(Received for publication, October 21, 1996, and in revised form, February 7, 1997)
From the Laboratory of Medicinal Chemistry (F. F. W.), Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium and the
§ Laboratory of Protein Biochemistry and Protein
Engineering, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
A random pentapeptide library composed of 14 D-amino acids, including two unusual amino acids,
thus representing 537,824 different peptide sequences anchored on
polystyrene beads was created with each bead bearing a single
pentapeptide sequence. This library was used for affinity screening
against the fructose-1,6-bisphosphate aldolase of Trypanosoma
brucei labeled with biotin as well as versus the
COOH-terminal labeled with fluorescein isothiocyanate. The thus
selected peptide beads were identified and the appropriate sequences
synthesized as peptide amides and evaluated for enzyme activity
inhibition. Screening against the whole enzyme did not result in
selection of an enzyme inhibitor. However, we demonstrate here that
screening against a part of the enzyme involved in the catalytic
activity may lead to the discovery of an enzyme inhibitor as well as an
enzyme activator. Two low affinity inhibitors, RRVKF-NH2 and KThiKAR-NH2, with an IC50 of 1
mM and
0.2 mM, respectively, were
identified. Two other pentapeptides with the sequence
SWChaKK-NH2 and SKChaKM-NH2 are able to
activate the enzyme fructose-1,6-bisphosphate aldolase. Thus,
successful screening of solid phase libraries can be accomplished using
selected sequences of the target enzyme.
The African trypanosomes are parasites of wild and domesticated animals and of humans (1). The existing chemotherapy is unsatisfactory and prospects for immunoprofylaxis are extremely poor. As the trypanosomes in the bloodstream form lack a functional Krebs cycle, a respiratory chain, and storage forms for metabolic energy such as carbohydrates or "high energy phosphate" molecules, the bloodstream form depends entirely on glycolysis for its energy supply. Moreover, the glycolysis in the bloodstream form is 50 times faster than the glucose consumption in mammalian cells. For these reasons, it is believed that compounds interfering with glycolysis should be able to stop the evolution of the disease.
We already started a program based on x-ray and modeling experiments to
design inhibitors of the enzyme glyceraldehyde phosphate dehydrogenase
(2). The consecutive enzyme glycosomal phosphoglycerate kinase has been
used as target to screen against a peptide library composed of the 19 natural L-amino acids except for cysteine (3). This
research led to the selection of a pentapeptide with sequence NWMMF
able to inhibit glycosomal phosphoglycerate kinase (IC50 80 µM). We now selected a third glycosomal enzyme,
fructose-1,6-bisphosphate aldolase of the African trypanosome. This
enzyme, until now fails to crystallize and is not accessible for
structure based drug design.
Fructose-1,6-bisphosphate aldolase of T. brucei is a class I
aldolase (4). It catalyses the cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in
glucose metabolism or the reverse reaction in gluconeogenesis. It
consists of four identical subunits each bearing an active site (Fig.
1). Each subunit also contains, at the COOH-terminal end, an extended polypeptide chain with a COOH-terminal tyrosine that
interacts with the cleft-like region. The active site, which contains a
lysine--amino group is located at the bottom of the cleft (5).
Analysis of T. brucei aldolase in view of selective inhibitor design results in the following features (6).1 Inspection of the active site of the aldolases with known structure combined with an analysis of the sequence alignment revealed several differences in amino acid content between the parasite and human enzymes (Table I). Moreover, these residues are located relatively close to the active site and are fully solvent accessible. Especially residues 54-55 look quite promising since their human counterparts have much larger side chains. This difference in active site between human and trypanosomal enzyme led us believe that selective inhibition of the parasitic enzyme might be possible.
