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Experimental Therapeutics |
Enact Pharma Plc, Porton Down Science Park, Salisbury SP4 0JQ, United Kingdom [R. J. K., R. G. M., P. J. B.]; Yorkshire Cancer Research Laboratory of Drug Design, Cancer Research Unit, University of Bradford, Bradford BD7 1DP, United Kingdom [T. C. J.]; CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom [S. M. H.]; and Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010-0269 [S. C.]
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ABSTRACT |
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Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as co-substrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4-dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development.
NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10,000 times less in cells able to
perform this conversion than in cells that cannot (reviewed in Ref.
1
). NQO1 is also present in human cells and appears to be an exploitable enzyme for inducing selective cytotoxicity as its levels are significantly raised (compared with surrounding normal tissue) in a number of human cancers such as colon and liver (2) . Furthermore, levels in bone marrow are very low (2) . However, the human form of NQO1 metabolizes CB 1954 much less efficiently than rat NQO1 (3) . Thus, even those cells that are high in human NQO1 are insensitive to CB 1954 (3 , 4) . This catalytic difference between the two forms of the enzyme is mainly accounted for by a single amino acid change at residue 104 (tyrosine in the rat enzyme and glutamine in the human enzyme; Ref. 5 ).
In view of the proven success of CB 1954 in the rat system, it would be highly desirable to recreate its antitumor activity in humans. In this respect, a gene therapy-based approach for targeting cancer cells and making them sensitive to CB 1954 has been proposed. GDEPT has been used to express an Escherichia coli nitroreductase in tumor cells (6, 7, 8, 9, 10) . This enzyme can bioactivate CB 1954 much more efficiently than even rat NQO1 (11 , 12) , and human tumor cells transduced to express this enzyme are very sensitive to CB 1954 (6, 7, 8, 9, 10) . The nitroreductase enzyme could also be targeted using a tumor-localizing antibody, an approach known as ADEPT (reviewed in Ref. 13 ).
However, it might be possible to use CB 1954 directly for prodrug therapy of human tumors without the complications associated with macromolecular targeting systems required for GDEPT and ADEPT. We have shown that there is an additional CB 1954-reducing activity in human tumor cells (14) . This activity is much greater than that attributable to NQO1. However, this activity is latent and only detectable in the presence of NRH. The agent responsible for this activity is the enzyme NQO2 (14) . NQO2 was originally identified by its homology to NQO1 (15) . However, the NQO2 protein is 43 amino acids shorter than NQO1 and lacked enzymatic activity (16) . This apparent lack of activity is because NQO2 uses NRH not NAD(P)H as an electron donor (14) , a novel and unique property. Interestingly, an NRH-metabolizing activity described in bovine kidney in the early 1960s (17 , 18) has now been ascribed to NQO2 (19) . In the presence of NRH, NQO2 can catalyze a two-electron reduction of quinones and the four-electron nitroreduction of CB 1954 (14) . NQO2 is 3000-fold more effective than human DT-diaphorase in the reduction of CB 1954. In this respect, NQO2 resembles the E. coli nitroreductase, but like DT-diaphorase, it selectively generates only the 4-hydroxylamine derivative (14) . There is a very conserved region between the NQO1 and NQO2 proteins (residues 94–115 in NQO1), with the only difference being at residue 104 (tyrosine in NQO2 and glutamine in NQO1; Ref. 14 ). This finding is identical to the difference seen between the rat and human forms of NQO1 in this region (5) ; thus, NQO2 is 100% homologous to rat NQO1 in this important region. This single amino acid change accounts for the enhanced ability of rat NQO1 to reduce CB 1954 [although NQO2 can still reduce CB 1954 much more rapidly than even rat NQO1; kcat = 360 min-1 (14) versus kcat = 4.1 min-1 (3 , 20) ].
NQO2 should more correctly be called NRH quinone oxidoreductase (14) and can also be considered as a human NRH-dependent nitroreductase. We show here that NRH produces a dramatic increase in the cytotoxicity of CB 1954 in both NQO2-transfected rodent and nontransfected human tumor cell lines. We also report on the synthesis and properties of a series of reduced pyridinium derivatives that, like NRH, act as co-substrates for NQO2. These compounds may improve on the pharmaceutical properties of NRH such that the NQO2 enzyme can be fully exploited as a new target for prodrug therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis of Co-substrates.
1-Alkylnicotinamide derivatives 1–12 (Fig. 1
) were prepared by condensation of nicotinamide with one to two
equivalents of the appropriate alkylating agent as described.
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): 4.41 (s, 3H,
CH3), 8.16 (s, 1H,
CONHa), 8.26 (dd, 1H,
J = 8.4 Hz, J = 6.2
Hz, H-5), 8.52 (s, 1H, CONHb), 8.91
(d, 1H, J = 8.4 Hz, H-4), 9.12 (d, 1H,
J = 6.2 Hz, H-6), 9.41 (s, 1H, H-2).
Analysis: found C, 31.62; H, 3.37; N, 10.42%.
C7H9N2OI
requires C, 31.84; H, 3.44; N, 10.61%.
1-Ethyl-3-carbamoylpyridinium iodide 2 was prepared by
similar treatment of nicotinamide and iodoethane. Pale yellow prisms
(82%), mp 202–203°C. NMR (DMSO-d6,
): 1.59 (t, 3H, J = 7.3 Hz,
CH2 CH3), 4.72
(q, 2H, J = 7.3 Hz,
CH2 CH3), 8.16
(br s, CONHa), 8.30 (br t, 1H,
J = 7.2 Hz, H-5), 8.53 (br s, 1H,
CONHb), 8.93 (d, 1H,
J = 8.1 Hz, H-4), 9.27 (d, 1H,
J = 6.2 Hz, H-6), 9.50 (s, 1H, H-2).
