural conformation was confirmed through high-performance liquid chromatographic (HPLC) analysis and proton nuclear magnetic resonance ('H NMR). TLC. TLC was performed on Baker-flex Cellulose F (J. T. Baker Chemical Co., Phillipsburg, N.J.) or 13254 Cellulose (Eastman Kodak Co., Rochester, N.Y.) by using a solvent system consisting of 70% ethanol and 30% 1 M ammonium acetate adjusted to pH 5.0 with concentrated HCl (19). This system was found to separate readily the dinucleotide, mononucleotide, pyridine riboside, and the corresponding pyridine compounds of each NAD analog described above. The Rf values for the nicotinamide and adenine compounds were as follows: NAD, 0.12; NMN, 0.31; NR, 0.62; Nam, 0.84; AMP, 0.24; adenosine, 0.62; and adenine, 0.57. This system was used to follow dinucleotide metabolism and degradation. Samples of 10 ,u were loaded in separate lanes on the TLC plates. Standards were run beside the samples on each plate for comparison. These plates were developed in 200 ml of the ethanol-ammonium acetate solvent for 3 h, dried, and then observed under UV light. For the production of [carbonyl-14C]NR, the lane for each sample was cut at intervals corresponding to the appropriate standard marker, and each section was placed in scintillation counting vials (Laboratory Product Sales, Rochester, N.Y.) with 5 ml of Filtron-X (National Diagnostics, Manville, N.J.) for measurement of radioactivity by liquid scintillation counting. 'H NMR. 'H NMR spectra were recorded at 500 MHz on a spectrometer (GN 500; General Electric Co., Schenectady, N.Y.). Samples of the nucleosides were lyophilized from 99.8% D20 twice and then dissolved in 100o D20 to a final concentration of 1 mg/ml. The sample volume was 0.5 ml. The results from the spectral analysis indicated that the structural conformations were consistent with published 'H NMR spectra obtained for NR and NMN (data not shown) (24). V-factor growth studies. H. influenzae was grown overnight at 37°C in BHI broth supplemented with 11.3 ,uM NAD and 15.8 ,uM hematin. A 1 in 3 dilution of the H. influenzae into fresh BHI broth was performed 2 h prior to the growth study. The cells were washed twice with 50 mM potassium phosphate buffer (pH 7.0) and suspended to a final concentration of 10' CFU/ml in BHI broth. The viable cell count of the initial inoculum was determined by titration on chocolate agar. Cells were diluted 1:10 into BHI broth supplemented with 15.8 ,uM hematin and the V-factor compound at concentrations from 3 to 3,000 ,uM. The final volume of each test sample was 1 ml. Reaction mixtures were incubated for 24 h at 37°C on a rotary shaker. Viable cell counts were determined in duplicate on chocolate agar. Growth was presented as the mean log number of CFU at 0 h and after 24 h of incubation. In the inhibition studies, NAD, NMN, or NR was added to a final concentration of 3 p.M to a reaction mixture containing 15.8 p.M hematin, BHI broth, and the compound to be evaluated. The potential inhibitors were added at molar ratios of 1, 10, and 100 compared with the amount of V factor. Cells were added to a final concentration of 105 CFU/ml. Viable cell counts were determined on chocolate agar after 24 h of incubation. Results were presented as the mean log number of CFU at 0 h and after 24 h of incubation compared with the mean log number of CFU in the NAD, NMN, or NR growth controls. Antimicrobial activity of 3-AmPR. Both Staphylococcus