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During glycolysis, a Schiff base is formed with fructose 1,6-bisphosphate (7, 10). This Schiff base is protonated under physiological conditions and acts as an electron sink to enhance the expulsion of glyceraldehyde phosphate and leaves an enamine carbanion bound in the active site. Stereospecific protonation of the chemically reactive enamine regenerates a ketamine intermediate which upon hydrolysis liberates dihydroxyacetone phosphate (11). The protonation or deprotonation of dihydroxyacetone phosphate is accelerated by the COOH-terminal tyrosine and becomes the rate-limiting step in the absence of COOH-terminal tyrosine. Callens and Opperdoes showed that T. brucei aldolase activity is markedly reduced in the absence of COOH-terminal tyrosine. The COOH-terminal tyrosine of these class I aldolases also plays a role in the active site in the interaction with the 6-phosphate group of fructose-1,6-bisphosphate. This interaction should enhance the stability or induce a more reactive conformation of the enzyme. The COOH-terminal residue is preceded by a flexible stretch of about 19 amino acids (12) (Table II). Preventing the insertion of the carboxyl terminus should interfere with catalysis. Inspection of the COOH-terminal sequence alignment reveals that many residues differ between human and parasite aldolase.
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Although the non-conserved amino acids, distal to the active site of the COOH-terminal residue have no specific function in the catalytic mechanism, the differences have a significant role since many differences in catalytic activity could be observed among aldolase enzymes and by point mutations (5). This critical function of the COOH-terminal peptide allows us to test the hypothesis whether only fragments of an enzyme can be used to discover inhibitors using a solid phase library approach.
A synthetic pentapeptide library, assembled according to the "one-bead one-peptide" approach (13) from 14 D-amino acids (consisting of beads with each bead bearing one single pentapeptide sequence, as well as the whole library, representing the universe of all possible sequences), has been used to find new lead compounds that inhibit the activity of the enzyme fructose-1,6-bisphosphate aldolase. With this library, pentapeptides which bind to the whole enzyme or to the COOH-terminal end can be identified. In this paper, we describe the selection procedure for peptide ligands which bound the target molecule and their evaluation as enzyme inhibitors. This process led to the discovery of two inhibitors which can be used for further lead optimization. The discovery of these ligands is of particular interest given the bad crystallization properties of the enzyme.
Fmoc2-D-Ala-OH (Fmoc,
fluoren-9-ylmethoxycarbonyl), Fmoc-D-Arg(Mtr)-OH (Mtr,
4-methoxy-2,3,6-trimethyl-benzene-sulfonyl),
Fmoc-D-Asn(Trt)-OH (Trt, Trityl),
Fmoc-D-Glu(O-tBu)-OH (tBu,
tert-butyl), Fmoc-D-Met-OH, Fmoc-D-Lys(Boc)-OH (Boc, tert-butyloxycarbonyl),
Fmoc-D-Phe-OH, Fmoc-D-Pro-OH,
Fmoc-D-Ser(tBu)-OH,
Fmoc-D-Tyr(tBu)-OH, Fmoc-D-Val-OH, Fmoc-L-Tyr(tBu)-OH,
Fmoc-L-Lys(Boc)-OH,
Fmoc-L-Thr(tBu)-OH,
Fmoc-L-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-L-Ala-OH,
Fmoc-L-Val-OH, Fmoc-L-Leu-OH,
Fmoc-L-Ser(tBu)-OH, Fmoc-L-Gln(Trt)-OH,
Fmoc-L-Asp(O-tBu)-OH,
Fmoc-L-Arg(Pmc)-OH
(Pmc,2,2,5,7,8-pentamethylchroman-6-sulfonyl), and HBTU
(2"-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) were purchased from Advanced ChemTech Europe. Fmoc-D-Trp(Boc)-OH,
Fmoc--(2-thienyl)-D-alanine,
Fmoc-D-cyclohexylalanine, Rink amide MBHA resin was from
Novabiochem (Läufelfingen, Switserland), and
TentaGel-NH2 was obtained from Rapp polymer, (Tubingen,
Germany). Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), phenylmethylsulfonyl fluoride, and protamine sulfate were from Sigma.
