Specificity of a prodrug-activating enzyme hVACVase: The leaving group effect

Article abstract of DOI:10.1021/mp100300k

Human valacyclovirase (hVACVase) is a prodrug-activating enzyme for amino acid prodrugs including the antiviral drugs valacyclovir and valganciclovir. In hVACVase-catalyzed reactions, the leaving group of the substrate corresponds to the drug moiety of the prodrug, making the leaving group effect essential for the rational design of new prodrugs targeting hVACVase activation. In this study, a series of valine esters, phenylalanine esters, and a valine amide were characterized for the effect of the leaving group on the efficiency of hVACVase-mediated prodrug activation. Except for phenylalanine methyl and ethyl esters, all of the ester substrates exhibited a relatively high specificity constant (k_cat/K_m), ranging from 850 to 9490 mM ^-1?s^-1. The valine amide Val-3-APG exhibited significantly higher K_m and lower k_cat values compared to the corresponding ester Val-3-HPG, indicating poor specificity for hVACVase. In conclusion, the substrate leaving group has been shown to affect both binding and specific activity of hVACVase-catalyzed activation. It is proposed that hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate toward amide prodrugs.

Full text of DOI:10.1021/mp100300k

brief articles
Specificity of a Prodrug-Activating Enzyme hVACVase:
The Leaving Group Effect
Jing Sun,† Arik Dahan,‡ Zachary F. Walls,† Longsheng Lai,§ Kyung-Dall Lee,†
and Gordon L. Amidon*,†
Department of Pharmaceutical Sciences, College of Pharmacy, UniVersity of Michigan, 428
Church Street, Ann Arbor, Michigan 48109-1065, United States, Department of Clinical
Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion UniVersity of
the NegeV, Beer-SheVa 84105, Israel, and Department of Molecular Biophysics and
Biochemistry, Yale UniVersity, New HaVen, Connecticut 06520-8114, United States
Received September 1, 2010; Revised Manuscript Received October 23, 2010; Accepted
October 28, 2010
Abstract: Human valacyclovirase (hVACVase) is a prodrug-activating enzyme for amino acid
prodrugs including the antiviral drugs valacyclovir and valganciclovir. In hVACVase-catalyzed
reactions, the leaving group of the substrate corresponds to the drug moiety of the prodrug,
making the leaving group effect essential for the rational design of new prodrugs targeting
hVACVase activation. In this study, a series of valine esters, phenylalanine esters, and a valine
amide were characterized for the effect of the leaving group on the efficiency of hVACVase-
mediated prodrug activation. Except for phenylalanine methyl and ethyl esters, all of the ester
substrates exhibited a relatively high specificity constant (kcat/Km), ranging from 850 to 9490
mM-1 ·s-1. The valine amide Val-3-APG exhibited significantly higher Km and lower kcat values
compared to the corresponding ester Val-3-HPG, indicating poor specificity for hVACVase. In
conclusion, the substrate leaving group has been shown to affect both binding and specific
activity of hVACVase-catalyzed activation. It is proposed that hVACVase is an ideal target for
R-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate
toward amide prodrugs.
Keywords: hVACVase; prodrug
prodrug must be activated, either chemically or enzymati-
cally, to the active parent drug. Historically, the activation
mechanism was often not investigated. A more modern
approach, on the other hand, would exploit the mechanism
behind the activation, enabling targeting of the location and
control over the extent of this essential process.5-8 We have
recently identified an amino acid ester prodrug-activating
Introduction
The prodrug strategy has proven to be a successful
approach for improving oral absorption of low-permeability
drugs by various mechanisms.1-4 Following absorption, the
* To whom correspondence should be addressed. Phone: 734-
764-2226. Fax: 734-764-6282. E-mail: glamidon@umich.edu.
Address: Department of Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, 428 Church Street, Ann
Arbor, Michigan 48109-1065, United States.
† University of Michigan.
(3) De Clercq, E.; Field, H. J. Antiviral prodrugssthe development
of successful prodrug strategies for antiviral chemotherapy. Br. J.
Pharmacol. 2006, 147 (1), 1–11.
(4) Li, F.; Maag, H.; Alfredson, T. Prodrugs of nucleoside analogues
for improved oral absorption and tissue targeting. J. Pharm. Sci.
2008, 97 (3), 1109–1134.
(5) Wu, J. Z.; Lin, C. C.; Hong, Z. Ribavirin, viramidine and
adenosine-deaminase-catalysed drug activation: implication for
nucleoside prodrug design. J. Antimicrob. Chemother. 2003, 52
(4), 543–546.
‡ Ben-Gurion University of the Negev.
§ Yale University.