Analysis: found C, 34.12; H, 3.91; N, 9.77%.
C8H11N2OI
requires C, 34.55; H, 3.98; N, 10.07%.
1-Propyl-3-carbamoylpyridinium iodide 3a was similarly
prepared by reaction of nicotinamide with 1-iodopropane in DMF at
90–95°C for 4 h. After cooling and addition of ethyl acetate,
the separated solid was filtered. Recrystallization gave from methanol
gave pale yellow crystals (48%), mp 183–184°C [lit. 180–182°C
(18)
]. NMR (DMSO-d6,
): 0.90 (t, 3H, CH2 CH2
CH3), 1.98 (sextet, 2H,
CH2CH2CH3),
4.64 (t, 2H,
CH2CH2CH3),
8.17 (br s, 1H,
CONHaHb), 8.30
(t, 1H, J = 7.0 Hz, H-5), 8.54 (br s, 1H,
CONHaHb), 8.94 (d,
1H, J = 8.1 Hz, H-4), 9.23 (d, 1H,
J = 6.2 Hz, H-6), 9.49 (s, 1H, H-2).
Analysis: found C, 37.05; H, 4.47; N, 9.49%.
C9H13N2OI
requires C, 37.01; H, 4.49; N, 9.59%.
1-Propyl-3-carbamoylpyridinium bromide 3b was prepared analogously using 1-bromopropane (79%), mp 171.5–172.5°C. Analysis: found C, 44.21; H, 5.36; N, 11.32%. C9H13N2OBr requires C, 44.10; H, 5.35; N, 11.43%.
1-(2-Propyl)-3-carbamoylpyridinium iodide 4a was prepared by analogous treatment of nicotinamide with 2-iodopropane to give yellow crystals (23%), mp 188–189°C. Analysis: found C, 37.16; H, 4.55; N, 9.53%. C9H13N2OI requires C, 37.01; H, 4.49; N, 9.59%.
1-(2-Propyl)-3-carbamoylpyridinium Bromide 4b.
A solution of nicotinamide (12.21 g, 0.10 mol) and 2-bromopropane
(12.30 g, 0.10 mol) in DMF (20 ml) was stirred and heated to 70°C for
10 h. Work-up and recrystallization from DMF gave colorless prisms
(16.81 g, 69%), mp 215.5–217°C: NMR
(DMSO-d6, ): 1.68 (d,
J = 7.0 Hz, 6H, N+
CH(CH3)2), 5.17
(septet, J = 7.0 Hz, 2H,
N+ CH
(CH3)2), 8.20 (br s, slow
D2O exchange, 1H,
CONHaHb), 8.31
(dd, J = 8.1 Hz, J = 6.2 Hz, 1H, H-5), 8.71 (br s, slow D2O
exchange, 1H,
CONHaHb), 8.98 (d,
J = 8.1 Hz, 1H, H-4), 9.43 (d,
J = 6.2 Hz, 1H, H-6), 9.62 (s, 1H, H-2).
Analysis: found C, 44.19; H, 5.34; N, 11.30%.
C9H13N2OBr
requires C, 44.10; H, 5.35; N, 11.43%.
1-Benzyl-3-carbamoylpyridinium bromide 5 was prepared
similarly, using benzyl bromide as the alkylating agent. Colorless
prisms (85%), mp 212–213°C. NMR
(DMSO-d6, ): 5.99 (s, 2H,
CH2 Ph), 7.40–7.68 (m, 5H,
CH2Ph), 8.22 (br, s, 1H,
CONHa), 8.32 (br t, 1H,
J = 7.2 Hz, H-5), 8.68 (br s, 1H,
CONHb), 9.03 (d, 1H,
J = 8.1 Hz, H-4), 9.39 (d, 1H,
J = 6.2 Hz, H-6), 9.74 (s, 1H, H-2).
Analysis: found C, 53.42; H, 4.47; N, 9.48%.
C13H13N2OBr
requires C, 53.26; H, 4.47; N, 9.56%.
1-(2-Phenylethyl)-3-carbamoylpyridinium Iodide 6.
Treatment of nicotinamide with (2-iodoethyl)benzene in DMF by a similar
procedure gave yellow prisms (65%), mp 188–189°C. NMR
(DMSO-d6, ): 3.32 (t, 2H,
J = 7.3 Hz, CH2
CH2Ph), 4.93 (t, 2H,
J = 7.3 Hz,
CH2 CH2Ph),
7.15–7.40 (m, 5H, CH2
CH2Ph), 8.17 (br s, 1H,
CONHa), 8.26 (br t, 1H,
J = 7.0 Hz, H-5), 8.52 (br s, 1H,
CONHb), 8.93 (d, 1H,
J = 8.1 Hz, H-4), 9.15 (d, 1H,
J = 6.2 Hz, H-6), 9.47 (s, 1H, H-2).
Analysis: found C, 47.35; H, 4.31; N, 7.85%.
C14H15N2OI
requires C, 47.48; H, 4.27; N, 7.91%.
1-(2-Hydroxyethyl)-3-carbamoylpyridinium iodide 7 was
prepared by stirring and heating a mixture of nicotinamide and
2-iodoethanol in DMF at 90–95°C for 4 h. Work-up done as
described previously gave colorless crystals (79%), mp 128–129°C.
NMR (DMSO-d6, ): 3.85–3.98 (m, 2H,
CH2CH2OH), 5.21 (t,
2H, J = 7.2 Hz,
CH2CH2OH), 8.17
(br s, 1H, CONHa), 8.34 (br t, 1H,
J = 7.2 Hz, H-5), 8.56 (br s, 1H,
CONHb), 8.98 (d, 1H,
J = 8.1 Hz, H-4), 9.16 (d, 1H,
J = 6.2 Hz, H-6), 9.43 (s, 1H, H-2).