Fructose-1,6-bisphosphate crystalline trisodium salt,
glycerol-3-phosphate dehydrogenase/triose-phosphate isomerase were
obtained from Boehringer (Mannheim, Germany). Dichloromethane (DCM),
N,N-dimethylformamide (DMF), acetic anhydride, and pyridine
were obtained from BDH. Trifluoroacetic acid, tetrahydrofuran, ammonium
sulfate, 1-methylimidazole, benzoic anhydride, fluorescein
isothiocyanate (FITC),
-nicotinamide adenine dinucleotide disodium
salt, reduced form (NADH), and dithiothreitol (DTT) and thioanisole
were supplied by ACROS (Geel, Belgium). Piperidine and dichloromethane
were distilled from calciumhydride. Tetrahydrofuran was distilled from
lithium aluminium hydride. Dynabeads M-280 were obtained from Dynal
International (Oslo, Norway).
Isopropyl-
-D-thiogalactopyranoside was supplied by ICN. Ethanedithiol was supplied by Aldrich. Diisopropylethylamine (DIEA) was
obtained form Applied Biosystems.
The peptide library
was synthesized using the split synthesis approach and assembled on a
TentaGel-S-NH2 support (14 g, 0.27 mmol g1,
130 µm) using Fmoc chemistry (14). One cycle consisted of
dividing the resin into 14 equal portions where each portion was
treated with 1 amino acid as follows: 2.5 mmol of derivatized amino
acid and 2.5 mmol of HOBt were dissolved in 2.5 ml of
N,N-dimethylformamide, 2.5 ml of a 1 M solution
of DIC in DCM was added to the above mentioned amino acid/HOBt
solution. The amino acid/HOBt/DIC solution was stirred for 2 min,
transferred to the resin, and incubated for 2 h at room
temperature while mixing at 200 rpm. The resin was filtered and rinsed
three times with DMF and three times with DCM. Free amino groups were
capped for 5 min with a solution of 10 ml of acetic anhydride in
pyridine (1:4) containing a catalytic amount of 1-methylimidazole. The
beads were repooled, mixed, rinsed three times with DCM and three times
with DMF. Deprotection was carried out by a 20-min treatment with 20%
piperidine/DMF, followed by filtration and rinsing three times with
DMF, three times with DCM, and three times with acetone after which the
beads were dried in vacuo.
This cycle was repeated 5 times to obtain a pentapeptide library. After synthesis was completed using 5 randomized coupling steps, side chain protecting groups attached to the amino acids were cleaved by stirring the peptide-bearing resin with the cleaving reagent consisting of 82.5% trifluoroacetic acid, 5% thioanisole, 5% m-cresol, 5% H2O, and 2.5% ethanedithiol for 3 h at 50 °C. The cleaving reagent was filtered and the beads were thoroughly washed with 50% trifluoroacetic acid in water.
Aldolase Expression and PurificationMutant strain
Escherichia coli BL21 (DE3) pLysS containing plasmid pTbALD1
(plasmid pTbALD1 is pET3A in which aldolase gene was inserted) (15) was
a generous gift from the laboratory of F. Opperdoes (ICP). Twenty-seven
mg of protein per liter of culture was obtained in LB medium
supplemented with 100 µg/ml ampicilline and 25 µg/ml
chloramphenicol, under vigorous agitation at 37 °C. Protein
expression was induced with 0.4 mM
isopropyl--D-thiogalactopyranoside at an OD (600 nm) of
0.7-0.8, and allowed to accumulate with shaking at 37 °C for 3 h. The cells were harvested at 4,000 rpm for 30 min at 4 °C. The
supernatant was removed. The cells were resuspended in a medium
(approximately 40 ml/liter of culture) containing 0.1 M
triethanolamine, pH 7.6, 1 mM DTT, 0.2 mM EDTA,
and 100 µM phenylmethylsulfonyl fluoride and opened using
a French Press. Cell fragments were removed by centrifugation for 20 min at 12,000 rpm. The supernatant was collected and sodium chloride
was added to a final concentration of 0.25 M. Nucleic acids
were further eliminated by incubation in the presence of 2000 units
Benzon nuclease (Merck, Darmstadt, Germany) for 30 min at 37 °C.