(1) Han, H. K.; Amidon, G. L. Targeted prodrug design to optimize
drug delivery. AAPS PharmSci 2000, 2 (1), E6.
(2) Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. Lessons
learned from marketed and investigational prodrugs. J. Med.
Chem. 2004, 47 (10), 2393–2404.
2362 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6, 2362–2368
10.1021/mp100300k  2010 American Chemical Society
Published on Web 10/28/2010

Specificity of a Prodrug-ActiVating Enzyme hVACVase
brief articles
enzyme, human valacyclovirase, hVACVase,9 which has
been shown to be one of the major enzymes for the activation
of the valine ester prodrugs valacyclovir and valganciclovir.
We have also shown that hVACVase effectively hydrolyzes
many other amino acid esters as well.10-12 Its potential to
serve as a prodrug-activating enzyme for amino acid ester
and amide prodrugs depends on its leaving group (drug)
specificity.
significant hydrolytic activity toward a series of amides,
including Lys-p-NA, Leu-p-NA, Pro-p-NA, Phe-p-NA, Val-
p-NA, and Gly-Pro-p-NA.10 This may be due to the much
more stable amide bond compared with the ester bond. The
10-fold difference in Km values of the prodrugs valacyclovir
and valganciclovir (0.19 and 1.90 mM, respectively) indicates
that the leaving groups may also affect the binding affinity.9
To better understand the hVACVase-catalyzed prodrug
activation and guide future prodrug design, an investigation
of the leaving group effect is essential.
hVACVase is a serine hydrolase with a catalytic triad
S122-H255-D227. The very specific preference for amino
acid analogues can be attributed to the electrostatic interaction
between the critical residue D123, adjacent to the active
S122, and the free amino group of the substrate.11 As
revealed by its crystal structure, hVACVase contains a large
leaving group-accommodating groove, which explains the
diversity of its substrate leaving groups (potential drugs),
including nucleoside analogues acyclovir, ganciclovir, floxu-
ridine, gemcitabine, zidovudine, 2-bromo-5,6-dichloro-1-(ꢀ-
D-ribofuranosyl) benzimidazole, and other alcohols such as
methanol, ethanol, benzyl alcohol, and [3-(hydroxymeth-
yl)phenyl]guanidine (3-HPG).10-12 Because the leaving
group corresponds to the drug moiety of the prodrug,
hVACVase, due to its expression in the intestinal epithelial
cells, is an ideal target for oral delivery and activation of
amino acid ester prodrugs and perhaps amide prodrugs as
well. While the phenylalanine benzyl and ethyl esters are
hVACVase substrates (specific activities are 358.3 and 75.3
units/mg, respectively), phenylalanine t-butyl ester is not a
substrate.11 In addition, the valine 5′-floxuridine has a higher
Vmax value compared with valine 3′-floxuridine ester (148
and 33 nmol/min/µg, respectively),10 thus indicating that the
alcohol leaving groups do have an effect on hVACVase
catalytic activity. Additionally, hVACVase does not exhibit
The purpose of this study was to investigate the effect of
leaving groups on the hVACVase-catalyzed prodrug activa-
tion in a systematic fashion. We have determined the kinetic
parameters of a series of valine esters (Val-3-HPG, valacy-
clovir, valine benzyl ester, valine p-nitrobenzyl ester),
phenylalanine esters (Phe-3-HPG, phenylalanine benzyl ester,
phenylalanine ethyl ester, phenylalanine methyl ester), and
valine amide of [3-(aminomethyl)phenyl]guanidine, Val-3-
APG (Scheme 1). This approach allows us to determine the
rate-limiting step of hVACVase-catalyzed hydrolysis and the
effect of leaving groups on the binding affinity and specific
activity. This will allow a more rational design of successful
amino acid ester and amide prodrugs.
Experimental Section
Materials. Boc-L-valine and N,N,N′,N′-tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) were
obtained from Calbiochem-Novabiochem (San Diego, CA).
Valacyclovir was a gift from GlaxoSmithKline, Inc. (Re-
search Triangle Park, NC). L-Valine p-nitrobenzyl ester
hydrobromide was obtained from Chem-Impex International,
Inc. (Wood Dale, IL). High-performance liquid chromatog-
raphy (HPLC) grade acetonitrile was obtained from Fisher
Scientific (St. Louis, MO). 3-Aminobenzonitrile, 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea, mercury(II) chlo-
ride, Pd/C, 2 M NH3/EtOH, trifluoroacetic acid (TFA), N,N-
diisopropylethylamine, L-valine benzyl ester hydrochloride,
L-phenylalanine benzyl ester hydrochloride, L-phenylalanine
methyl ester hydrochloride, L-phenylalanine ethyl ester
hydrochloride, and all other reagents and solvents were
purchased from Sigma-Aldrich Co. (St. Louis, MO). Cell
culture reagents were obtained from Invitrogen (Carlsbad,
CA), and cell culture supplies were obtained from Corning
(Corning, NY) and Falcon (Lincoln Park, NJ). All chemicals
were either analytical or HPLC grade.
