Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer
Synthesis of fluorogenic peptide substrate of HIV-I protease Dns-SQNYPIVWL which corresponds to the p17/p24 cleavage site for HIV-l protease have been performed. This fluorogenic substrate was based on the fluorescence resonance energy transfer between donor – Trp residue, and acceptor – dansyl grou...
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Інститут молекулярної біології і генетики НАН України
1995
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| Цитувати: | Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer / A.L. Kornelyuk, A.G. Terentiev, S. Fisher, T. Porter // Биополимеры и клетка. — 1995. — Т. 11, № 6. — С. 57-61. — Бібліогр.: 18 назв. — англ. |
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Kornelyuk, A.I. Terentiev, A.G. Fisher, S. Porter, T. 2019-06-18T09:50:45Z 2019-06-18T09:50:45Z 1995 Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer / A.L. Kornelyuk, A.G. Terentiev, S. Fisher, T. Porter // Биополимеры и клетка. — 1995. — Т. 11, № 6. — С. 57-61. — Бібліогр.: 18 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000404 https://nasplib.isofts.kiev.ua/handle/123456789/156188 577.325 Synthesis of fluorogenic peptide substrate of HIV-I protease Dns-SQNYPIVWL which corresponds to the p17/p24 cleavage site for HIV-l protease have been performed. This fluorogenic substrate was based on the fluorescence resonance energy transfer between donor – Trp residue, and acceptor – dansyl group in the intact peptide. Hydrolysis of substrate by recombinant HIV-I protease resulted in the time-dependent increase of Trp fluorescence and decrease of dansyl fluorescence measured at 350 and 500 nm, respectively, due to the break of resonance energy transfer between donor and acceptor fluorophors. Hydrolysis of fluorogenic peptide substrate was studied also by reversed phase HPLC and two peptide fragments after cleavage of substrate have been detected. Kinetic constants of hydrolysis for this fluorogenic peptide substrate by HIV-I protease were calculated from Lineweaver – Burk plots: KM - 29mkM, kcat =5.4 s⁻¹ and kcat / KM -180000 M⁻¹s⁻¹. Проведено хімічний синтез флюорогенного пептидного субстрату ВІЛ-1 протеази, що має структуру Dns-SQNYPIVWL і відповідає сайту розщеплення р17/р24 у gag-поліпротеіні вірусу імунодефіциту людини.Принцип використання даного субстрату базується на резонансному переносі енергії збудження між донором – залишком Trp і акцептором – дансильною групою. Встановлено, що гідроліз флюорогенного пептидного субстрату рекомбінантною ВІЛ-1 протеазою призводить до падіння інтенсивності флюоресценції дансильної групи і одночасного зростання триптофанової флюоресценції внаслідок порушення резонансного переносу енергії між донором і акцептором. За допомогою високоефективної рідинної хроматографії в оберненій фазі зафіксовано появу пептидів, які є продуктами гідролізу субстрату. Визначено кінетичні параметри гідролізу флюорогенного пептидного субстрату ВІЛ-1 протеазою: КМ – 29 мкМ, kcat– 5,4 с⁻¹ та kcat/KM – 180 000 M⁻¹c⁻¹. Проведен химический синтез флюорогенного пептидной субстрата ВИЧ -1 протеазы , имеющей структуру Dns – SQNYPIVWL и соответсвует сайтурасщепления р17/р24 в gag – полипротеини вируса иммунодефицита людини. Принцип использования данного субстрата базируется на резонансном переносе энергии возбуждения между донором - остатком Trp и акцептором – дансильною группой. Установлено, что гидролиз флюорогенного пептидной субстрата рекомбинантной ВИЧ -1 протеазой приводит к падению интенсивности флюоресценции дансильнои группы и одновременного роста триптофановой флюоресценции вследствие нарушения резонансного переноса энергии между донором