Interaction of chloramphenicol tripeptide analogs with ribosomes

Article abstract of DOI:10.1134/S000629791604009X

Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory “stop peptides”–MRL, IRA, IWP–were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin an

Full text of DOI:10.1134/S000629791604009X

ISSN 0006ꢀ2979, Biochemistry (Moscow), 2016, Vol. 81, No. 4, pp. 392ꢀ400. © Pleiades Publishing, Ltd., 2016.
Published in Russian in Biokhimiya, 2016, Vol. 81, No. 4, pp. 538ꢀ547.
Originally published in Biochemistry (Moscow) OnꢀLine Papers in Press, as Manuscript BM15ꢀ322, January 31, 2016.
Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes
A. G. Tereshchenkov1, A. V. Shishkina2, V. N. Tashlitsky1,
G. A. Korshunova2, A. A. Bogdanov1,2, and N. V. Sumbatyan1*
1Lomonosov Moscow State University, Faculty of Chemistry, 119991 Moscow, Russia; Eꢀmail: sumbtyan@belozersky.msu.ru
2Lomonosov Moscow State University, Belozersky Institute of PhysicoꢀChemical Biology, 119991 Moscow, Russia
Received September 24, 2015
Revision received November 18, 2015
Abstract—Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory “stop peptides” –
MRL, IRA, IWP – were synthesized. The ability of the compounds to form ribosomal complexes was studied by displaceꢀ
ment of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramꢀ
phenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3ꢀ10 μM, which is 100ꢀfold lower
than the corresponding values for chloramphenicol amine–ribosome complex. Interaction of the chloramphenicol peptide
analogs with ribosomes was simulated by molecular docking, and the most probable contacts of “stop peptide” motifs with
the elements of nascent peptide exit tunnel were identified.
DOI: 10.1134/S000629791604009X
Key words: ribosome, “stop peptides”, chloramphenicol, peptide derivatives, nascent peptide exit tunnel
One of the important achievements of Xꢀray analysis atively short segments of the growing polypeptide chain
of ribosome was the proof of the existence of a tunnel called “stop peptides”, which are usually encoded in the
located in its large subunit [1ꢀ3]. The start of the ribosome leader strands of the regulated genes [9ꢀ12]. It appeared
tunnel (RT) overlaps with the ribosome peptidyl transꢀ that RT elements are able to interact not only with the
ferase center (PTC). The main function of the RT is to nascent peptide chain, but also with peptides coming from
ensure unhindered exit of newly synthesized polypeptide the outside. This was established by Xꢀray and biochemiꢀ
chain from the ribosome during translation and its delivꢀ cal data for bacterial ribosomal complexes with antimiꢀ
ery to the place where functionally complete protein molꢀ crobial peptides, particularly oncocines. The structures of
ecule formation begins [4ꢀ6]. The binding sites of many their ribosomal complexes have recently been resolved by
antibiotics are located in the RT, and consequently, modꢀ two scientific groups [13, 14].
ification of rRNA nucleotide residues and protein amino
In recent years, the study of the ribosome and its
acid residues forming RT walls leads to bacterial resistꢀ complexes with antibiotics and peptides as ligands was
ance to antibiotics [7, 8]. RT walls take part in amino acid studied using biochemical methods [15ꢀ19], Xꢀray analyꢀ
sequence monitoring of polypeptide chain moving along sis [13, 14], and cryoelectron microscopy [12, 20, 21]. It
them and, in some cases, polypeptides come into strong has been revealed that the RT is a dynamic functional sysꢀ
interactions with RT elements, which results in translaꢀ tem where specific interactions between the peptide chain
tion arrest [4, 9]. The translation arrest is caused by the and certain structural elements of the ribosome affect its
inhibition of ribosome peptidyl transferase activity by relꢀ functioning allosterically. One approach to the study of
the principles of peptide chain recognition by the RT, as
Abbreviations: Bhoc, Nꢀbenzhydryloxycarbonyl; Boc, tertꢀ
butyloxycarbonyl; BODIPY, (4,4ꢀdifluoroꢀ4ꢀboraꢀ5,7ꢀ
dimethyl)ꢀ3a,4aꢀdiazaꢀsꢀindaceneꢀ3ꢀpentanoic acid; Caeg, 3ꢀ
(2ꢀaminoethyl)ꢀ3ꢀ[2ꢀ(cytosinꢀ1ꢀyl)acetyl]glycine; DCC, 1,3ꢀ
dicyclohexylcarbodiimide; DIPEA, diisopropylethylamine;
Ery, erythromycin; Fmoc, fluorenylmethyloxycarbonyl; LCꢀ
MS, liquid chromatographyꢀmass spectrometry; PNA, pepꢀ
tideꢀnucleic acids; PTC, peptidyl transferase center; RT, riboꢀ
somal tunnel; TFA, trifluoroacetic acid.