Subsequently, 80 mg of protamine sulfate was added to the suspension,
which was stirred for 15 min at room temperature followed by
centrifugation for 20 min at 12,000 rpm, 4 °C. Aldolase was
precipitated from the supernatant by addition of 75% ammonium sulfate
and incubation overnight at 4 °C. The precipitate was collected by
centrifugation (12,000 rpm, 20 min, 4 °C) and the pellet resuspended
in 100 ml of 0.1 M triethanolamine buffer, pH 7.6, 1 mM DTT, 0.2 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride and further diluted in the same buffer
until the suspension had an ionic strength corresponding to that of
0.05 M NaCl.
Purification of the enzyme was performed on a S-Sepharose exchange
chromatography column with a linear gradient of solvent A to solvent B. Solvent A was 0.1 M triethanolamine, pH 7.6, 1 mM DTT, 0.2 mM EDTA, 200 mM NaCl;
solvent B was 0.1 M triethanolamine, pH 7.6, 1 mM DTT, 0.2 mM EDTA, 500 mM NaCl.
Fractions of 2 ml were collected and enzyme fractions were checked by
activity measurement. Purity of the enzyme was checked by
SDS-polyacrylamide gel electrophoresis (16). The pooled fractions from
the S-Sepharose column were concentrated by ultrafiltration using a
Ultrafree-15 Centrifugal filter device (Millipore). The enzyme was
stored in solution at 20 °C or as an ammonium sulfate
precipitate.
Protein was measured by the Bradford test (17) using bovine serum albumin as a standard.
Labeling Aldolase with BiotinFour ml of the ammonium
sulfate suspension was centrifuged. The pellet was dissolved in 1 ml of
biotin labeling buffer, pH 8.3 (0.2 M NaHCO3,
0.5 M NaCl). The solution was desalted using Sephadex G-25
medium of DNA grade (NAP-10, Pharmacia) equilibrated with biotin
labeling buffer. The enzyme was eluted with 1.5 ml of biotin labeling
buffer. Fructose 1,6-bisphosphate was added to a final concentration of
90 µM to protect the active site of the enzyme during
labeling and 18 µl of a 50 mg ml1 biotin in
Me2SO (freshly prepared). The solution was vortexed for a
moment and labeling was continued overnight at room temperature. After
labeling, unbound biotin was separated from the conjugate by gel
filtration on a NAP-10 column equilibrated with PBS (phosphate-buffered saline; 8 g of NaCl, 0.2 g of KCl, 1.43 g of
Na2HPO4·H2O, 0.2 g of
KH2PO4 per liter, adjusted to pH 7 with
HCl).
After
side chain deprotection, the peptide library (2 g resin beads) was
washed three times with 10% DIEA in DMF, three times with DMF, three
times with PBS, three times with T-PBS (0.05% Tween 20 in PBS), and
three times with PBS. Labeled enzyme was incubated with the peptide
library overnight at 4 °C. The library was washed three times with
PBS and incubated with streptavidin-coated magnetic beads for 1 h
at 4 °C. Magnetic beads and indirectly peptide beads with affinity
for the enzyme were isolated from the library with a magnet. The
library was placed in the MPC for at least 15 min. Supernatant and
unbound peptide beads were removed by aspiration while the tube
remained in the Dynal MPC. The tube was removed from the Dynal MPC, PBS
was added, and Dynabeads were gently resuspended. The tube was placed
in the Dynal MPC and supernatant (with unbound peptide beads) removed.
This was repeated until no peptide beads remained in the supernatant.
The remaining peptide beads were freed from the magnetic beads by
incubation with 1% trifluoroacetic acid in water for 4 h.
Magnetic beads were separated with the Dynal MPC. The supernatant
(containing the selected peptide beads) was aspirated, transferred to a
microcentrifuge tube filter, and the solvent removed by centrifugation.