и акцептором. С помощью высокоэффективной жидкостной хроматографии в обратной фазе зафиксировано появление пептидов, которые являются продуктами гидролиза субстрата. Определены кинетические параметры гидролиза флюорогенного пептидной субстрата ВИЧ -1 протеазой : КМ – 29 мкм , kcat - 5,4 с ⁻¹ и kcat / KM – 180 000 M⁻¹c ⁻¹. en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer Синтез та характеристика флюорогенного пептидного субстрату ВІЛ-1 протеази на основі флюоресцентного резонансного переносу енергії Синтез и характеристика флюорогенного пептидной субстрата ВИЧ-1 протеазы на основе флюоресцентного резонансного переноса энергии Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer |
| spellingShingle |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer Kornelyuk, A.I. Terentiev, A.G. Fisher, S. Porter, T. |
| title_short |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer |
| title_full |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer |
| title_fullStr |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer |
| title_full_unstemmed |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer |
| title_sort |
synthesis and characterization of fluorogenic peptide substrate of hiv-1 protease based on fluorescence resonance energy transfer |
| author |
Kornelyuk, A.I. Terentiev, A.G. Fisher, S. Porter, T. |
| author_facet |
Kornelyuk, A.I. Terentiev, A.G. Fisher, S. Porter, T. |
| publishDate |
1995 |
| language |
English |
| container_title |
Биополимеры и клетка |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Синтез та характеристика флюорогенного пептидного субстрату ВІЛ-1 протеази на основі флюоресцентного резонансного переносу енергії Синтез и характеристика флюорогенного пептидной субстрата ВИЧ-1 протеазы на основе флюоресцентного резонансного переноса энергии |
| description |
Synthesis of fluorogenic peptide substrate of HIV-I protease Dns-SQNYPIVWL which corresponds to the p17/p24 cleavage site for HIV-l protease have been performed. This fluorogenic substrate was based on the fluorescence resonance energy transfer between donor – Trp residue, and acceptor – dansyl group in the intact peptide. Hydrolysis of substrate by recombinant HIV-I protease resulted in the time-dependent increase of Trp fluorescence and decrease of dansyl fluorescence measured at 350 and 500 nm, respectively, due to the break of resonance energy transfer between donor and acceptor fluorophors. Hydrolysis of fluorogenic peptide substrate was studied also by reversed phase HPLC and two peptide fragments after cleavage of substrate have been detected. Kinetic constants of hydrolysis for this fluorogenic peptide substrate by HIV-I protease were calculated from Lineweaver – Burk plots: KM - 29mkM, kcat =5.4 s⁻¹ and kcat / KM -180000 M⁻¹s⁻¹.
Проведено хімічний синтез флюорогенного пептидного субстрату ВІЛ-1 протеази, що має структуру Dns-SQNYPIVWL і відповідає сайту розщеплення р17/р24 у gag-поліпротеіні вірусу імунодефіциту людини.Принцип використання даного субстрату базується на резонансному переносі енергії збудження між донором – залишком Trp і акцептором – дансильною групою. Встановлено, що гідроліз флюорогенного пептидного субстрату рекомбінантною ВІЛ-1 протеазою призводить до падіння інтенсивності флюоресценції дансильної групи і одночасного зростання триптофанової флюоресценції внаслідок порушення резонансного переносу енергії між донором і акцептором. За допомогою високоефективної рідинної хроматографії в оберненій фазі зафіксовано появу пептидів, які є продуктами гідролізу субстрату. Визначено кінетичні параметри гідролізу флюорогенного пептидного субстрату ВІЛ-1 протеазою: КМ – 29 мкМ, kcat– 5,4 с⁻¹ та kcat/KM – 180 000 M⁻¹c⁻¹.