well as related mechanisms of translation regulation, is
the creation of molecular probes based on ribosomal
antibiotics, whose structure includes amino acid and pepꢀ
tide residues simulating the nascent polypeptide chain
[19, 22ꢀ24].
In this work, novel peptide analogs of chlorampheniꢀ
col modeling the 3′ꢀterminal region of the peptidylꢀtRNA
were synthesized. The dissociation constants of the comꢀ
plexes of compounds obtained with E. coli ribosomes
* To whom correspondence should be addressed.
392

PEPTIDE CHLORAMPHENICOL ANALOGS
393
were measured by displacement of a fluorescent erythroꢀ ence of antibiotics cause translational stalling of signal
mycin analog from its ribosomal complexes, and molecuꢀ polypeptides encoded in the gene operon of the proteins
lar docking based modeling of these complexes was responsible for resistance to macrolides [25]. The IRA
accomplished for determination of probable contacts of motif is presented in the leader strand of SecM peptide
the peptides and RT. In the chloramphenicol analog that is responsible for regulation of SecA protein, playing
structure, the dichloroacetic fragment is replaced by short an important role in E. coli protein secretion and memꢀ
regulatory “stop peptide” sequences (Fig. 1a), which will brane transport [26]. The IWP motif is characteristic for
be oriented towards the RT when interacting with the leader sequences regulating translation of E. coli proteins,
ribosome (similarly to the growing polypeptide chain), including polyproline “stop peptides” defined by genetic
with the amphenicol ring serving as an “anchor” for the selection [18].
molecule binding in the PTC Aꢀsite (Fig. 1b).
The conjugate containing peptideꢀnucleic acid
As the peptide fragments, short conserved peptide (PNA) [27ꢀ29] in the 3′ꢀterminal tRNA region and “stop
motifs were selected: MRL, IRA, IWP (Fig. 1a), that are peptide” MRL in the peptide part (Fig. 2a) was syntheꢀ
met in the “stop peptide” Cꢀterminal sequences, located sized as another type of compound modeling peptidylꢀ
at the PTC during translation (Table S1; see Supplement tRNA for comparison. The conjugate of dipeptideꢀ
to this paper on the site of the journal (http://protein. nucleotide, whose skeleton consists of Nꢀ(2ꢀ
bio.msu.ru/biokhimiya) and on Springer site (Link. aminoethyl)ꢀglycine moieties (aeg), with 2′ꢀdeoxyadenoꢀ
springer.com)) [11, 18, 25].
sine – CaegCaegdA – was designed as the PNA to
In particular, the MRLꢀmotif is found in sequences address binding with ribosomal PTC. When interacting
that when arranged in the ribosomal tunnel in the presꢀ with the ribosome, the cytosine bases of this compound
a
b
Fig. 1. a) Structures of chloramphenicol peptide analogs: AcMRLꢀCAM (I), AcIRAꢀCAM (II), AcIWPꢀCAM (III). b) Imposition of strucꢀ
tures of E. coli ribosome complex with poly(Ala)ꢀtRNA (PDB – 2WWQ) and ribosomal complex with AcIRAꢀCAM modeled by molecular
docking. PeptidylꢀtRNA and 23S rRNA are dark, and AcIRAꢀCAM is light colored.
BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

394
TERESHCHENKOV et al.
a
b
23S rRNA
Fig. 2. a) Structure of peptidylꢀtRNA analog: AcMetArgLeuꢀdACaegCaeg (IV). b) Static modeling of PNA CaegCaegdA interaction with PTC
Pꢀsite of H. marismortui ribosome (PDB – 1VQ6).
should be complementary to 23S rRNA nucleotide
Fluorescence polarization was measured with a VICꢀ
residues G2251 and G2252 that are used by peptidylꢀ TOR™ X5 Multilabel Plate Reader (Perkin Elmer, USA).
tRNA for a specific binding with PTC Pꢀsite (Fig. 2b).
MATERIALS AND METHODS
The excitation wavelength was 485 nm, and the emission
wavelength was 530 nm.
Analytical reverse phase HPLC was performed on a
Milichrom Aꢀ02 chromatograph (Econova, Russia) with
ProntoSILꢀ120ꢀ5ꢀC18 AQ (2.0 × 75 mm, 5 μm) column
Chemicals used in this work were as follows: chloramꢀ in gradient of acetonitrile (MeCN) in 0.1% trifluoꢀ
phenicol from Sigma (China); BODIPYꢀOSu from roacetic acid (TFA) with elution rate 100 μl/min, and
Invitrogen (USA); amino acids, their derivatives, and detection at 214 and 260 nm.
reagents for peptide synthesis from Fluka (Switzerland),
Analytical and preparative reverse phase HPLC was
Merck (USA), and Reanal (Hungary); FmocCaegꢀ performed on a Knauer semipreparative chromatograph
(Bhoc)OH from Panagene (Korea). The fluorescent (Germany) with Beckman Coulter Ultrasphere ODS (10 ×
erythromycin derivative BODIPYꢀEry was obtained previꢀ 250 mm, 5 μm) column in gradient of MeCN with elution
ously [24]. The tripeptides AcMetArg(Pbf)LeuOH, rate 5 ml/min, and detection at 214, 260, and 500 nm.
AcIleArg(Pbf)AlaOH, and AcIleTrp(Boc)ProOH were
Optical rotation of substances was measured on a
synthesized and kindly provided by M. N. Zhmak VNIEKIProdmash EPO 1A instrument (Russia) at
(Department of Molecular Bases of Neurosignalizaꢀ 598 nm.
tion, Shemyakin–Ovchinnikov Institute of Bioorganic
Chemistry, Russian Academy of Sciences).
MALDIꢀTOF massꢀspectra were recorded on an
UltrafleXtreme MALDIꢀtimeꢀofꢀflightꢀmassꢀspectroꢀ
TLC was performed on Kieselgel 60 F254 plates from meter (Bruker Daltonics, Germany) equipped with UVꢀ
Merck (Germany), and Silica gel 60 (0.063ꢀ0.200 and laser (Nd), in the positive ion mode with reflectron.
0.040ꢀ0.063 mm) from Merck was used for column chroꢀ
LCꢀMS (liquid chromatography with massꢀspectroꢀ
matography. The compounds containing UVꢀabsorbing metry) was carried out with a UPLC/MS/MS system
groups were detected with UVꢀcabinet Camag (England); containing Acquity UPLC System chromatograph
the substances with free or Bocꢀprotected amino groups (Waters, USA), Acquity BEH C18 (2.1 × 50 mm, 1.7 μm)
were found by ninhydrin reactive; Sakaguchi and Ehrlich column (0.5 ml/min, 20 mM formic acid, gradient 5ꢀ
reagents were used for detection of compounds containꢀ 100% MeCN for 3 min), and quadrupole massꢀspectromꢀ
ing arginine with free guanidine group and tryptophan eter TQD (Waters) (ESI MS1 in the positive ion mode).
correspondingly.
1H NMR spectra were registered on a Bruker Avance
UV absorption spectra were registered using a Cary 400 NMR spectrometer (Bruker, USA) with 400 MHz
50 Bio spectrophotometer from Varian (Australia).
operating frequency for 1H nuclei.