The remaining beads were selected individually and placed on a small
filter.
The COOH-terminal end of
the enzyme (Table II) was synthesized by solid phase peptide synthesis
on a model 431A peptide synthesizer (Applied Biosystems Inc., Foster
City, CA) using Fmoc chemistry with p-benzyloxybenzyl
alcohol resin (Wang Resin, 0.72 mmol g1, 347 mg) as the
support. The carboxyl-terminal tyrosine was loaded to the resin after
activation of the amino acid with dicyclohexylcarbodiimide to form a
symmetric anhydride. Dimethylaminopyridine was added to the resin as
coupling catalyst. After coupling of the COOH-terminal tyrosine,
remaining hydroxyl functions on the resin were capped with benzoic
anhydride in the presence of dimethylaminopyridine. The Fmoc group was
removed using 20% piperidine in DMF. The next amino acids were coupled
to the amino acid resin by in situ activation in the
presence of HBTU, HOBt, and DIEA. After each coupling step, the free
amino termini of nonreacted peptides were capped with acetic anhydride
(infra). The Fmoc removal, coupling and capping steps were repeated for
each amino acid to obtain the desired oligomer.
The resin bound COOH-terminal end (295 mg of resin) was treated with 180 mg of FITC in DMF for 3 days at room temperature. The excess FITC was removed by filtration. The labeled peptide was side chain deprotected and cleaved from the resin with 10 ml of trifluoroacetic acid containing 0.25 ml of EDT, 0.5 ml of H2O, 0.5 ml of m-cresol, and 0.5 ml of thioanisole for 2 h at room temperature. The mixture was filtered and the resin washed with 2 × 1 ml of trifluoroacetic acid. The filtrate was immediately collected in a 250-ml flask containing 50 ml of ice-cold diisopropylether to precipitate the peptide. The precipitate was obtained by centrifugation (9,000 rpm, 20 min, 4 °C) and the pellet was dissolved in 5% CH3CN/H2O (0.1% trifluoroacetic acid) from the resin and purified as described.
Screening and Affinity Selection with FITC-COOH-terminal FragmentThe protected library (1.6 g) was treated with 20% piperidine in DMF for 20 min. The resin was washed three times with DMF, three times with DCM, and dried in vacuo for 3 h. Side chain protecting groups were removed by treating the resin twice with a solution containing 20 ml of trifluoroacetic acid, 0.5 ml of EDTA, 1 ml of H2O, 1 ml of m-cresol, and 1 ml of thioanisole for 2 h. The resin was filtered, washed with trifluoroacetic acid (3 times) with 100 ml of PBS and 100 ml of T-PBS (0.05% Tween 20 in PBS). FITC-COOH-terminal end was added to the beads and incubated at 4 °C for 4 h. Beads were washed with PBS and fluorescent beads were selected under fluorescent microscope. Selected beads were treated with 8 M guanidine hydrochloride and 1% trifluoroacetic acid at 50 °C for 1.5 h and subjected to a second round of affinity selection with the FITC-COOH-terminal end. Beads were washed with T-PBS and T-PBS (2 times, 16 g of NaCl/liter). Fluorescent beads were selected and treated with T-PBS (4 times, 32 g of NaCl/liter). The remaining fluorescent beads were isolated and treated with T-PBS (8 times, 64 g of NaCl/liter). The beads were washed with 1% trifluoroacetic acid in water and placed individually on a small filter.
Identification of the Peptide BeadsSequence analysis was
performed on the model 476A gas-pulsed liquid phase sequenator with
on-line phenylthiohydantoin (PTH) analysis on a 120A analyzer (Applied
Biosystems, Foster City, CA). Sequencing reagents and solvents were
obtained from the same company. Determination of the retention times of
the PTH-derivatives of the non-natural amino acids, -(2-thienyl)-Ala
and
-cyclohexylalanine, was performed by eluting their
PTH-derivative (400 pmol for Thi) together with the standard containing
the PTH-derivatives of the gene-coded (L) amino acids. Solvent A was
3.5% tetrahydrofuran/H2O with PremixTM where
solvent B was CH3CN with 12% isopropyl alcohol.