Analysis: found C, 33.06; H, 3.85; N, 9.57%.
C8H11N2O2I
requires C, 32.67; H, 3.77; N, 9.53%.
1-(3-Hydroxypropyl)-3-carbamoylpyridinium bromide 8 was
prepared by the general procedure using 3-bromo-1-propanol.
Recrystallization from DMF gave colorless prisms (74%), mp
119–120°C. NMR (DMSO-d6, ): 2.14 (br
quintet, J 6.2 Hz, 2H, N+
CH2
CH2CH2 OH), 3.48
(t, J = 5.7 Hz, 2H, N+
CH2CH2CH2OH),
4.77 (t, J = 7.0 Hz, 2H,
N+
CH2CH2CH2OH),
8.18 (br s, slow D2O exchange, 1H,
CONHa Hb), 8.28
(dd, J = 8.1 Hz, J = 5.9 Hz, 1H, H-5), 8.63 (br s, slow D2O
exchange, 1H,
CONHaHb), 8.97 (d,
J = 8.1 Hz, 1H, H-4), 9.26 (d,
J = 5.9 Hz, 1H, H-6), 9.56 (s, 1H, H-2).
Analysis: found C, 40.07; H, 5.17; N, 10.16%.
C9H13N2O2Br
·0.5H2O requires C, 40.02; H, 5.22; N, 10.37%.
1-(2-Carboxyethyl)-3-carbamoylpyridinium Iodide 9.
Treatment of nicotinamide with 3-iodopropionic acid by the same method
gave the salt as colorless crystals (46%), mp 185–186°C. Analysis:
found C, 33.80; H, 3.45; N, 8.57%.
C9H11N2O3I
requires C, 33.56; H, 3.44; N, 8.70%.
1-(Carbamoylmethyl)-3-carbamoylpyridinium iodide 10 was prepared by stirring and heating a mixture of nicotinamide and 2-iodoacetamide in DMF at 55–60°C for 3 h. After cooling to room temperature, ethyl acetate was added, and the mixture was stirred for 30 min. Filtration and recrystallization from aqueous ethanol afforded colorless crystals (66%), mp 210–211°C. Analysis: found C, 31.37; H, 3.34; N, 13.77%. C8H10N3O2I requires C, 31.29; H, 3.28; N, 13.68%.
1-(Carbamoylmethyl)-3-(N-methyl)carbamoylpyridinium iodide
11 was prepared by stirring and heating a mixture of
N-methylnicotinamide (2.0 g, 14.7 mmol) and 2-iodoacetamide
(2.7 g, 14.6 mmol) in DMF (5 ml) at 55–60°C for 4 h.
Ethyl acetate (50 ml) was added to the cooled solution, and the mixture
was stirred at room temperature for 30 min. The solid was filtered,
dried at the pump, and recrystallized from aqueous methanol to give
yellow prisms (4.2 g, 89%), mp 233–235°C. NMR
(D2O, ): 3.01 (s, 3H,
CONHCH3), 5.64 (s, 2H,
CH2CONH2), 8.28
(br t, 1H, J = 7.2 Hz, H-5), 8.91–9.06 (m,
2H, H-4 and H-6), 9.28 (s, 1H, H-2). Analysis: found C, 33.23; H, 3.65;
N, 12.83%.
C9H12N3O2I
requires C, 33.66; H, 3.76; N, 13.09%.
1-(3-Sulfonatopropyl)-3-carbamoylpyridinium 12 was prepared
by adding 1,3-propanesultone (12.21 g, 0.10 mol) in one portion to a
stirred solution of nicotinamide (12.21 g, 0.10 mol) in DMF (20 ml).
The clear solution was heated to 100°C for 1 h, during which
time (>5 min) a heavy colorless solid separated. The reaction mixture
was cooled to room temperature and filtered, and the solid was washed
serially with cold DMF (2 x 25 ml) and then dry diethyl
ether (2 x 30 ml). Recrystallization from aqueous DMF
gave colorless prisms (88%), mp 300–302°C. NMR
(D2O, ): 2.52 (quintet, J = 7.3 Hz, 2H, N+
CH2CH2CH2SO3–),
3.04 (t, J = 7.3 Hz, 2H,
N+
CH2CH2CH2SO3–),
4.89 (t, J = 7.3 Hz, 2H,
N+
CH2CH2CH2SO3–),
7.95 (br s, slow exchange, CONH2),
8.24 (br t, J 7.2 Hz, 1H, H-5), 8.94 (d,
J = 8.1 Hz, 1H, H-4), 9.10 (d,
J = 6.2 Hz, 1H, H-6), 9.40 (s, 1H, H-2).
Analysis: found C, 43.38; H, 4.95; N, 10.94%.
C9H12N2O4S·0.25H2O
requires C, 43.45; H, 5.06; N, 11.26%.
The reduced dihydronicotinamide derivatives
1R–12R were synthesized from the corresponding
nicotinamide salts 1–12 (Fig. 1
) by reduction
with aqueous sodium hydrosulfite, as illustrated for
1-methyldihydronicotinamide 1R in the following example.
General Procedure.