(6) Rooseboom, M.; Commandeur, J. N.; Vermeulen, N. P. Enzyme-
catalyzed activation of anticancer prodrugs. Pharmacol. ReV. 2004,
56 (1), 53–102.
(7) Landowski, C. P.; Lorenzi, P. L.; Song, X.; Amidon, G. L.
Nucleoside ester prodrug substrate specificity of liver carboxy-
lesterase. J. Pharmacol. Exp. Ther. 2006, 316 (2), 572–580.
(8) Shi, D.; Yang, J.; Yang, D.; LeCluyse, E. L.; Black, C.; You, L.;
Akhlaghi, F.; Yan, B. Anti-influenza prodrug oseltamivir is
activated by carboxylesterase human carboxylesterase 1, and the
activation is inhibited by antiplatelet agent clopidogrel. J. Phar-
macol. Exp. Ther. 2006, 319 (3), 1477–1484.
(9) Kim, I.; Chu, X. Y.; Kim, S.; Provoda, C. J.; Lee, K. D.; Amidon,
G. L. Identification of a human valacyclovirase: biphenyl hydro-
lase-like protein as valacyclovir hydrolase. J. Biol. Chem. 2003,
278 (28), 25348–25356.
(10) Kim, I.; Song, X.; Vig, B. S.; Mittal, S.; Shin, H. C.; Lorenzi,
P. J.; Amidon, G. L. A novel nucleoside prodrug-activating
enzyme: substrate specificity of biphenyl hydrolase-like protein.
Mol. Pharm. 2004, 1 (2), 117–127.
(11) Lai, L.; Xu, Z.; Zhou, J.; Lee, K. D.; Amidon, G. L. Molecular
basis of prodrug activation by human valacyclovirase, an alpha-
amino acid ester hydrolase. J. Biol. Chem. 2008, 283 (14), 9318–
9327.
Synthesis. NMR spectra were obtained on a Bruker
AVANCE DRX500 NMR spectrometer. Electronspray ion-
ization mass spectra were obtained on a Micromass LCT
time-of-flight mass spectrometer. The purity of all synthe-
sized test compounds was at least 95% as determined by
HPLC.
3-[[Bis[[(1,1-dimethylethoxy)carbonyl]amino]methylene]-
amino]benzonitrile (2). To a stirred suspension of 3-ami-
nobenzonitrile (1, 118 mg, 1.0 mmol), 1,3-bis(tert-butoxy-
carbonyl)-2-methyl-2-thiopseudourea (290 mg, 1 mmol), and
mercury(II) chloride (353 mg, 1.3 mmol) in 5 mL of dry
(12) Sun, J.; Dahan, A.; Amidon, G. L. Enhancing the intestinal
absorption of molecules containing the polar guanidino functional-
ity: a double-targeted prodrug approach. J. Med. Chem. 2010, 53
(2), 624–632.
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Sun et al.
Scheme 1. Structures of Valine and Phenylalanine Analogues
tetrahydrofuran (THF), 405 mg (4.0 mmol) of triethylamine
was added dropwise under argon. After 2 h, the reaction was
stopped and reaction mixture was diluted with ethyl acetate
and filtered through Celite. Solvents were removed and the
residue was dissolved in 40 mL of ethyl acetate and washed
with 10% w/v citric acid, saturated NaHCO3 and brine. The
organic phase was dried over anhydrous MgSO4 and
concentrated in Vacuo. Product 2 was purified by column
chromatography (hexanes:ethyl acetate, 20:1) to give 327.9
mg white powder. Yield: 91.0%. 1H NMR (CDCl3) δ 11.65
(1H, br), 10.55 (1H, br), 8.11 (1H, s), 7.77 (1H, d, J ) 7.8
Hz), 7.42 (2H, m), 1.56 (9H, s), 1.54 (9H, s). ESI-MS: 383.1
(M + Na)+.