Identified pentapeptides
were synthesized and HPLC purified. Pentapeptide amides were
synthesized on a Rink Amide Resin (510 mg, 0.49 mmol g1)
using Fmoc chemistry on a model 431A peptide synthesizer (Applied Biosystems Inc.). The resin was washed several times with NMP. The Fmoc
group was removed using 10 ml of 20% piperidine in NMP for 15 min.
After washing the resin five times with 8 ml of NMP, 1 mmol of
derivatized amino acid was dissolved in approximately 2 ml of DMF
containing 0.45 M HOBt and HBTU. DIEA in NMP (0.5 ml, 2 M) was added to the amino acid solution and the amino acid solution was transferred to the resin. The mixture was vortexed for 18 min at room temperature and the resin was filtered and washed several
times with NMP. Unreacted amino functions were acetylated with 10 ml of
NMP containing 0.5 M acetic anhydride, 0.125 M
DIEA, and 0.015 M HOBt. This complete cycle of Fmoc
removal, coupling and capping was repeated five times to obtain the
desired pentapeptides. After assembly of the pentapeptides and
deprotection of the last Fmoc group, side chain deprotection and
cleavage from the resin was done with 10 ml of trifluoroacetic acid
containing 0.25 ml of EDT, 0.5 ml of H2O, 0.5 ml of
m-cresol, and 0.5 ml of thioanisole for 2 h at room
temperature. The mixture was filtered and the resin washed with
trifluoroacetic acid (2 × 1 ml). The filtrate was immediately
collected in a 250-ml flask containing 50 ml of ice-cold
diisopropylether to precipitate the peptide. The precipitate was
collected by centrifugation (9,000 rpm, 20 min, 4 °C) and the pellet
was dissolved in 5% CH3CN/H2O (0.1% trifluoroacetic acid).
Purification of the synthesized peptides was performed
by high performance liquid chromatography on a PLRP-S semi-preparative column (250 × 9 mm). Peptides were eluted at 2-4 ml
min1 with a linear gradient of solvent A to solvent B,
solvent A being 5% CH3CN/H2O (0.1%
trifluoroacetic acid) and solvent B 80%
CH3CN/H2O (0.1% trifluoroacetic acid). The
eluate was monitored at 220 nm. Identification of the lyophilized
pentapeptide was performed by mass spectrometry on a Kratos Concept 1H
mass spectrometer (Kratos Analytical, Manchester, United Kingdom).
Liquid secondary ion mass spectrometry was used as ionization
(Cs+ as primary ion beam, accelerated at 9 kV). Glycerol
with the addition of trifluoroacetic acid (0.1% w/v) was used as
matrix. The sequence was determined by collisionally activated
dissociation using helium gas in the collision cell located in the
first field free region. The spectra were interpreted using a computer
program. Details about the mass spectrometric study have been published elsewhere (9).
Inhibition of the
fructose-1,6-bisphosphate aldolase enzyme activity by the identified
peptides was assayed at 25 °C in a final volume of 1 ml using
plastic cuvettes (Kartell-Milan) in parallel with a control. A 0.2 M glycine Tris buffer, pH 8.3, was used whereas the other
reactants (except auxiliary enzymes) were added as a 20-fold
concentrated mixture that was kept stored frozen at 20 °C. The
concentrations of the reactants in the assay where as follows: 1 mM EDTA, 0.42 mM NADH, 1 mM
fructose-1,6-bisphosphate, and 3 mM NaHCO3 plus
25 µg of glycerol-3-phosphate dehydrogenase/triose-phosphate isomerase (10:1) ml
1. The auxiliary enzymes
glycerol-3-phosphate dehydrogenase and triose-phosphate isomerase were
added as a crystalline suspension supplied from Boehringer. The
peptides were preincubated with aldolase in the assay buffer. After 10 min at room temperature, the other reactants were added to start the
reaction.