To a solution of 1 (20 mg) in water (5 ml) was added
anhydrous sodium carbonate (50 mg), sodium bicarbonate (50 mg), and
sodium hydrosulfite (50 mg), and the stoppered solution was allowed to
stand at 37°C for 30 min. The reduction product was then separated
from the reaction mixture by preparative HPLC. Reaction mixture (5 ml)
was injected onto a Dynamax Macro C18 (21.4 x 250-mm)
reverse-phase column (Rainin; obtained from Anachem, Luton, United
Kingdom) and eluted by a gradient of acetonitrile in water (0–50%
over 30 min) at 20 ml/min. The eluate was continually monitored by
absorbance at 340 nm and by fluorescence (excitation, 340 nm; emission,
450 nm), and a fraction corresponding to a peak of fluorescence was
collected. The eluate was collected and freeze-dried to afford
compound 1R (12 mg, 62%) as an homogeneous yellow powder.
The derivative was used without further analysis to prevent degradation
effects. Other compounds 2R–2R were prepared as
yellow/orange amorphous powders by an analogous reduction procedure;
characterization details for selected compounds are collected below.
Compound 3R, mp 92–92.5°C [lit. 92–93°C
(18)
]. NMR (CDCl3, ): 0.90 (t,
J = 7.3 Hz, 3H,
NCH2CH2CH3),
1.56 (sextet, J = 7.3 Hz, 2H,
NCH2CH2CH3),
3.05 (t, J = 7.3 Hz, 2H,
NCH2CH2CH3),
3.16 (dd, J = 3.6 Hz, J = 1.8 Hz, 2H, 4-CH2), 4.72 (dt,
J = 8.1 Hz, J = 3.6
Hz, 1H, H-5), 5.35 (br s, 2H, slow D2O exchange,
CONH2), 5.72 (dq, J = 8.1 Hz, 1.8 Hz, 1H, H-6), 7.04 (d, J = 1.8
Hz, H-2); MS (FAB) m/z 166 ([M
]+); MS (EI): found 166.1115
(C9H14N2O
requires 166.1106).
Compound 8R. NMR (D2O, ): 1.89 (br
quintet, J 6.6 Hz, 2H,
NCH2CH2CH2OH),
3.17 (br t, J = 1.8 Hz, 2H,
4-CH2), 3.38 (t,
J = 6.9 Hz, 2H,
NCH2CH2CH2OH),
3.74 (t, J = 6.2 Hz, 2H,
NCH2CH2CH2OH),
4.95–5.05 (m, 1H, H-5), 6.01 (dm, J = 8.1
Hz, 1H, H-6), 7.13 (s, 1H, H-2); MS (FAB) m/z 183
([M + H]+).
Compound 9R. NMR (D2O, ): 2.46 (t,
J = 6.9 Hz, 2H,
NCH2CH2CO2H),
2.96 (t, J = 6.9 Hz, 2H,
NCH2CH2CO2H),
4.85–4.95 (m, 1H, H-5). Spectral overlap attributable to H-2 and H-6
ring protons prevented assignment of the diagnostic
4-CH2 protons.
Compound 10R, mp 178–180°C [lit. 179–182°C
(23)
]. NMR (D2O, ): 3.00 (br t,
J = 1.8 Hz, 2H,
4-CH2), 3.90 (s, 2H,
CH2 CONH2),
4.82–4.90 (m, 1H, H-5), 5.76 (dm, J = 8.1
Hz, H-6), 6.89 (s, 1H, H-2); MS (FAB) m/z 181
([M]+). Analysis: found C, 52.87; H, 6.09; N,
23.17%.
C8H11N3O2
requires C, 53.03; H, 6.12; N, 23.19%. MS (EI): found 181.0814
(calculated 181.0851).
Compound 12R. NMR (D2O, ):
1.95–2.08 (m, J 7.3 Hz, 2H,
NCH2CH2CH2SO3–),
2.94 (t, J = 7.7 Hz, 2H,
NCH2CH2CH2SO3–),
3.04 (br t, J = 1.8 Hz, 1H,
4-CH2), 3.34 (t,
J = 6.8 Hz, 2H,
NCH2CH2CH2SO3–),
4.88–4.98 (m, 1H, H-5), 5.95 (dd, J = 8.1
Hz, J = 1.5 Hz, 1H, H-6), 7.04 (s, 1H, H-2).
Plasmid Vector Construction.
A bicistronic eukaryotic expression vector containing the coding
regions for human NQO2 and puromycin acetyl transferase (conferring
puromycin resistance) driven from a single cytomegalovirus promoter was
constructed as follows. An intermediate plasmid was used, consisting of
pBluescript II SK(+) (Stratagene, Amsterdam, the Netherlands)
containing an insert that started with the sequence
CCTCGAGTCACCATGGATATCANNN ... blunt cloned
in the forward direction at the unique MCS EcoRV site
(7)
. This introduced extra XhoI and
NcoI sites (underlined) and an adenine three bases before
the NcoI ATG site to ensure good eukaryotic translation
initiation (24)
. The unwanted remainder of the insert was
removed by digestion with NcoI and HindIII. The
coding region containing the full-length NQO2 open reading frame was
excised as an NcoI-HindIII fragment from the
bacterial expression vector pKK-hNQO2 (14)
and cloned into
the modified pBluescript to attach the XhoI site and Kozak
sequence to the 5'-end of the open reading frame, producing the vector
H1. The bicistronic vector pIRES-P (EMBL:Z75185; Ref. 25
)
was used for expression of NQO2 in eukaryotic cells. It was prepared by
digestion with XbaI and partial fill-in with Klenow and
dCTP/dTTP. The Klenow was heat killed and removed, and the vector was
then digested with XhoI. The NQO2 insert with
NH2-terminal Kozak sequence was excised from H1
by digestion with HindIII and partial fill-in with Klenow
and dATP/dGTP, followed by a separate XhoI digest as before.