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PEPTIDE CHLORAMPHENICOL ANALOGS
395
Synthesis of peptide derivatives of chloramphenicol λmax 279 nm; LCꢀMS, m/z calculated for
–
amine. (1R,2R)ꢀ2ꢀaminoꢀ1ꢀ(4ꢀnitrophenyl)propaneꢀ1,3ꢀ C28H47N8O8S+ – 655.3; found – 655.7; tR = 1.10 min.
diol (chloramphenicol amine) (V) [30]. A solution of 1.0 g TFA·AcIleArgAlaꢀCAM (II) was obtained as I, from
(3.1 mmol) of chloramphenicol in 15 ml of 1 M aqueous 50 mg (77 μmol) of AcIleArg(Pbf)AlaOH, 9 mg
HCl was heated on a boiling water bath for 2 h, and after (77 μmol) of Nꢀhydroxysuccinimide, 19 mg (92 μmol) of
cooling to room temperature the mixture was extracted DCC, 13 μl (77 μmol) of DIPEA, and 16 mg (77 μmol) of
with ether (4 × 20 ml). The aqueous layer was evaporated V. Yield: 31 mg (57%); TLC: Rf (BuOH–H2O–AcOH,
to dryness in vacuo. The target product was isolated from 4 : 1 : 1) 0.19, Rf (CHCl3–H2O–MeOH, 65 : 4 : 25) 0.11;
the residue by column chromatography on silica gel elutꢀ UV (H2O): λmax – 280 nm; LCꢀMS, m/z calculated for
ing with CHCl3–19% NH4OH–MeOH, 65 : 5 : 25 (v/v). C26H43N8O8+ – 595.3; found – 595.7; tR = 0.89 min.
Yield: 0.61 g (93%); TLC: Rf (CHCl3–19% NH4OH–
AcIleTrpProꢀCAM (III) was obtained as I, from
MeOH, 65 : 5 : 25) 0.54, Rf (CHCl3–H2O–MeOH, 65 : 50 mg (90 μmol) of AcIleTrp(Boc)ProOH, 10 mg
4 : 25) 0.19; m.p. 162ꢀ164°C (160ꢀ162°C [30]); (90 μmol) of Nꢀhydroxysuccinimide, 22 mg (108 μmol)
[α]2D0 –18.2 (c = 3; MeOH) (–22.8 (c = 3; MeOH) [30]); of DCC, 15.6 μl (90 μmol) of DIPEA, and 19 mg
1HꢀNMR: (DMSOꢀd6, 294K, 400 MHz), δ (ppm): 1.74 (90 μmol) of V. Yield: 51 mg (87%); TLC: Rf
(bs, 2H, ꢀNH2), 2.72 (m, 1H, ꢀCHNH2), 3.18 (dd, Jdd
=
(CHCl3–MeOH, 9 : 1) 0.33; UV (H2O): λmax – 224 and
a
+
10.4 Hz, 6.0 Hz, 1H, ꢀCH2 OH), 3.35 (dd, Jdd = 10.4 Hz, 280 nm; LCꢀMS, m/z calculated for C33H43N6O8
–
b
5.7 Hz, 1H, ꢀCH2 OH), 4.68 (d, Jd = 4.2 Hz, 1H, 651.3; found – 651.8; tR = 1.53 min.
ꢀCHOH), 7.59 (d, Jd = 8.6 Hz, 2H, mꢀHꢀNO2ꢀPhe), 8.18
(d, Jd = 8.6 Hz, 2H, oꢀHꢀNO2ꢀPhe).
Syntheses of AcMRLꢀdACaegCaeg (IV) and MRLꢀ
OMe peptide (VII) are described in the Supplement (see
AcMetArg(Pbf)LeuꢀCAM (VI). To a cold solution of Figs. S1 and S2).