A peptide library (Fig. 2), consisting of beads,
with each bead containing one single pentapeptide was generated using
the split synthesis approach (13). Fourteen D-amino acids
were used: D-alanine, D-arginine,
D-asparagine, D-glutamic acid,
D-methionine, D-lysine,
D-phenylalanine, D-proline,
D-serine, D-tyrosine, D-valine, D-tryptophan, -(2-thienyl)-D-alanine, and
-cyclohexylalanine. D-Amino acids were chosen, as
peptides composed of D-amino acids have a better stability
against enzymatic degradation. These amino acids represent main group
of side chain functionality. Together with the use of 14 g of
resin beads (
130 µm), these 14 amino acids allow the
preparation of a library representing 537,824 (145)
different pentapeptide sequences in roughly equimolar proportion. In a
first attempt, part of this library was subjected to affinity selection
with fructose-1,6-bisphosphate aldolase labeled with biotin. A 2.5 M excess of biotin in biotin labeling buffer, pH 8.3, was
used in the presence of fructose 1,6-bisphosphate to avoid labeling of
the active site lysine. Peptide beads with affinity for the
biotin-labeled enzyme were selected with streptavidin-coated paramagnetic beads. In this selection protocol, the library was incubated with biotin-labeled aldolase, washed with PBS, and exposed to
a second incubation round of 1 h with streptavidin-coated
paramagnetic beads. Peptide beads that bound the biotin-labeled enzyme
were (by streptavidin-biotin affinity) indirectly bound to the
paramagnetic beads allowing the use of a magnet for the isolation of
these beads. After several rounds of separation by simple decanting while holding versus a magnet (separating the affinity beads
from the other library members), the obtained beads were freed of
biotin-labeled enzyme by treatment with 1% trifluoroacetic acid in
H2O. Nineteen beads were isolated. They were individually
placed on a small filter incubated with Biobrene®.
Fourteen peptide sequences could be clearly identified by Edman degradation. Determination of the retention times of the
PTH-derivatives of the non-natural amino acid,
-(2-thienyl)-Ala and
-cyclohexylalanine, was performed by eluting their PTH-derivative
(400 pmol for Thi) together with the standard containing the
PTH-derivatives of the gene-coded (L) amino acids. The PTH-derivative
of
-(2-thienyl)-D-alanine elutes between dptu and Trp.
Derivatized
-cyclohexyl-D-alanine did not elute during
the normal retention time. Since cysteine was not used during synthesis
of the library, a blank cycle could be considered as Cha.
These peptides were synthesized as soluble peptide amides on a Rink amide MBHA resin using standard Fmoc chemistry on a peptide synthesizer (ABI 431A). The peptide amides were purified by HPLC on a reversed phase column (Bio-Gel®). The identity of the peptides was verified by mass spectrometry and inhibition of the enzyme activity was measured at the 1 mM level. However, none of these peptides showed any significant inhibition at the 1 mM level (Table III).
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From the analysis of the mechanism of catalytic activity, it is clear
that the COOH-terminal end of the enzyme is important for catalytic
activity and compounds that prevent the insertion of the COOH-terminal
tyrosine should interfere with catalysis. As many interactions are
possible with a large enzyme such as aldolase
(Mr 160,000) without interfering with the
enzymatic activity, and considering the above mentioned function of the
COOH-terminal end, this region was subsequently selected as target for
screening. The COOH-terminal end (20 amino acids, Table II) was
synthesized chemically and labeled at the NH2 terminus
using FITC. Efforts to determine the mass of the labeled COOH-terminal
end failed. Selection of the appropriate HPLC fraction was done on the
basis of peak shifting as compared with the unlabeled peptide (MS
correctly determined as 2294.24) and UV absorption at 495 nm. After
purification, the labeled COOH-terminal end was incubated with the
peptide library and fluorescent beads were selected under a
fluorescence microscope. The isolated beads were subjected to a several
washing and isolation steps with increasing NaCl concentration until
the washing step resulted in a complete removal of the fluorescence on
the thus remaining beads. Twenty beads were selected of which 19 peptide sequences successfully were identified (Table
IV). After synthesis of the corresponding pentapeptide
amides, the enzyme inhibitory activity was investigated in triplicate
at the 1 mM level. Four interesting pentapeptides were
upheld: two of them with significant inhibitory activity, and
surprisingly likewise two sequences were found that significantly
increased the enzyme activity at the 1 mM level
(SWChaKK-NH2 and SKChaKM-NH2, Table IV). The
IC50 value was determined at pH 7.3. For the inhibitory
pentapeptide RRVKF-NH2, an IC50 1 mM was observed (Fig. 3), the pentapeptide
KThiKAR-NH2 displayed an IC50
0.2 mM (Fig. 4).