Проведен химический синтез флюорогенного пептидной субстрата ВИЧ -1 протеазы , имеющей структуру Dns – SQNYPIVWL и соответсвует сайтурасщепления р17/р24 в gag – полипротеини вируса иммунодефицита людини. Принцип использования данного субстрата базируется на резонансном переносе энергии возбуждения между донором - остатком Trp и акцептором – дансильною группой. Установлено, что гидролиз флюорогенного пептидной субстрата рекомбинантной ВИЧ -1 протеазой приводит к падению интенсивности флюоресценции дансильнои группы и одновременного роста триптофановой флюоресценции вследствие нарушения резонансного переноса энергии между донором и акцептором. С помощью высокоэффективной жидкостной хроматографии в обратной фазе зафиксировано появление пептидов, которые являются продуктами гидролиза субстрата. Определены кинетические параметры гидролиза флюорогенного пептидной субстрата ВИЧ -1 протеазой : КМ – 29 мкм , kcat - 5,4 с ⁻¹ и kcat / KM – 180 000 M⁻¹c ⁻¹.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/156188 |
| citation_txt |
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer / A.L. Kornelyuk, A.G. Terentiev, S. Fisher, T. Porter // Биополимеры и клетка. — 1995. — Т. 11, № 6. — С. 57-61. — Бібліогр.: 18 назв. — англ. |
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ISSN 0233-7657. Биополимеры н клетка. 1995, Т. 11. N» 6
UDC 577.325
A. L Kornelyuk, A. G. Terentiev, S. Fisher, T. Porter
Synthesis and characterization of fluorogenic peptide
substrate of HIV-1 protease based on fluorescence
resonance energy transfer
Synthesis of fluorogenic peptide substrate of HIV-1 protease Dns-SQNYPIVWL which corresponds to the
pi?7p24 cleavage site forHIV-J protease havebeen performed. This fluorogenic substrate was based onthe
fluorescence resonance energy transfer between donor — Trp residue, and acceptor — dansyl group in the
intact peptide. Hydrolysis of substrate by recombinant HIV-1 protease resulted in the time-dependent
increase ofTrp fluorescence and decrease of dansyl fluorescence measured at 350 and 500 nm, respectively,
due to the break of resonance energy transfer between donor and acceptorfluorophors. Hydrolysis of fluoro
genic peptide substrate was studied also by reversed phase HPLC and two peptide fragments after cleavage
of substrate have been detected. Kinetic constants of hydrolysis for this fluorogenic peptide substrate by
HIV-1 protease were calculated from Lineweaver— Burk plots: KM -29pM, kcat - 5.4 s'1 and kcailKM -
180 000 hf V і . -
Introduction* Human immunodeficiency virus protease (HIV-1 protease) per
forms the proteolytic processing of viral gag and gag-pol polyproteins precursors
during the replication cycle of retrovirus [1, 2]. It has been shown using site
directed mutagenesis and also by use of specific inhibitors of HIV-1 protease
that disruption of this function produces morphologically immature and
noninfectious viral particles [3]. Thus, HIV-1 protease represents an important
therapeutic target in the development of treatment for AIDS [4 ]. The number
of specific inhibitors of HIV-1 protease have been synthesized and studied and
results are summarized in reviews [5, 6 J. The antivirus synergy between HIV-1
protease inhibitors and nucleoside analogue inhibitors of HIV reverse transcrip
tase (AZT, ddC) has been also revealed [7 J. •
Evaluation of efficiency of novel HIV-1 protease inhibitors requires the
rapid and sensitive method of measurement of protease activity. Recently
chromophoric peptide substrate for the spectrophotometric assay of HIV-1
protease have been reported [8], which exhibits an absorbance increase upon
its hydrolysis by protease. Fluorogenic peptide substrates of HIV-1 protease
[9—11 ] are most efficient and sensitive in the monitoring of specific hydrolysis
reaction.
In this work we report the synthesis and characterization of fluorogenic
peptide substrate of HIV-1 protease based on fluorescence resonance energy
transfer (FRET). Resonance energy transfer between donor and acceptor
chromophores is widely used as a tool for the measurement of intramolecular
distances in biopolymers [12] and also to monitor the enzyme activity [9—11,
13]. Our substrate was based on the FRET between two fluorescent probes,
tryptophan and dansyl group, introduced into the oligopeptide substrate, which
corresponds to the pi 7/p24 cleavage site for HIV-1 protease.