20 mg (28 μmol) of AcMetArg(Pbf)LeuOH and 3.2 mg
BODIPYꢀMetArgLeuꢀOMe (VIII). To a solution of
(28 μmol) of Nꢀhydroxysuccinimide in 300 μl of anhyꢀ 0.5 mg (0.76 μmol) MetArgLeuꢀOMe × 2TFA in 100 μl of
drous CH2Cl2, 7 mg (34 μmol) of DCC was added at 0°C. 0.1 N aqueous NaHCO3, 0.58 mg (1.39 μmol) BODIPYꢀ
The mixture was stirred for 2 h at 0°C, then overnight at OSu in 80 μl MeCN was added, and the resulting mixture
4°C. The formed precipitate was filtered off, and solvent was stirred for 3 h at room temperature. The target comꢀ
was removed in vacuo. The residue was dissolved in 250 μl pound was isolated by HPLC (Beckman Coulter
of DMF and added to a solution of 6 mg (28 μmol) of V Ultrasphere ODS; 10 × 250 mm, 5 μm; 5 ml/min, 0ꢀ
and 4.9 μl (28 μmol) of DIPEA in 40 μl of DMF. The 100% MeCN at 20 min). Yield: 0.32 mg (57%): TLC: Rf
resulting mixture was stirred for 7 h at room temperature, (CHCl3–MeOH, 9 : 1) 0.05, Rf (CHCl3–MeOH, 3 : 1)
then overnight at 4°C. The reaction mixture was diluted 0.37; HPLC: tR = 16.4 min; UV (H2O): λmax – 227, 365,
with 3 ml of CHCl3, washed with water (3 × 2 ml). and 505 nm; LCꢀMS, m/z calculated for
Organic layer was dried with Na2SO4, and the volatiles C34H54BF2N8O5S+ – 735.4, found – 735.8.
were evaporated in vacuo. The target product was isolated
Molecular docking was performed in AutoDock Vina
from the residue by purification on a silica gel column [31]. The crystal structure of the E. coli ribosome with
eluting with CHCl3–MeOH, 9 : 1. Yield: 20 mg (78%); chloramphenicol (PDB: 3OFC) [32], from which the
TLC: Rf (CHCl3–MeOH, 9 : 1) 0.24, Rf (CHCl3– water molecules, inorganic ions, and low molecular weight
EtOAc–MeOH, 6 : 3 : 0.5) 0.11, Rf (CHCl3–19% ligands were removed, was used as a receptor. The molecuꢀ
NH4OH–MeOH, 65 : 5 : 25) 0.86, Rf (CHCl3–MeOH, lar docking region was determined as a cube with the cenꢀ
4 : 1) 0.91; UV (H2O): λmax – 224 nm, 258 nm; LCꢀMS, ter in the chloramphenicolꢀbinding site and with 34 Å
+
m/z calculated for C41H63N8O11S2 – 907.4; found – sides. The initial conformations of ligands were preꢀoptiꢀ
908.0; tR = 1.96 min.
mized by a semiempirical method in MOPAC2012 with
TFA·AcMetArgLeuꢀCAM (I). A mixture of 2.48 ml of PM7 Hamiltonian [33]. The “exhaustiveness” parameter,
TFA, 150 μl of phenol, 150 μl of water, 150 μl mꢀcresol, characterizing the docking accuracy, was equal to 100. The
and 75 mg of dithiothreitol was deaerated by bubbling data were graphically displayed using PyMOL 1.5.
with nitrogen for a few minutes. The resulted mixture was
Investigation of ligand binding to bacterial E. coli riboꢀ
added to 20 mg (22 μmol) of VI and stirred for 4 h at room somes [34]. 70S ribosomes of E. coli strain MREꢀ600 were
temperature under nitrogen. The volatiles were evaporatꢀ kindly provided by A. L. Konevega (Laboratory of Protein
ed in vacuo, and the residue was dissolved in a minimal Biosynthesis, Molecular and Radiation Biophysics
amount of water and treated with ether. The resulting preꢀ Division, Petersburg Nuclear Physics Institute (PNPI)).
cipitate was filtered. The target product was isolated by Ribosome concentration was determined by optical denꢀ
HPLC. Yield: 14.5 mg (86%); TCX: Rf (CHCl3– sity at 260 nm (A260 = 1 a.u. at C(Rs) = 24 nM). BIND
H2O–MeOH, 65 : 4 : 25) 0.16, Rf (CHCl3–19% buffer was used with the following composition: 20 mM
NH4OH–MeOH, 65 : 5 : 25) 0.29; HPLC: tR = 16 min HEPES, pH 7.5, 50 mM NH4Cl, 10 mM MgCl2, 0.05%
(Beckman Coulter Ultrasphere ODS (10 × 250 mm, Tween 20. Escherichia coli 70S ribosomes were thawed at
5 μm), 5 ml/min, 0ꢀ60% MeCN at 20 min); UV (H2O): 0°C, followed by incubation for 15 min at 37°C.