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Screening of the labeled enzyme aldolase against a random pentapeptide library to find peptide ligands possessing biological activity against the target enzyme was not successful. Although 14 sequences could be identified with apparent high affinity for the enzyme (as deduced from the thorough washing procedures during isolation), none of these showed biological activity. This is, most probably, due to the difficulties in bringing the active site in close proximity to the solid-phase bound oligopeptide. These results stimulated us to test the hypothesis whether a fragment of an enzyme may be used to increase the success of the solid-phase library screening procedure.
Because many interactions are possible with such a large enzyme as aldolase without interfering with the catalytic activity, and because the COOH-terminal end has a key function in the catalytic activity, in the second experiment, only the COOH-terminal end of the aldolase enzyme was used for screening against the random pentapeptide library. After affinity selection against the COOH-terminal end, 19 peptide sequences could be identified. From the primary structure analysis of the pentapeptides, it was obvious that, except for four sequences, all the pentapeptides were bearing two or three basic amino acids. This can partly be explained by the primary structure of the COOH-terminal end which contains three successive aspartic acids followed by a lysine and another aspartic acid. Ionic interactions between these residues and the basic side chains are very likely to occur. After screening the identified peptides for inhibition of the enzyme activity, two peptides showed a significant inhibition at the 1 mM level. With fluorescence quenching technique, we tried to determine binding constants toward the enzyme and the COOH-terminal end of the enzyme. A fluorescein fluorophore was attached to the pentapeptides at the NH2 terminus and the affinity of the enzyme for the labeled pentapeptides was measured. Unfortunately, no quenching of fluorescence nor shifting of wavelength maximum could be observed. Also with the COOH-terminal end no fluorescence quenching could be observed. This was partly expected due to the low IC50 values measured for the two inhibitory peptides. On the other hand, due to the high fluorescence of fluorescein, only small quantities of labeled pentapeptide can be used which makes this technique less sensitive than the direct activity measurement where only a small amount of enzyme and large excess of pentapeptide are used. The improvement of the catalytic activity of fructose-1,6-bisphosphate aldolase, observed in the presence of the peptide amides SWChaKK-NH2 and SKChaKM-NH2 could be explained by forcing of the enzyme into a more reactive conformation by the latter two amides.
Combinatorial chemistry for the rapid generation of a vast collection of compounds may be used to find a product compound with biological activity against a certain target, without knowing the structure of the target enzyme. The library approach may then be viewed as a competitive or alternative approach to structure-based design. From these and the previous studies (3) it is clear that screening of a solid-phase library, constructed by ignoring every structural information about the enzyme or known ligands, mostly results in the selection of low affinity ligands. The method is primarily useful for lead generation.
The present work demonstrates that using only the COOH-terminal sequence of the target enzyme (proposed to play a significant but no specific role during catalysis) for solid-phase screening was beneficial to increase the success of identifying molecules interfering with the catalytic activity of the whole enzyme. Screening with the whole enzyme did not result in finding any significant inhibitor, while screening with the COOH-terminal end on the other hand resulted in identifying four compounds that clearly influence the catalytic activity of fructose-1,6-bisphosphate aldolase.
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