Materials and Methods. Purification of HIV-1 protease. The expression
system in Escherichia coli strain AR58 was as previously described by Meek et
aL [14 ]. Cells (300 g) were thawed in 1500 ml of 50 mM Tris buffer containing
© A. I- KORNELYUK, A. C. TERENTIEV, S. FISHER, T. PORTER, 1995
57
A. I. KORNELYUK, A. G. TBRENTmY, S. FISHER, T. PORTER
5 mM EDTA, 10 mM DTT and 1 mM PMSF, pH 8.0 and lysed with one pass
through a Rainie cell disrupter at 6000 psi. Following centrifugation of the
lysate at 30.000 * g for 60 min, the supernatant was drawn off and solid
ammonium sulfate was added slowly to 35 % final concentration. After stirring
for 60 min at 4 °С, the mixture was allowed to settle for 30 min prior to
centrifugation at 30.000 * g for 60 min. The pellet was resuspended in a
minimal volume of Superose 12 chromatography buffer (50 mM Tris buffer
containing 5 mM EDTA, 10 mM DTT, 200 mM NaCl and 10 % glycerol, pH
8.0) and dialyzed overnight at 4 °С against the same buffer. The dialysate was
then clarified by centrifugation at 30.000 *g for 30 min and applied to a
Superose 12 column (5 liters). Fractions were collected and assayed for HIV-1
protease by reversed phase HPLC (RP-HPLC) and peak activity fractions were
pooled, made 40 % v/v with glycerol and stored at -70 °С. The protein was
judged to be greater than 95 % pure by SDS-PAGE and RP-HPLC analysis.
Protease activity assay was conducted according to [14 J with substrate
Ac-RASQNY • P W-NH2 .
Synthesis of fluorogenic peptide substrate. Protected amino acids Boc-Leu,
Boc-Ile, Boc-Val, Вое-Pro, Boc-Asn, Boc-Gln, N(a)-Boc-0-benzyl-L-Ser,
N(a)-Boc-0-benzyl-L-Tyr and N(a)-Boc-(indolylformyl)-LrTrp were synthe
sized as described [15].
Synthesis of fluorogenic peptide substrate of DnsSerGInAsnTyrProlleVal-
TrpLeu-OH was performed by peptide synthesizer using Merrifield resin
(chloromethylated copolymer of styrol and divinylbenzole, particle size of
200—400 mesh and active chlorine content of 0.7 mmole per 1 g of resin)
obtained from Fluka (Switzerland). For synthesis convenience N (a)-Boc-Leu
has been attached to the polymer as C-terminal amino acid by means of
potassium salt in the presence of 18-crown-6-ether as catalyst [16].
H2N-SerGlnAsnTyrProIleVaITrpLeu-resin was dansylated by Dns-Cl.
Dansylated peptide was isolated by pass of bromide hydrogen for 90 min
through the resin suspension in trifluoroacetic acid (TFA), containing thio-
anizole. Formylaticm protection of Tip was removed by 0.1 M triethylamine in
H20 for 10 min at 0 °С. The crude peptide (52 mg) was then purified to
homogeneity by RP-HPLC.
HPLC. Substrate purification, as well as the analysis of peptide fragments
after cleavage of substrate by HIV-1 protease were performed on a Gold System
(Beckinan), using either Ultrasphere C-18 column (10*250 mm) or Ultra-
sphere C-18 IP column (4.6 * 45 mm). Linear elution gradient of 0 to 80 %
acetonitrile in 0.1% TFA was used in HPLC experiments.
Fluorescence measurements. All measurements were performed on the
Hitachi Model 850 fluorescence spectrophotometer (Japan), with a thermostated
cell compartment at 20 eC. Fluorescence measurements were performed in
0.5 * 0.5 cm2 quartz microcuvette.