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TERESHCHENKOV et al.
The dissociation constants of the investigated comꢀ
Peptide derivatives and dissociation constants of their
complexes with ribosomes ( 95% confidence interval)
pounds were determined by replacement of fluorescently
labeled erythromycin (BODIPYꢀEry) from the complex
with ribosomes. BODIPYꢀEry (4 nM) was incubated with
ribosomes (24 nM) for 30 min at 25°C in 384ꢀwell plate.
The solution of rival ligand in concentrations from 1 μM
to 1 mM was added to the formed ribosomal complex.
The mixture was incubated for 2 h until equilibrium was
reached, and the values of the polarization of fluoresꢀ
cence were measured and then converted to anisotropy
(mA). The calculations of dissociation constants of the
complexes of peptide derivatives with ribosomes were
performed basing on two replicate measurements. The
confidence interval (95%) was calculated for the presentꢀ
ed dissociation constant values.
Compound
KD, μM
AcMRLꢀCAM (I)
4.7 0.6
4.3 0.6
AcIRAꢀCAM (II)
AcIWPꢀCAM (III)
AcMRLꢀdACaegCaeg (IV)
MRLꢀOMe (VII)
10
5
1
1
250 140
2.8 0.5*
710 170
Chloramphenicol
Chloramphenicol amine (V)
* The corresponding KD value determined by [14C]chloramphenicol
replacement is 1.7 μM [19].
With the goal to obtain BODIPYꢀMetArgLeuꢀOMe
binding curves, solutions of ribosomes in different conꢀ
centrations (from 1 to 7000 nM) in BIND buffer were hydroxyꢀ2ꢀ(4ꢀnitrophenyl)ꢀ1ꢀhydroxymethylꢀethyl]ꢀ
incubated with the fluorescently labeled peptide (4 nM) amide (AcIRAꢀCAM, II), NꢀacetylꢀLꢀisoleucylꢀLꢀtrypꢀ
solution at 25°C for 1 h to reach equilibrium, and then the tophylꢀLꢀprolylꢀ[(1R,2R)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀnitrophenyl)ꢀ
fluorescence polarization values were measured.
1ꢀhydroxymethylꢀethyl]ꢀamide (AcIWPꢀCAM, III). The
The data for binding and fluorescent substrate synthetic scheme comprised chloramphenicol hydrolysis
replacement experiments were processed using the to chloramphenicol amine [30], acylation of the latter by
GraphPad Prizm 6 computer program.
Nꢀacetylꢀpeptides with the side groups protected, and
final deprotection (Fig. 3).
The conjugate of PNA with “stop peptide”
AcMetArgLeuꢀdACaegCaeg (IV) was also obtained synꢀ
thetically. Its structure is shown in Fig. 2a, and its syntheꢀ
RESULTS
Synthesis. Novel chloramphenicol amine analogs sis is depicted in Fig. S1 in the Supplement.
were synthesized: NꢀacetylꢀLꢀmethionylꢀLꢀarginylꢀLꢀ
The fluorescent derivative BODIPYꢀMetArgLeuꢀ
leucylꢀ[(1R,2R)ꢀ2ꢀhydroxyꢀ2ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ OMe (VIII) was synthesized by a reaction between sucꢀ
hydroxymethylꢀethyl]ꢀamide (AcMRLꢀCAM, I), Nꢀ cinimide ester BODIPYꢀOSu and peptide MRLꢀOMe
acetylꢀLꢀisoleucylꢀLꢀarginylꢀLꢀalanylꢀ[(1R,2R)ꢀ2ꢀ (VII), obtained according to the scheme shown in Fig. S2
in the Supplement.