This insert was then cloned into the prepared pIRES-P to produce the
final NQO2 expression vector H6. Insert orientation and identity were
confirmed by diagnostic restriction digests and dideoxy sequencing
using a Sequenase II kit (Amersham Pharmacia Biotech, St. Albans,
Herts, United Kingdom).
Cell Culture and Transfection.
V79 Chinese hamster lung fibroblasts were grown in monolayer culture in
DMEM containing 10% FCS and 4 mM glutamine (all from Life
Technologies, Ltd., Paisley, Scotland, United Kingdom). Cells were
maintained in a humidified atmosphere at 37°C with 5%
CO2 and subcultured twice weekly by
trypsinization. All cells were determined to be free of
Mycoplasma. Plasmid vectors were transfected into cells by
calcium phosphate coprecipitation (Profection; Promega Corp.,
Southampton, Hampshire, United Kingdom), and positive clones were
selected in growth medium containing 10 µg/ml puromycin and
maintained under selective pressure.
Four human cancer cell lines were used in this study: PC-3, a prostate carcinoma, U373-MG and U87-MG, both glioblastoma multiforme, and T98G, a glioblastoma. All cell lines were obtained from the European Collection of Cell Cultures (Porton Down, Wiltshire, United Kingdom). Cells were grown as monolayers in Eagle’s MEM with Earle’s salts without L-glutamine (EMEM), supplemented with 10% (v/v) heat-inactivated FCS, 1% (v/v) L-glutamine (200 mM), 1% (v/v) nonessential amino acids (all from Life Technologies, Inc.), and 10,000 units of penicillin and streptomycin (Sigma). Cells were routinely passaged twice weekly by trypsinization.
Cytotoxicity Analysis in Vitro by SRB Assay.
Cells in exponential phase of growth were trypsinized, seeded in
96-well plates at a density of 1000 cells/well (2000 cells/well for
U373-MG; 100 µl), and permitted to recover for 24 h. The medium
was replaced with fresh medium containing co-substrate (100
µM). Serial dilutions of the drug solution (8 of
3.66-fold) were performed in situ, giving final
concentrations of 1000–0.46 µM. Cells were
then incubated with drug for either 3 days (V79), 8 days (U373-MG), 5
days (U87-MG), or 6 days (T98G and PC-3) at 37°C. The plates were
fixed and stained with SRB, before reading optical absorption at 590
nm; results were expressed as percentage of control growth. The
IC50s were evaluated by interpolation.
Co-substrate cytotoxicity was assessed in V79 cells for a dose range of
10,000–4.6 µM.
Determination of NQO2 Activity in Cell Lines.
Cells were grown to confluence in T80-cm2 tissue
culture flasks then trypsinized and pelleted by centrifugation. The
cell pellet was extensively washed (five times) with ice-cold PBS and
stored at -80°C until required. The sample was defrosted into 1 ml
of lysis buffer (1% NP40, 1% aprotinin in PBS) and disrupted with
four to five strokes of a homogenizer in a 1.5-ml microcentrifuge tube.
The lysate was cleared by centrifugation at 13,000 x g (3 min), after which the supernatant was taken and assayed
immediately as below. Protein concentration was determined by the
Bradford method (assay kit supplied by Sigma) after appropriate
dilution (usually 1/1000) in PBS.
The assay was started by addition of 200 µl of supernatant to a mixture of CB 1954 (100 µM) and NRH (500 µM) in sodium phosphate buffer (pH 7) to give a final volume of 1 ml. The mixture was incubated at 37°C and aliquots (10 µl) were taken every 6 min and assayed immediately by HPLC [Partisil 10 SCX (4.2 x 150-mm); Whatman, Maidstone, Kent, United Kingdom] and eluted isocratically with 0.13 M sodium phosphate (pH 5) at 1.5 ml/min. The concentration of CB 1954 was determined in each sample by reference of the corresponding peak area with an external standard, quantified by absorbance at 325 nm. Initial rates were calculated by curve fitting (FigP; Biosoft, Cambridge, United Kingdom). As a control for NQO1 and nonspecific activity, NADH was substituted for the NRH and the assay repeated. NQO1 can also use NRH as a co-substrate, but the rate of CB 1954 reduction is the same as with NADH (21) . This rate was subtracted from the rate obtained with NRH to calculate NQO2 activity. In cells expressing NQO2, the rate in the presence of NADH was <5% of that obtained with NRH. One unit of NQO2 enzyme will convert 1 µmol of CB 1954 to its 4-hydroxylamine per minute under these conditions. Assays were performed in triplicate with a SD <10%; the limit of sensitivity was 10-4 units.
Determination of NQO1 Activity in Cell Lines.
NQO1 (DT-diaphorase) activity was assayed in cell lysates, obtained as
above, as described previously (26)
using menadione (10
µM) as substrate and cytochrome c (70
µM) as terminal electron acceptor. Activity was
defined as the cytochrome c reduction inhibited by 1
µM dicoumarol and expressed as µmol
cytochrome c reduced per minute per mg of protein (1
µmol/min = 1 unit).
Purification of NQO2.
Recombinant NQO2 was expressed in E. coli and purified as
described previously (14)
. Purity was >95% and gave a
specific activity of about 15 units/mg using NRH as co-substrate.
Assay of Purified NQO2.
The purified NQO2 was assayed as in the supernatants described above,
except that the assay was started by addition of 0.5 µg/ml NQO2, and
no control for nonspecific activity was necessary. For assays using
other potential co-substrates, these compounds were substituted for the
NRH.
Measurement of Co-substrate Uptake into Cells.