3-[[Bis[[(1,1-dimethylethoxy)carbonyl]amino]methylene]-
amino]benzylamine (3). A mixture of 2 (150 mg, 0.416 mmol)
and Pd/C (10 wt % palladium on activated carbon, 150 mg)
in 100 mL of 2 M NH3/EtOH was hydrogenated in a Parr
apparatus at 45 psi and room temperature for 9 h. The
reaction mixture was filtered through Celite. The solvents
were removed and product was purified by column chroma-
tography (dichloromethane:methanol, 15:1) to give 80.0 mg
light-yellow gum. Yield: 52.7%. 1H NMR (CDCl3) δ 11.65
(1H, br), 10.37 (1H, br), 7.56 (1H, s), 7.52 (1H, d, J ) 7.8
Hz), 7.32 (1H, dd, J ) 7.8 Hz, 7.8 Hz), 7.10 (1H, d, J ) 7.8
Hz), 3.90 (2H, s), 1.56 (9H, s), 1.53 (9H, s). ESI-MS: 365.2
(M + H)+.
acid (TFA):CH2Cl2 (1:1.2) and stirred at room temperature
for 2 h. Then solvents were removed and the residue was
dissolved in 0.1% TFA, filtered and lyophilized. The raw
product was further purified by semiprep HPLC to give 15.4
1
mg white solid. Yield: 57.7%. H NMR (CD3OD) δ 7.57
(1H, dd, J ) 7.9 Hz, 7.9 Hz), 7.44 (2H, m), 7.36 (1H, d, J
) 7.9 Hz), 4.18 (2H, s). ESI-MS: 165.1 (M + H)+.
(2S)-2-Amino-3-methyl-N-[3-[(aminoiminomethyl)ami-
no]phenyl]methylbutanamide (Val-3-APG, 6). To a stirred
solution of 3 (12.6 mg, 0.035 mmol), Boc-L-valine (9.1 mg,
0.042 mmol), and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-
1-yl)uronium hexafluorophosphate (HBTU, 16.0 mg, 0.042
mmol) in 1 mL of anhydrous CH2Cl2, 18 µL of N,N-
diisopropylethylamine were added dropwise under argon. The
reaction mixture was stirred at ambient temperature for 3 h,
and then the solvents were removed. The residue was
dissolved in 30 mL of CH2Cl2 and washed with 10% w/v
citric acid, saturated NaHCO3, and brine. The organic phase
was dried over anhydrous MgSO4 and concentrated in Vacuo.
The mixture was then chromatographed on silica gel (hex-
anes:ethyl acetate, 5:1) to obtain 5 as colorless oil. 1H NMR
(CDCl3) δ 11.65 (1H, br), 10.35 (1H, br), 7.52 (1H, d, J )
7.8 Hz), 7.47 (1H, s), 7.28 (1H, dd, J ) 7.8 Hz, 7.8 Hz),
7.03 (1H, d, J ) 7.8 Hz), 6.37 (1H, br), 5.06 (1H, m), 4.43
(2H, d, J ) 5.6 Hz), 3.92 (1H, m), 2.19 (1H, m), 1.42-1.53
(27 H, m), 0.96 (3H, d, J ) 6.8 Hz), 0.91 (3H, d, J ) 6.8
Hz). ESI-MS: 564.2 (M + H)+.
[3-(Aminomethyl)phenyl]guanidine (3-APG, 4). Com-
pound 3 (24.8 mg) was dissolved in 2.2 mL of trifluoroacetic
2364 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

Specificity of a Prodrug-ActiVating Enzyme hVACVase
brief articles
out in 50 mM HEPES (pH 7.4) buffer at 37 °C. After
preincubation of the buffer for 5 min, hVACVase was added
and then the reaction was initiated by the addition of
substrate. Aliquots were taken at different time points and
quenched by adding to same volume of 10% (v/v) trifluo-
roacetic acid. Initial velocities were calculated from the linear
time course for the product formation. The kinetic parameters
Km and Vmax were determined by fitting the initial velocity
data to the Michaelis-Menten equation by the nonlinear
least-squares regression analysis in GraphPad Prism software
Compound 5 was treated with 2.2 mL of TFA:CH2Cl2 (1:
1.2) for 2 h. Solvents were removed and residue was
dissolved in 0.1% TFA, filtered, and lyophilized to give 12.86
mg of 6 as light-brown syrup. Yield of two steps: 74.8%.
1H NMR (CD3OD) δ 7.48 (1H, dd, J ) 7.9 Hz, 7.8 Hz),
7.35 (1H, d, J ) 7.9 Hz), 7.28 (1H, s), 7.23 (1H, d, J ) 7.8
Hz), 4.49 (2H, m), 3.70 (1H, d, J ) 5.7 Hz), 2.21 (1H, m),
1.06 (6H, m). ESI-MS: 264.1 (M + H)+.