Study of binding of chloramphenicol analogs and PNA
to E. coli ribosomes. The interaction between peptide
derivatives and ribosomes was studied via replacement of
a
BODIPYꢀEry from its complexes with E. coli ribosomes
by these compounds. There was a decrease in fluoresꢀ
cence polarization, indicating competition of the tested
b
c
compounds with erythromycin for the corresponding
binding site in RT (Fig. 4).
To quantify the affinity of the peptide derivatives to
the ribosomes, the data on BODIPYꢀEry replacement
was approximated using a function corresponding to the
solution of the cubic equation that describes the competꢀ
itive binding of two ligands in one site [35]. We choose
this model based on the fact that the ribosomal binding
sites of chloramphenicol peptide derivatives and erythroꢀ
mycin overlap. The binding areas of chloramphenicol and
Fig. 3. Scheme of synthesis of peptide chloramphenicol analogs.
erythromycin differ spatially; however, it appears that
both antibiotics cannot simultaneously be complexed
with the ribosome. This indicates the allosteric nature of
chloramphenicol displacement of fluorescently labeled
erythromycin, for which the same mathematical descripꢀ
tion can be applied. The dissociation constant value of
AcMRLꢀCAM (I): a) 1 M HCl, 100°C; b) AcMetArg(Pbf)Leuꢀ
OSu, DMF, DIPEA; c) TFA, PhOH, H2O, mꢀcresol, DTT, 33 :
2 : 2 : 2 : 1 (v/v). AcIRAꢀCAM (II): a) 1 M HCl, 100°C; b)
AcIleArg(Pbf)AlaꢀOSu, DMF, DIPEA; c) TFA, PhOH, H2O, mꢀ
cresol, DTT, 33 : 2 : 2 : 2:1 (v/v). AcIWPꢀCAM (III): a) 1 M HCl,
100°C; b) AcIleTrp(Boc)ProꢀOSu, DMF, DIPEA; c) TFA,
PhOH, H2O, mꢀcresol, DTT, 33 : 2 : 2 : 2 : 1 (v/v).
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397
a
b
150
125
100
75
150
125
2
4
7
6
1
100
75
5
3
50
50
1
10
100
1
10
100
1000
Ligand concentration, μM
Fig. 4. Curves of replacement of BODIPYꢀEry from its complex with 70S E. coli ribosomes by the investigated compounds. a: 1) chloramꢀ
phenicol; 2) AcIRAꢀCAM (II); 3) AcMRLꢀCAM (I); 4) AcIWPꢀCAM (III); b: 5) AcMRLꢀdACaegCaeg (IV); 6) MRLꢀOMe (VII); 7) chloꢀ
ramphenicol amine (V). BODIPYꢀEry concentration was 4 nM, ribosomes – 24 nM. For each point the mean value s.d. is given.
the BODIPYꢀEry ribosomal complex used for this calcuꢀ with atom O1P G2505 (not shown), while the carbonyl
lation was measured previously and was 16.4 nM [36]. oxygen atom of the amino acid can form hydrogen bonds
The KD obtained for the test compounds are presented in with bases C2611 and C2612. The arginine guanidine
the table.
group is close to the C2611 phosphate and forms hydroꢀ
As seen from the table, the dissociation constants of gen bonds with the O2 atom of C2610 and O6 atom of
chloramphenicol peptide analogs lie in the range of 4.3ꢀ G2581.
10 μM. The data suggests that the peptide fragment
In the case of the AcIRAꢀCAM (II) derivative, the
increases the affinity of the compounds to the ribosome arginine side chain is surrounded by nucleotides A2062,
around two hundred times more to the level characterisꢀ U2585, U2586, and its guanidine group can form hydroꢀ
tic for chloramphenicolamine. At the same time, the gen bonds with O4′, O5′, and O2P atoms of U2586
MRLꢀOMe (VII) peptide itself does not exhibit any affinꢀ (Fig. 5b). The αꢀN hydrogen atom of isoleucine forms a
ity to RT, underscoring the importance of the anchor role hydrogen bond with the U2506 O4 atom (not shown),
of the chloramphenicol amine. This is confirmed by the and the amino acid side chain is in close proximity to the
fact that the fluorescently labeled peptide BODIPYꢀ C2610 base.