Chinese hamster V79 cells were grown to confluence in
T25-cm2 tissue culture flasks. The culture medium
was replaced with 5 ml of PBS at 37°C. Co-substrate solution (250
µl of 10 mM in PBS) was added, and the flask was
incubated at 37°C for either 10 or 30 min. The co-substrate solution
was then removed, and the cell monolayer was extensively washed with
ice-cold PBS (6 x 10 ml). Excess PBS was removed by
flicking, and 2 ml of lysis solution were added. The cells were allowed
to lyse for 30 min with gentle agitation. Cell debris was removed by
centrifugation (13,000 x g, 3 min), and
co-substrate concentration was determined by fluorimetry. Fluorescence
measurements were carried out on a PE-500 (Perkin-Elmer) fluorimeter
(excitation and emission wavelengths of 340 and 450 nm, respectively)
thermostated to 37°C and calibrated with the appropriate standards.
All measurements were carried out in triplicate. Untreated cell
monolayers were used to measure the background fluorescence, which was
less than the equivalent reading from a lysate containing 5
µM NRH. Further untreated cell monolayers were
used to obtain the cell number. Cells were detached by trypsinization
and counted in a hemacytometer.
|
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As shown in Fig. 2
and quantified in Table 1
, expression of NQO2 had no effect on the
cytotoxicity of CB 1954 alone against these cell lines. All had a
similar sensitivity to nontransfected V79 cells with an
IC50 of 250 µM after a 72-h
continuous exposure to CB 1954. Addition of NRH did not increase the
sensitivity of nontransfected V79 cells toward CB 1954, but all of the
transfected cell lines showed a very large increase in sensitivity
toward this prodrug (Table 1
; Fig. 2
). On the basis of the
IC50s, this increased sensitivity ranged from
100-fold (TM7) to >3000-fold (TM6). NQO2 activity could not be
detected in nontransfected V79 cells, but activity ranged between
4.9 x 10-4 units/mg (TM7) and
0.83 units/mg (TM6) in the clones (Table 1)
.
|
|
) were prepared and
tested for their ability to act as a co-substrate for the reduction of
CB 1954 by NQO2. The specific activities obtained by this assay are
shown in Table 2
. In contrast, none of the oxidized forms of these compounds had any
activity in this assay (data not shown). Under the assay conditions, a
number of compounds were more active than NRH. However, there were
clear structure-dependent effects on the specific activity, such that
little activity was found with either a 1-benzyl or 1-(2-phenylethyl)
substitution (i.e., 5R or 6R). The two
nicotinic acid derivatives examined (NARH and NAMNH) were similarly
inactive.
|
).
Only two further derivatives, 11R
(T1/2 = 3.68 h) and
12R (T1/2 = 4.6 h), had half-lives >2 h (Table 2)
.
Enzyme Kinetics.
The kinetic properties of NQO2 for NRH, 10R and
12R, were assayed at 37°C by measuring the initial rates
of production of the 4-hydroxylamine derivative of CB 1954. As shown in
Table 3
, NQO2 had the lowest apparent Km for
NRH (29 µM), but the enzyme also showed its
lowest kcat using this co-substrate
(364 min-1). These figures are in good agreement
with values published previously (14)
.
kcat was more than doubled using
12R as co-substrate, but the enzyme had a 6-fold higher
apparent Km for this compound. An
intermediate result was obtained with 10R. The variation of
both Km and
kcat was not predicted, but the enzyme
exhibited classic hyperbolic Michaelis-Menten kinetics with all three
co-substrates (data not shown).
|
). However, 12R had no significant effect in any cell line.
In PC-3 cells, the IC50 of CB 1954 was <0.1
µM in the presence of either NRH or 10R.
However, this cell line was relatively sensitive to CB 1954 alone
(IC50, 15 µM), and the potentiation
factor is similar to that seen in the other cell lines (Fig. 3A
).
|
, all four human cell lines used in the cytotoxicity study had
measurable levels of both the enzymes NQO1 and NQO2. The PC-3 cell line
showed the highest levels of both NQO1 (1.17 units/mg) and NQO2
(2.25 x 10-3 units/mg). However,
there was little spread in activity, and the lowest level of NQO1
activity, found in the U87-MG line, was only half that found in the
PC-3. Similarly, the lowest NQO2 activity (also in the U87-MG line) was
25% of that in the PC-3 cells (Table 4)
.
|
30
nmol/107 cells). However, there was no
significant uptake of 12R over 30 min (<1
nmol/107 cells; data not shown). |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, to exploit this enzyme, macromolecular targeting systems such as ADEPT or GDEPT need to be used (reviewed in Ref. 13 ). We have now shown that an endogenous human enzyme does exist that can bioactivate CB 1954, and this rekindles the concept of using CB 1954 in a simple but selective antitumor therapy without the need for targeting systems. The enzyme is NQO2, where its activity is latent and not normally detectable. A co-substrate, such as NRH, is required that acts with this new enzyme to reduce CB 1954 (14) . NQO2 resembles the E. coli nitroreductase in terms of its size and rate of reduction, although there is no significant sequence homology between them. Furthermore, NQO2 resembles NQO1 in that, unlike the bacterial enzyme, it forms only the more cytotoxic 4-hydroxylamine reduction product of CB 1954.
Introduction of the gene for NQO2 into Chinese hamster V79 cells
increased the cytotoxicity of CB 1954 by 100-3000-fold but only in the
presence of NRH. In the absence of an NQO2 co-substrate, required as a
source of hydride equivalents in the bioreductive process, the enzyme
had no effect on CB 1954 cytotoxicity. This demonstrates that no
significant levels of endogenous co-substrates for NQO2 are present in
these cells. The enzyme activity in these transfected cells varied from
0.82 unit/mg to 4.9 x 10-4
units/mg, representing a 1000-fold range However, the increase in
cytotoxicity varied over only a 30-fold range (i.e.,
100-3000-fold). A significant potentiation was seen at the lower levels
of NQO2 activity, and similar levels of NQO2 activity were observed in
human tumor cell lines (Table 3)
. At NQO2 activities greater than
0.01 unit/mg, there was little increase in the potentiation of CB
1954 cytotoxicity by NRH. A plateau effect is not unexpected in these
cytotoxicity assays and probably indicates that this level of enzyme
activity is such that it can metabolize all of the prodrug present in
the well of the assay plate. Therefore, no further effect is seen in
cells containing higher levels of NQO2.