Hydrolysis of Valine and Phenylalanine Esters in
pH 7.4 HEPES Buffer. Hydrolysis in pH 7.4 HEPES buffer
was determined at 37 °C. The hydrolysis reaction was
initiated by adding 0.75 µL of test compound solution (200
mM in DMSO) to a reaction tube containing 749.25 µL of
pH 7.4 HEPES buffer. At various time points, 100 µL of
the reaction mixture was removed and added to a quenching
plate containing 100 µL of 1% TFA (in water) and stored in
ice. Following the collection of all samples, the quenching
plate was filtered (2000 rpm, 4 °C, 10 min). The filtrate was
removed and assayed by HPLC.
version 4.01. The kcat value was calculated from Vmax
/
[enzyme]0 based on the 28.83 kDa molecular mass of
hVACVase and 33.38 kDa molecular mass of 6×His-tagged
hVACVase. Specific activity of valacyclovir was routinely
monitored to normalize active protein concentration.
HPLC Analysis. The concentrations of test compounds
were determined on a Waters HPLC system (Waters Inc.,
Milford, MA) consisting of two Waters pumps (model 515),
a Waters autosampler (WISP model 712), and a Waters UV
detector (model 996 photodiode array detector). The system
was controlled by Waters Millennium 32 software (version
3.0.1). Samples were resolved in an Agilent ZORBAX
Eclipse XDB-C18 column (3.5 µm, 4.6 mm × 150 mm)
equipped with a guard column and the flow rate was 1.0
mL/min. For all the test compounds except for valine
p-nitrobenzyl ester, the mobile phase consisted of 0.1% (v/
v) TFA in milli-Q water (solvent A) and 0.1% (v/v) TFA in
acetonitrile (solvent B) with the solvent B gradient changing
from 2% to 30% at a rate of 2%/min during a 25 min run.
The retention times for 3-HPG, Val-3-HPG, Phe-3-HPG,
acyclovir, valacyclovir, 3-APG, Val-3-APG, benzyl alcohol,
Val-OBn, phenylalanine, Phe-OMe, Phe-OEt, and Phe-OBn
were 6.7, 9.7, 12.1, 4.9, 7.9, 4.2, 8.4, 11.3, 15.7, 8.2, 10.7,
13.0, and 18.9 min, respectively. For valine p-nitrobenzyl
ester, the mobile phase consisted of 70:30 (v/v) 0.1%
trifluoroacetic acid in milli-Q water: 0.1% trifluoroacetic acid
in methanol. The retention times were 8.5 min for p-
nitrobenzyl alcohol and 16.4 min for valine p-nitrobenzyl
ester. The detection wavelength was 235 nm for 3-HPG,
3-APG, Val-3-HPG, Phe-3-HPG, and Val-3-APG, 254 nm
for acyclovir and valacyclovir, 275 nm for p-nitrobenzyl
alcohol and L-valine p-nitrobenzyl ester, and 256 nm for
phenylalanine, benzyl alcohol, Val-OBn, and all the pheny-
lalanine esters except Phe-3-HPG.
The apparent first-order degradation rate constants were
determined by plotting the natural logarithm of test com-
pound remaining as a function of time. The slope of this
plot equals to negative rate constant (k). The degradation
half-life was then estimated by the equation:
t1/2 ) 0.693/k
Hydrolysis of Val-3-APG in pH 7.4 HEPES Buffer. The
hydrolysis reaction was initiated by adding 20 µL of Val-
3-APG solution (200 mM in DMSO) to a reaction tube
containing 380 µL of pH 7.4 HEPES buffer and incubated
at 37 °C. At various time points (up to 800 h), 85 µL of the
reaction mixture was removed and added to a quenching tube
containing 85 µL of 10% TFA (in water), filtered (2000 rpm,
4 °C, 10 min), and the filtrate was immediately assayed by
HPLC.
The apparent first-order degradation rate constants were
determined by plotting the natural logarithm of Val-3-APG
percentage (1-[3-APG]/([3-HPG] + [Val-3-APG])) as a
function of time. The slope equals to negative rate constant
(k). The degradation half-lives were then estimated by the
equation:
t1/2 ) 0.693/k
Statistical Analysis. All the experiments were performed
in triplicate unless stated otherwise. The data are presented
as mean ( SEM. Statistical difference between groups with
equal sample size was determined using the Tukey test (p <
0.01). Statistical difference between groups with unequal
sample size was determined using the Tukey-Kramer test
(p < 0.05). Letters designate groups with statistical signifi-
cance from one another.
hVACVase-Mediated Hydrolysis. hVACVase was over-
expressed and purified from Escherichia coli as described
previously11 and used for all the test compounds except
Val-3-APG. 6×His-tagged hVACVase (M-S tag-
PDLGTLVPRGSMGM-hVACVase-AAALE-His tag) was
produced in bacteria, purified by affinity chromatography,
and used for Val-3-APG. The results from the two proteins
are considered to be comparable due to their very similar
specific activity toward valacyclovir activation. The protein
concentration was determined by Bio-Rad DC assay (Her-
cules, CA) with bovine serum albumin as a standard. The
kinetic parameters of hVACVase-catalyzed hydrolysis were
determined as follows. Kinetic measurements were carried
Results
Synthesis of 3-APG and Val-3-APG. The synthesis of
3-APG and its valine amide is summarized in Scheme 2.