Fluorogenic peptide substrate Dns-SQNYPIVWL-OH was dissolved in
50 % isopropanol. Then peptide solution was diluted by addition of reaction
buffer, containing 0.05 M sodium acetate, pH 5J, 0.01 M dithiotreitol and 10 %
glycerol.
Kinetic analysis. Kinetic constants were determined from the fluorescence
assay data of hydrolysis of fluorogenic substrate by HIV-1 protease. Initial rate
of hydrolysis was calculated according to [171 as: v * FtlS]/(^'F0)ty where v
— initial rate of hydrolysis (umole/min), [S] — concentration of substrate
</*mole)r Ft — fluorescence intensity of substrate at the time t (t — time of
hydrolysis, min), F — fluorescence intensity after complete hydrolysis and F0
— initial fluorescence intensity. The kinetic parameters were obtained from the
Lineweaver — Burk plots after linearization procedure performed using wEnz-
fitter" program.
58
SYNTHESIS AND CHARACTERIZATION OF FLUOROGENIC PEPTIDE
Results. Design of structure of fluorogenic substrate. The amino acid
sequence which corresponds to the pJ7/p24 cleavage site for HIV-1 protease
was selected. This sequence was selected because it had been shown to be a
better substrate for cleavage by HIV-1 protease than synthetic peptides based
on other cleavage sites [18].
Trp residue was introduced into C-terminus of oligopeptide as a donor
fluorophor Clmw at 355 nm) and dansyl group at N-terminus served as an
acceptor fluorophor (Лшах at 520 nm). Substrate design was based on the FRET
between donor and acceptor groups in the intact peptide. Proteolytical cleavage
of substrate by HIV-1 protease at the Tyr-Pro site should release two
fluorophor-peptide fragments and break resonance energy transfer between Trp
and Dns with concomitant changes of fluorescence intensities both of donor and
acceptor fluorophors.
Fluorescence properties of substrate. Fluorescence of fluorogenic substrate
was excited at 290 nm and corrected emission spectra were recorded. Two
emission bands at 355 and 520 nm (Trp and Dns emissions) were observed in
the spectra. The ratio of fluorescence intensity of Dns to Trp fluorescence was
about 1.8—2.0 in the intact substrate.
Hydrolysis of substrate by HIV-J protease. Addition of HIV-1 protease
(1.9 • 10~7 M) to substrate solution in the reaction buffer resulted in the changes
of intensities of both fluorescence emission bands. Time-dependent increase of
relative emission intensity of Trp fluorescence and decrease of dansyl fluore
scence measured at 350 and 500 nm, respectively, are shown in Figure. These
fluorescence changes were apparently due to the break of FRET between donor
and acceptor fluorophors.
Fluorescence intensities changes (excitation wavelength
290 nm) of Dns-SQNYPIVWL-OH (concentration of
2.5 -l<f5 M) in the course of its hydrolysis by HTV-1
protease. Protease concentration was 1.9-10"' M. J —
relative intensity change of tryptophan fluorescence
measured at 350 nm; 2 — relative intensity change of
dansyl fluorescence measured at 500 nm
Hydrolysis ої fluorogenic peptide substrate was studied also by TtP-HPLC.
Action of HIV-1 protease on Dns-SQNYPIVWL resulted in the appearance of
two new peaks with retention times of 12.5 and 15.2 min. These species were
characterized by fluorescence spectroscopy and identified as substrate fragments
Dns-SQNY and PIVWL. These results suggest that fluorogenic substrate had
been cleaved by HIV-1 protease at Tyr-Pro site.