That the cytotoxicity of CB 1954 toward human cells was greatly
enhanced by the presence of NADH (when FCS was present in the culture
medium) has been reported (30)
. It was shown that the
molecule responsible for this effect is NRH (30)
. NRH is a
co-substrate for NQO1, although the rate of reduction is the same as in
the presence of NADH (21)
. NRH is generated from NADH by
enzymes in FCS (31)
. It was proposed that NRH (unlike
NADH) can enter the cell and stimulate the activity of NQO1 toward CB
1954 (30)
. This was supported by the fact that the
cytotoxicity experienced by human cancer cell lines after exposure to
CB 1954 and either NADH or NRH was proportional to their levels of NQO1
(30)
. However, although NQO1 can use NRH as a
co-substrate, the kinetics are such that NADH or NADPH would be
expected to be used in almost total preference to NRH
(21)
. Because these latter two biogenic NQO1 co-substrates
are readily available in cells, any stimulation in enzyme activity by
addition of NRH would not be predicted. It is now obvious that these
observations were attributable to the action of NQO2. The correlation
with NQO1 levels would suggest that the expressions of these two
enzymes are linked. Introduction of the NQO2 expression vector into
Chinese hamster V79 cells had no effect on their NQO1 activity (which
is very low; Table 1
). However, the human cancer cell lines all had
similar relative levels of NQO1 and NQO2 (Table 4)
that would support
linked expression. Sequence analysis of the 5'-flanking region of the
NQO2 gene has revealed the presence of a single copy of the
antioxidant response element and three copies of the xenobiotic
response element. Antioxidant response element and xenobiotic response
element elements have been found previously in the promoter for the
NQO1 gene and mediate increases in expression in response to
polycyclic aromatic compounds, phenolic antioxidants, and
2,3,7,8-tetrachlorodibenzo-p-dioxin (16)
.
Early work in the early 1960s on a bovine kidney enzyme, now ascribed
as NQO2 (19)
, showed that it could also oxidize some
derivatives of dihydronicotinamide (18)
. We have shown
that other reduced pyridinium compounds can act as co-substrates for
human NQO2. The simplest quaternary (and therefore reducible)
derivative of nicotinamide, 1-methylnicotinamide salt 1, in
its reduced form 1R, was a co-substrate for NQO2 with a
specific activity 30% that of NRH. Increased chain length and bulk
at the 1-position of the nicotinamide ring improved specific activity
and several compounds more active than NRH were found (Table 2)
.
However, there was a limit to this effect, and little activity was seen
with either the corresponding 1-benzyl (5R) or
1-(2-phenylethyl) (6R) derivatives. This suggests that the
co-substrate binding site of NQO2 may be sterically constrained. Little
activity was seen with any nicotinic acid derivatives, suggesting that
a negatively charged substituent at the 3-position on the pyridine ring
is poorly tolerated in the binding pocket of the enzyme.
Most of the reduced pyridinium compounds examined were less chemically
stable than NRH and in aqueous solution rapidly oxidized to regenerate
the parent pyridinium salts (e.g., 1R 
1, and others), which are not co-substrates for the NQO2
enzyme. However, the 1-carboxamidomethyl derivative 10R was
more stable than NRH, suggesting that stereoelectronic effects or
intramolecular hydrogen bonding may stabilize the molecule to oxidative
degradation. Qualitative support is provided by the deleterious effect
of N-methyl substitution of the 3-carboxamide residue on
chemical stability (i.e., 10R 11R
in Table 2
). Two compounds, the uncharged 10R and the
anionic 1-(3-sulfonatopropyl) derivative 12R, were analyzed
further because of their specific activity and inherent stability. With
respect to kcat values, both these
compounds were superior to NRH, although NQO2 shows a higher apparent
Km for each co-substrate. The increase
in kcat with
Km explains why both 10R
and 12R (and certain other analogues) appear superior to NRH
as co-substrates in the reduction of CB 1954 by NQO2 (Table 2)
, even if
the enzyme has an apparent lower affinity for these two compounds
compared with NRH. This result was not predicted because it was
expected that the kcat values would be
the same irrespective of the co-substrate. NQO2 exhibited classic
hyperbolic Michaelis-Menten kinetics with all three co-substrates.
However, it is possible that there is product inhibition in this system
and that NQO2 is being inhibited by the accumulation of the oxidized
co-substrate. In support of this theory, we have found that the
reduction of CB 1954 by NQO2 and NRH is inhibited by 10, the
oxidized form of 10R (data not shown).
In cytotoxicity assays, both 10R and 12R were
noncytotoxic, similar to NRH. In four human cancer cell lines,
10R potentiated the effect of CB 1954 to about the same
extent as NRH (Fig. 3
). All of the cell lines had similar measurable
levels of NQO2 (Table 4)
. These levels were not as high as those
obtained in the majority of the transfected cells but were comparable
with levels measured in the TM2 and TM7 transfectants (Table 1)
. On the
basis of these data, the 100–200-fold increase in sensitivity to
CB 1954 observed in these human cell lines in the presence of either
NRH or 10R would be predicted from their intracellular NQO2
levels.