VOL. 34, 1990



aureus and Escherichia coli were grown overnight in BHI broth. A 1 in 3 dilution into fresh broth was performed 2 h prior to the study. Cells were diluted to a final concentration of approximately 105 CFU/ml. Three reaction mixtures were run for each organism, including a control and 30 and 300 ,uM 3-AmPR. Viable cell counts were determined on chocolate agar after 24 h of incubation. Results were expressed as the mean log number of CFU. [carbonyl-'4C]NR uptake studies. The cells were cultured and prepared as described above for the V-factor growth studies. Cells were suspended to a final concentration of approximately 108 CFU/ml in 50 mM potassium phosphate buffer (pH 7.0) supplemented with 5 mM glucose and 10 mM MgSO4 (5). After incubation for 5 min at 37°C on a rotary shaker, [carbonyl-14C]NR was added to a final concentration of 0.15 ,uM (specific activity, 44 mCi/mmol); and duplicate 0.5-ml samples were removed after 0, 2, and 4 minutes. The samples were vacuum filtered on 0.2-jim-pore-size membrane filters (SUPOR-200; Gelman Sciences, Inc., Ann Arbor, Mich.) which were soaked in 50 mM potassium phosphate buffer (pH 7.0) with 0.15 jiM NR. The filters were washed twice with 5 ml of the NR-containing phosphate buffer, placed in scintillation vials containing 10 ml of Filtron-X, and counted. These studies were also performed in the presence of the NR analogs. All NR analogs were incubated with H. influenzae for 5 min prior to the addition of radiolabeled NR. Uptake of the [carbonyl-'4C]NR was expressed as picomoles per minute per 108 CFU. 3-AmPAD metabolism. H. influenzae was suspended to a final concentration of 2.5 x 1010 CFU/ml in phosphate buffer supplemented with 10 mM MgSO4, 5 mM glucose, and 3 mM 3-AmPAD. The suspension was incubated at 37°C; and 0.1-ml samples were removed at 0, 4, and 6 h. Samples were pelleted in an Eppendorf centrifuge (model 5412) at maximum speed for 15 min at 5°C. The supernatant was removed and analyzed by TLC and HPLC. The metabolism of 3-AmPAD was also investigated by comparing the extracellular levels of 3-AmPAD metabolites from cells under uptake conditions with those from cells under nonuptake conditions. Two suspensions of H. influenzae were prepared at final concentrations of 2.4 x 109 CFU/ml in phosphate buffer with 10 mM MgSO4 and 0.15 mM 3-AmPAD. Glucose (5 mM) was present for uptake conditions, whereas 0.2 mM DNP was present for nonuptake conditions (5). The suspensions were incubated; and 0.1-ml samples were removed at 0, 2, and 4 h. Cells were pelleted, and the supernatant was analyzed by HPLC. HPLC. HPLC was performed by using a Waters Associates system that was equipped with a U6K injector, a model M-6000A pump, and a model 440 absorbance detector (Waters Associates, Inc., Milford, Mass.). The column used in the initial 3-AmPAD metabolism study was a jiBondapakNH2 column (PN 099511; Waters Associates), and the mobile phase consisted of 50 mM KH2PO4 with 10% methanol at pH 3.0. The pump speed was 1 ml/min with a column pressure of 1,000 lb/in2. The A254 was detected. Peaks were recorded on a recorder (OmniScribe series D5000; Houston Instruments); and the retention times (in minutes) were as follows: 3-aminopyridine, 2.2; 3-AmPR, 2.3; NR, 2.3; adenosine, 2.4; NMN, 3.5; NAD, 5.0; and 3-AmPAD, 6.0. A ,uBondapak C18 column (PN 27324; Waters Associates) was used in the uptake versus nonuptake extracellular analysis and had a mobile phase that consisted of 5 mM ammonium formate with 10% (vol/vol) methanol, because of the presence of DNP. The pump speed was 1.5 ml/min with a column pressure of 2,000 lb/in2. The A254 was detected.

TABLE 1. Growth studies with NAD analogs Analog

(concn, 3 ,uM) NAD NADP NADH 3'-Pyridine alteration ThioNAD 3-AcPAD 3-PAAD 3-AmPAD 3-lodoPAD NAAD

Purine alteration NHD NGD NED

Log CFU (mean ± Oh

SD)W 24 h

4.43 ± 0.63 4.06 ± 0.01 4.06 ± 0.01

9.56 + 0.31 9.67 + 0.05 9.56 ± 0.05

+ ± ± ± ± ±

0.01 0.01 0.01 0.29 0.01 0.01

6.54 ± 0.03 5.42 + 0.08

4.06 ± 0.01 4.06 ± 0.01 4.06 ± 0.01

8.64 ± 0.49 8.07 ± 0.17 7.04 ± 0.03

4.06 4.06 4.06 43.82 4.06 4.06

In vitro evaluation of nicotinamide riboside analogs against Haemophilus influenzae.

Exogenous NAD, nicotinamide mononucleotide, or nicotinamide riboside is required for the growth of Haemophilus influenzae. These compounds have been d...
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