Kinetic parameters of hydrolysis. Kinetic studies of the hydrolysis reaction
were performed at nine different concentrations of fluorogenic substrate. The
kinetic constants of HIV-1 protease-catalyzed cleavage were calculated from
Lineweaver — Burk plots. For this fluorogenic peptide substrate the values
KM • 29 ^M and £ttt - 5.4 s"1 were obtained. These kinetic constants are close
to the values obtained by Matayoshi et al. [9 ] for the hydrolysis of fluorogenic
substrate based on the same p!7/p24 cleavage site (Кы - 103 ^M and
*«i • 4.9 s~\ respectively).
59
A. I. KORNELYUK, A. G- TERENTIEV, S. FISHER, T. PORTER
Discussion. Different methods have been used for the measurement of
proteolytic activity of HIV-proteases, including HPLC analysis of cleavage
fragments [141, protein immunoblot analysis, spectrophotometric assay using
chromophoric peptide substrate [8 ]. Most of these methods are time-consuming
and unacceptable for screening of large number of HIV-protease inhibitors,
Fluorogenic peptide substrates of HIV-1 protease are based on fluorescence
assay of its proteolytic activity. This method is very sensitive and provides an
accurate determination of kinetic parameters of hydrolysis reaction.
Fluorogenic peptide substrate of HIV-1 protease Dns-SQNYPIVWL
described in this work was based on the FRET between Trp as a donor and
Dns as an acceptor molecules. This donor-acceptor pair is very suitable for
energy transfer measurements. It is known that FRET measurements are most
sensitive to distance variation when donor-acceptor separation is close to their
Fbrster distance [12]. The value of Forster distance using Trp as a donor and
Dns as an acceptor of resonance energy transfer R0 --21 A [12], which
corresponds well to the length of peptide separating two fluorescent probes in
fluorogenic substrate. Our fluorogenic substrate is similar to substrate described
by Geoghegan et al. [11 ] but it has much better ratio кш/Кч « 180 000 M'V1.
The solubility of this fluorogenic substrate may be increased by addition of
other hydrophilic groups into its structure, e.g. by incorporation of Arg residues
at the C- and N-ends of substrate.
Fluorogenic peptide substrate of HIV-1 protease based on FRET may be
used not only in steady-state fluorescence measurements but also in liquid
chromatography assays, microscopy and other biological and clinical
applications.
A. I. Корнелюк, А Г. Терентьсв, С. Фішер, Т. Портер
СИНТЕЗ ТА ХАРАКТЕРИСТИКА ФЛЮОРОГЕННОГО
ПЕПТИДНОГО СУБСТРАТУ ВІЛ-1 ПРОТЕАЗИ НА ОСНОВІ
ФЛЮОРЕСЦЕНТНОГО РЕЗОНАНСНОГО ПЕРЕНОСУ ЕНЕРГВ
Резюме
Проведено хімічний синтез флюорогенного пептидного субстрату ВІЛ-1 протеази, що має
структуру Dns-SQNYPIVWL і відповідає сайту розщеплення рІ7Ір24 у gag-поліпротеіні вірусу
імунодефіциту людини.Принцип використання даного субстрату базується на резонансному
переносі енергії збудження між донором — залишком Т ф і акцептором — дансильною групою.
Встановлено, що гідроліз флюорогенного пептидного субстрату рекомбінантною ВІЛ-1 протеа
зою призводить до падіння інтенсивності флюоресценції дансильної групи і одночасного
зростання триптофанової флюоресценції внаслідок порушення резонансного переносу енергії
між донором і акцептором. За допомогою високоефективної рідинної хроматографії в оберненій
фазі зафіксовано появу пептидів, які є продуктами гідролізу субстрату. Визначено кінетичні
параметри гідролізу флюорогенного пептидного субстрату ВІЛ-1 протеазою: Кц - 29 мкМ, Jfê t —
- 5,4 с та *ои'Км - 180 000 M'V1 .
REFERENCES
1. Krausslich H.-G.t Wimmer E Viral proteinases / / Ашш. Rev. Biochem.—1988.— 57.—
P. 701—754.
2. Wlodaver A., Miller M.y Jaskolski M. et al Concerved folding in retroviral proteases: crystal
structure of a synthetic HIV-1 protease / / Science.—1989.—245, N 4918.—f. 616—621.