3-Aminobenzonitrile was treated with 1,3-bis(tert-butoxy-
VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2365

brief articles
Sun et al.
Scheme 2. Synthesis of 3-APG and Its Valine Amide
Table 1. Estimated Half-Life Values of Valine and
Phenylalanine Analogues in pH 7.4 HEPES Buffer (Mean
( SEM; Esters, n ) 3; Val-3-APG, n ) 1)
Phe-3-HPG and phenylalanine benzyl ester exhibited high
specificity constants as shown in Table 2, indicating that they
are good hVACVase substrates. However, the specificity
constants of phenylalanine methyl and ethyl esters were much
lower, which was mainly attributable to the higher Km value.
phenylalanine
valine analogues
t1/2 (min)a
analogues
t1/2 (min)a
valine p-nitrobenzyl 412.3 ( 11.9
ester
Phe-3-HPG
230.0 ( 6.5
Discussion
Val-3-HPG
valacyclovir
Val-OBn
486.9 ( 6.6
742.1 ( 5.5
857.8 ( 7.6
6.2 × 107
Phe-OBn
Phe-OMe
Phe-OEt
365.0 ( 9.4
470.6 ( 12.6
1006.4 ( 26.0
Rational design of prodrugs includes a thorough investiga-
tion of the activation process, which releases the free active
drug. hVACVase, a novel prodrug-activating enzyme, has
been shown to mediate the hydrolytic activation of various
amino acid ester prodrugs including the significant antiviral
drugs valacyclovir and valganciclovir. In the hVACVase-
mediated prodrug activation process, the leaving group
corresponds to the drug moiety of the prodrug molecule,
hence, investigating the leaving group effect is essential for
designing new prodrugs targeting this enzyme for activation.
Val-3-APG
a All the groups are statistically different.
carbonyl)-2-methyl-2-thiopseudourea to convert the free
amino group to the Boc-protected guanidino group. Then
the cyano group was reduced by hydrogenation to afford
intermediate 3. Deprotection of 3 gives 3-APG. Valine-3-
APG was synthesized by coupling intermediate 3 and Boc-
valine followed by deprotection.
Hydrolysis in Buffer and Cell Homogenates. The half-
life values of valine and phenylalanine analogues in pH 7.4
HEPES buffer are shown in Table 1. Valacyclovir and valine
benzyl ester exhibited approximately 2-fold longer t1/2 than
valine p-nitrobenzyl ester and Val-3-HPG. Compared with
Val-3-HPG, the hydrolysis of Val-3-APG was 5 orders of
magnitude slower. For the phenylalanine esters, the half-
life values ranked in the following order: Phe-3-HPG < Phe-
OBn < Phe-OMe < PheOEt.
In contrast to the fast hydrolysis of Val-3-HPG (t1/2 < 5
min),12 the valine amide Val-3-APG was fairly stable in
Caco-2 cell homogenates. During the 2 h incubation time,
no degradation of Val-3-APG was detected.
hVACVase-Mediated Hydrolysis. The Michaelis-Menten
kinetic parameters of valine derivatives for hVACVase-
mediated hydrolysis are listed in Table 2. All the valine esters
showed relatively low Km values ranging from 14 to 68 µM.
The kcat values of valine p-nitrobenzyl ester and Val-3-HPG
were about 2-fold higher than valacyclovir and valine benzyl
ester, similar to the trend shown for buffer hydrolysis rates.
The valine amide Val-3-APG exhibited significantly higher
Km and lower kcat values compared with the esters, indicating
that it is a very poor substrate of hVACVase.
As a serine hydrolase, the hydrolysis process catalyzed
by hVACVase is presumably a three-step reaction as shown
in the following scheme, where E stands for enzyme, S stands
for substrate, and AA stands for amino acid:13
For this kinetic scheme the Michaelis-Menten equation
can be expressed as:
k2k3
k2 + k3
V ) [E]0[S]
Ksk3
+ [S]
k2 + k3
in which
(13) Fersht, A. The Basic Equations of Enzyme Kinetics. In Structure
and Mechanism in Protein Science: A Guide to Enzyme Catalysis
and Protein Folding. 1st ed.; Fersht, A., Ed.; W. H. Freeman &
Co.: New York, 1999; pp 107-108.