However, 12R had no significant effect on the cytotoxicity of CB 1954. Although this compound is less chemically stable than either NRH or 10R, this would not be expected to have such a dramatic effect on cytotoxicity. The compound is negatively charged at pH 7, and this charge prevents it from entering cells. Lack of cellular permeability of this co-substrate probably accounts for its inability to potentiate the cytotoxicity of CB 1954. Use of a charged co-substrate of this type would allow NQO2 to be used in targeted therapies such as ADEPT where a prodrug activating enzyme is localized to the outside of a cancer cell (13) . In the case of NQO2, an anionic co-substrate would be administered with CB 1954. Because the targeted NQO2 would be external, the co-substrate would allow the tumor-specific bioactivation of CB 1954 but would not facilitate activation by the endogenous enzyme.
Most importantly, the use of permeable NQO2 co-substrates allows the direct use of prodrug therapy without the complications associated with macromolecular targeting systems. NQO1 is potentially a source of selectivity in cytotoxic chemotherapy by prodrug activation (2) . High activity has been reported in human tumor cell lines of breast (32, 33, 34) , brain (35) , colon (32 , 33 , 36 , 37) , lung (32, 33, 34) , and liver (32 , 33 , 38 , 39) origin. There is a marked increase in the activity of NQO1 in human colonic carcinomas when compared with the enzymatic activity of the surrounding normal colonic mucosa (40) . Furthermore, NQO1 levels in bone marrow are low (2 , 30) , directing toxicity away from tissues that are usually sensitive to conventional cytotoxic chemotherapy. That NQO2 expression may be related to that of NQO1 suggests that a selective antitumor effect could be achieved using CB 1954 in conjunction with a suitable NQO2 co-substrate. In this respect, 10R is superior to NRH because, although they have similar kinetic properties, 10R is both more stable and amenable to synthesis.
The physiological role of NQO2 is not known. It seems unlikely that an enzyme would have a role using the co-substrates described here. In theory, NRH could be generated in cells by reaction of NADH or NADPH with phosphodiesterases and phosphatases. This reaction does occur in serum (30) , but NRH is not detected in cells. Furthermore, in cell lines, there was no effect on CB 1954 cytotoxicity unless a co-substrate is also administered. This confirms that endogenous co-substrates for the NQO2 catalyzed reduction of CB 1954 are not present. It is possible that both the physiological electron donor and substrate for this enzyme have yet to be identified. The crystal structure of NQO2 shows a specific metal binding site that may be redox active (41) . Such a site is not present in NQO1 and suggests different roles for these two enzymes. The possible involvement of a metal in the redox action of NQO2 might explain why this enzyme can reduce CB 1954 much more efficiently than NQO1.
Although in many respects CB 1954 is an ideal prodrug, it is probable that other compounds will be substrates for NQO2, and these could form the basis of alternative prodrug systems. The fact that an effective nitroreductase enzyme has been found in human tumor cells suggests a novel target for prodrug development. This approach is unique and represents the first example of a latent enzyme-prodrug system switched on by synthetic co-substrate. This ternary system is inactive if any one of the components is absent.
In summary, the NQO2 enzyme has been shown to activate the prodrug CB 1954 to its cytotoxic form in both transfected rodent and nontransfected human cancer cell lines. NQO2 levels in these cell lines are capable of increasing the cytotoxicity of CB 1954 by more than 100-fold. However, this effect is only manifest when a suitable reduced pyridinium compound is administered as a co-substrate. Using NRH as the lead compound, a series of such co-substrates has been synthesized and systematically evaluated. The 1-carbamoylmethyl derivative 10R was found to be superior to NRH in terms of inherent stability and potentiated the cytotoxicity of CB 1954 to a similar extent. NQO2 represents a novel target for prodrug therapy and offers a unique activation mechanism reliant upon a synthetic co-substrate to activate an otherwise apparently latent enzyme. Our findings reopen the potential use of CB 1954 for the therapy of human malignant disease, 30 years after it was first synthesized.
|
FOOTNOTES |
|---|
1 T. C. J. is funded by Yorkshire Cancer
Research, S. M. H. is supported by the Cancer Research Campaign (UK),
and S. C. is a member of the City of Hope Cancer Center (Grant
CA33572). 
2 To whom requests for reprints should be
addressed, at Enact Pharma Plc, Building 115, Porton Down Science Park,
Salisbury SP4 0JQ, United Kingdom. Phone: 44-(0)-1980-613272; Fax:
44-(0)-1980-613713; E-mail: rknox{at}enactpharma.com
3 The abbreviations used are: CB 1954,
5-(aziridin-1-yl)-2,4-dinitrobenzamide; NRH, dihydronicotinamide
riboside [nicotinamide riboside (reduced)]; NARH, nicotinic acid
riboside (reduced); NAMNH, nicotinic acid mononucleotide (reduced);
GDEPT, gene-directed enzyme prodrug therapy; ADEPT, antibody-directed
enzyme prodrug therapy; SRB, sulforhodamine B; NQO1, NAD(P)H quinone
oxidoreductase 1, DT-diaphorase (EC1.6.99.2); NQO2, NAD(P)H quinone
oxidoreductase 2; EI, electron impact; FAB, fast atom bombardment;
lit., literature value; mp, melting point; DMF, dimethylformamide; NMR,
nuclear magnetic resonance; s, singlet; d, doublet; dd, double doublet;
t, triplet; q, quartet; m, multiplet; br, broadened.
Received 11/ 8/99. Accepted 6/ 2/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, untransfected
V79; 
, clone TM1; 
, TM2; •, TM3; 
, TM4; 
, TM5; *, TM6;
, TM7. Drug exposure was 72 h, and relative growth was measured
by the SRB assay.