3. Kohl N.t Emini E.f Schleif W. et al. Active human immunodeficiency virus protease is required
for viral infectivity / / Proc. Nat. Acad. Sci. USA.—1988.—85.—P. 4686—4690.
4. Mitsuva # . , Yarchoan FL, Broder S. Molecular targets for AIDS therapy / / Science.—1990.—
249—P. 1533—1544.
5. Meek T. D. Inhibitors of HIV-1 protease / / J. Enzyme Inhibition—1992.—6.—P. 65—98.
6. Wlodawer A , Erickson J. W, Structure-based inhibitors of HTV-1 protease / / Алии. Rev.
Biochem.—1993.—62.—P. 543—585.
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SYNTHESIS AND CHARACTERIZATION OF FLUOROGBNIC PEPTIDE
7. Craig J. С Duncan I. B.f Whittaker L, Roberts N. A Antiviral synergy between inhibitors of
HIV proteinase and reverse transcriptase / / Antiviral Chem. and Chemother.—1993.—4.—
P. 161—166.
8. Tomaszek T. A., Magaard V. W.t Bryan H. G. et al. Chromophoric peptide substrates for the
spectrophotometric assay of HIV-1 protease / / Btochem. and Biophys. Res. Communs —
1990.—168, N 1.— P. 274—280.
9. Matayoshi E. D., Wang G. Т., Krafft G. A., Erickson J. Novel fluorogemc substrates for
assaying retroviral proteases by resonance energy transfer / / Science.—1990.—247.—P. 954—
958.
10. Wang G. T.t Matayoshi E. D., Huffaker # . , Krafft G. A. Design and synthesis of new
fluorogenic HIV protease substrates based on resonance energy transfer / / Tetrahedron
Lett.—1990,— 31, N 45.—P. 6493—6496.
11. Gcoghegan К P., Spencer R. W., Donley D. E. et al. Fluorescence-based continuous assay for
the aspartyl protease of human immunodeficiency virus-1 / / FEBS Lett.—1990.—262,
N 1.—P. 119—122.
12. Wu P., Brand L. Resonance energy transfer methods and applications / / Analyt. Biochem.—
1994.—218,-P. 1—13.
13. Latt S. A , Auld D. S., Vallee B. L Fluorescence determination of carboxypeptidase A activity
based on electronic energy transfer / / Ibid.—1972.—50, N 1.—P. 56—62.
14. Meek T. D., Dayton B. D.> Metcalf B. W. et al. Human immunodeficiency virus 1 protease
expressed in Е.СОІІ behaves as a dimeric aspartic protease / / Proc. Nat. Acad. Sci.
USA. —1989—86, N 6.—P. 1841—1845.
15. Gershkovich A A , Kibirev V. K. Synthesis of peptides. Reagents and methods.— Kiev: Naukova
dumka, 1987—264 p.
16. Roeske R., Gesellchen P. Reaction of potassium salts of Boc amino acids with chloromethyl
polystyrene catalyzed by 18-crown-6 / / Tetrahedron Lett—1976.—N 38.—P. 3369—3372.
17. Pilippova 1. YIL, Lysogorskaya E. N., Oksenoit E. S. et al. Intramolecular quenched fluorescent
substrates for aspartic proteinases / / Bioorg. Khimiya,—1986.—12, N 9.—P. 1172—1180.
18. Darke P. L., Nutt R. P., Brady S. P. et al HIV-1 protease specificity of peptide cleavage is
sufficient for processing of gag and pol polyproteins / / Biochem. and Biophys. Res.
Communs.—1988.—156, N 1.—P. 297—303.
Inst. Мої. Biol, and Genet., Nat. Acad, of Sci. of Ukraine, Kyiv Received
SmithKline Beecham Pharmaceuticals, USA 23.08.1995
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