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Specificity of a Prodrug-ActiVating Enzyme hVACVase
brief articles
Table 2. Michaelis-Menten Kinetic Parameters of Valine and Phenylalanine Derivatives for hVACVase-Mediated Hydrolysis
(Mean ( SEM; Phe-OMe, n ) 2; others, n ) 3)
c
valine analogues
Km (µM)b
Vmax (nmol/min/µg)
kcat (s-1
)
kcat/Km (mM-1 ·s-1
)
valine p-nitrobenzyl ester
Val-3-HPGa
valacyclovira
14 ( 1
46 ( 5
68 ( 4
272 ( 3
321 ( 9
120 ( 2
130 ( 2
154 ( 4
58 ( 1
9490 ( 580
3370 ( 460
850 ( 66
Val-OBn
60 ( 15
1810 ( 80
Km (µM)d
125 ( 7
60 ( 3
992 ( 300
Val-3-APG
(4.12 ( 0.08) × 10-3
(2.29 ( 0.04) × 10-3
(1.26 ( 0.08) × 10-3
e
phenylalanine analogues
Vmax (nmol/min/µg)
kcat (s-1
)
kcat/Km (mM-1 ·s-1
)
Phe-3-HPGa
Phe-OBn
Phe-OMe
Phe-OEt
207 ( 16
99 ( 26
5140 ( 1120
6700 ( 560
718 ( 16
822 ( 54
474 ( 49
193 ( 8
345 ( 8
395 ( 26
228 ( 24
93 ( 4
1660 ( 160
3980 ( 1310
44 ( 14
14 ( 2
a Previously published by Sun et al.12 b Statistically different groups: (A) valine p-nitrobenzyl ester, Val-3-HPG, valacyclovir, Val-OBn; (B)
Val-3-APG. c Statistically different groups: (A) valine p-nitrobenzyl ester; (B) Val-3-HPG; (C) valacyclovir, Val-OBn; (D) Val-3-APG.
d Statistically different groups: (A) Phe-3-HPG, Phe-OBn; (B) Phe-OMe, Phe-OEt. e Statistically different groups: (A) Phe-3-HPG, Phe-OBn;
(B) Phe-OMe; (C) Phe-OEt.
Table 3. Kinetic Parameters for N-Acetyltryptophanyl
k3
Km ) K
Substrates of Chymotrypsin (Adapted from Bender and
sk2 + k3
Kezdy14
)
and
k2k3
kcat
)
k2 + k3
It can be seen that the effect of the leaving groups depends
on the rate-limiting step, which can be either acylation or
deacylation. Because the leaving group (alcohol or amine)
is released from the enzyme in the acylation step, it will
only have an effect on the k2 value. If the acylation step is
rate-limiting (k2 , k3), then the kcat value will be affected
by the leaving group (kcat ≈ k2) and the Km value will
approach Ks. On the other hand, if the deacylation step is
rate-limiting (k2 . k3), the kcat value will be independent of
the leaving group (kcat ≈ k3) and Km value will be a function
of Ks, k2, and k3. For a series of esters or amides with the
same acyl group but different leaving groups with diverse
lability, k2 values should be different but k3 values should
be the same because the deacylation step is identical for all
of the substrates. Thus, the rate-limiting step can be
determined by comparing kcat (or Vmax) values for the ester
and amide substrates. A classical example of this strategy is
the case of chymotrypsin, where the amide exhibits a ∼3
orders of magnitude lower kcat value compared with the
corresponding esters, clearly indicating that the acylation step
is rate-limiting for the amide substrate (Table 3).14 Further,
for chymotrypsin-mediated hydrolysis of esters, while p-
nitrophenol is a much better leaving group than methanol
and ethanol, the very similar kcat values suggested that the
deacylation step was rate-limiting (Table 3). In the present
study, we have applied this classical strategy to investigate
hVACVase-mediated prodrug activation.
hydrolysis. The buffer hydrolysis rates of valine p-nitrobenzyl
ester and Val-3-HPG were 2-fold faster than valine benzyl
ester, consistent with the electron withdrawing effect of the
nitro and guanidino groups (Table 1). The order of pheny-
lalanine esters hydrolysis rate was also in correspondence
to the leaving group lability (Table 1). The very low acidity
of 3-APG makes it a poor leaving group, explaining the 5
orders of magnitude slower buffer hydrolysis of Val-3-APG
compared with Val-3-HPG.
The 4-5 orders of magnitude difference between the kcat
values of Val-3-HPG and Val-3-APG indicates that for the
amide Val-3-APG, the acylation step (k2) was rate-limiting,
similar to chymotrypsin-catalyzed hydrolysis (Table 2). For
the valine esters, the kcat values were also different and
matched the trend of buffer hydrolysis rate. This observation
suggests that the lability of the leaving groups did affect the
kcat and, unlike chymotrypsin, the acylation step (k2) is rate-
limiting. Similar phenomenon was observed for the rat liver
carboxylesterase-catalyzed hydrolysis of acetate esters, where
the chain length of the alkyl alcohol leaving group signifi-
cantly affects the reaction rate.15 However, because the kcat
differences in the current study were relatively small (up to
2.5-fold), more compounds with a broader range of labilities
will be necessary to conclusively confirm this analysis. For
the phenylalanine benzyl, methyl, and ethyl esters, the kcat
value also matched the buffer hydrolysis rate, suggesting that
The buffer hydrolysis rate reflects the lability of the leaving
groups and therefore the k2 value of the hVACVase-catalyzed
(15) Arndt, R.; Krisch, K. Catalytic properties of an unspecific
carboxylesterase (E1) from rat-liver microsomes. Eur. J. Biochem.
1973, 36 (1), 129–134.
(14) Bender, M. L.; Kezdy, J. Mechanism Of Action Of Proteolytic
Enzymes. Annu. ReV. Biochem. 1965, 34, 49–76.
VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2367

brief articles
Sun et al.
Scheme 3. Ester and Amide Substrates Binding to hVACVase
the acylation step is rate-limiting. Despite the faster chemical
hydrolysis of Phe-3-HPG, the kcat values of Phe-3-HPG and
phenylalanine benzyl ester were similar. It may indicate that
the deacylation rate becomes similar to acylation rate, but
because k2 depends on multiple factors other than the leaving
group lability, this hypothesis needs further evidence.
Assuming acylation as the rate-limiting step, the Km value
should be close to the binding affinity Ks. As shown in Table
2, all the amino acid esters showed similarly high affinity to
hVACVase except phenylalanine methyl and ethyl esters,
likely because methyl and ethyl groups are too small to have
favorable interactions such as hydrophobic interactions with
the leaving group binding pocket. Despite the negative
electrostatic potential in the leaving group-accommodating
groove, Val-3-HPG and Phe-3-HPG with the positively
charged leaving group exhibited similar binding affinity to
valine and phenylalanine benzyl esters, respectively (Table
2). This may be due to the water molecules in the leaving
group-accommodating groove interfering with expected
electrostatic interaction. The relatively similar Km values of
amino acid ester substrates suggest that the requirement of
the alcohol leaving groups for the binding is not very
stringent, in accordance with the large open leaving group-
accommodating groove. Although structurally similar to Val-
3-HPG, the amide Val-3-APG showed much lower affinity
to hVACVase (Table 2). The difference of ester and amide
could be due to the unfavorable interaction between the
amide hydrogen and the hydroxyl hydrogen of S122 (Scheme
3). The high Km combined with the low kcat value explains
why amides are poor substrates of hVACVase.10
of substrates have a significant effect on both the binding
and reaction rate of hVACVase-catalyzed hydrolysis. A good
hVACVase substrate should have a leaving group labile
enough for a reasonable activation rate but not too labile to
be chemically unstable. Alcohol leaving groups with reason-
able sizes and labilities are preferred by hVACVase. On the
other hand, the amino acid amide is a very poor substrate of
hVACVase due to both low affinity and the much stronger
amide bond. Therefore, amide prodrugs are more likely to
circumvent hVACVase-catalyzed hydrolysis. This can be
further exploited in rational prodrug design: if comparatively
quick intraenterocyte activation is desired, an amino acid
ester prodrug would be a better choice. However, if stability
of the prodrug in the enterocyte and activation at a later step
with a different mechanism is desired, amino acid amide may
be more suitable.
Abbreviations
hVACVase, human valacyclovirase; 3-HPG, [3-(hydroxym-
ethyl)phenyl] guanidine; p-NA, p-nitroanilide; 3-APG, [3-(ami-
nomethyl)phenyl]guanidine; Boc, tert-butyloxycarbonyl; TFA,
trifluoroacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-
1-ethanesulfonic acid; PNP, p-nitrophenyl.
Acknowledgment. We thank Dr. Deepak Gupta for
designing the synthetic method and Peter Ung for help
explaining the data. This research was supported by grant
2R01GM037188-21A2 awarded by the National Institutes
of Health.
In summary, these results indicate that the leaving groups
MP100300K
2368 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6