MetArgLeuꢀOMe (VIII) also very poorly interacts with E.
It was revealed for compound AcIWPꢀCAM (III)
coli ribosomes (see Fig. S3 in Supplement). Binding of that its tryptophan indole ring comes into stacking with
compound IV with the RT is flushed with chlorampheniꢀ the G2505 base (Fig. 5c).
col analogs I and II, which is apparently due to similarity
in structures of the peptide moieties.
Molecular docking. Molecular docking in 70S riboꢀ
somes was performed for peptide derivatives of chloramꢀ
phenicol amine. The Xꢀray data of the ribosomal complex
DISCUSSION
It is known that the removal of the dichloroacetyl
with chloramphenicol bound in the PTC Aꢀsite (PDB: moiety from the chloramphenicol molecule results in a
3OFC) were used for the investigation. From the molecꢀ significant decrease in biological activity [30] that is
ular docking, 20 structures for every compound were apparently due to a sharp decrease in the ability of the
obtained and ranged by the energy of binding with the chloramphenicol amine to bind to the ribosome (table).
ribosome. The structures where peptide fragments were These results confirm our hypothesis that a short peptide
oriented towards the RT and the nitrophenyl group was chain administration instead of dichloroacetyl fragment
located in the chloramphenicolꢀbinding site were chosen into the chloramphenicol analog molecular structure can
for further analysis.
compensate interactions critical for antibiotic binding to
According to the molecular docking results, the the ribosome (table) [37, 38]. It has been earlier elucidatꢀ
peptide fragment of the AcMRLꢀCAM molecule (I) is ed that pentapeptide chloramphenicol analogs, different
surrounded by residues C2610ꢀ2612, G2505, U2506, in amino acid sequence from the known “stop peptides”,
and G2581 (Fig. 5a). Thus, the hydrogen atom at the are able to bind to the E. coli ribosome in a way that the
methionine αꢀnitrogen atom forms a hydrogen bond peptide fragment of the molecule is located in the RT
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398
TERESHCHENKOV et al.
a
b
c
Fig. 5. Structures of E. coli ribosome complexes with peptide derivatives of chloramphenicol amine obtained by molecular docking. a)
AcMRLꢀCAM (I); b) AcIRAꢀCAM (II); c) AcIWPꢀCAM (III). The dotted lines show possible hydrogen bonds.
[19]. The values of the dissociation constants of complexꢀ
es of these compounds with ribosomes were in the range ular docking results, in most structures the peptide fragꢀ
of 2.5ꢀ36 μM. ment of the synthesized compounds locates analogously
It should also be noted that according to the molecꢀ
Greater or lesser affinity of the investigated derivaꢀ to the nascent polypeptide chain in ribosomal complexes
tives to RT elements is likely explained by the nature of of the regulatory “stop peptides” TnaC (PDB: 4V5H)
the amino acid side chains. As seen from Fig. 1b, the first [39] and MifM (PDB: 3J9W) [40], whose structure was
amino acid residue of the peptide attached to chloramꢀ obtained by cryoelectron microscopy.
phenicol amine corresponds to the position of the third
The affinity of PNA conjugate with MRL “stop pepꢀ
amino acid from Cꢀterminus of the peptidylꢀtRNA, tide” (IV) to the ribosome is flush with the corresponding
which is bound to the ribosome. Thus, the MRL, IRA, analog of chloramphenicol (I), which likely mimics a
and IWP peptides linked to chloramphenicol amine similar orientation of peptide residues of investigated
amino group will correspond to peptides MRLXX, compounds in the RT.
IRAXX, and IWPXX attached to the tRNA. With this in
Thus, using tripeptide analogs of chloramphenicol
mind, and from comparison of the dissociation conꢀ with the “stop peptides” it was demonstrated that short
stants of compounds II and III, it follows that the presꢀ peptide fragments are able to provide the interaction of
ence of the Arg residue at the second position from the these compounds with E. coli ribosomes at a level close to
Cꢀterminus (at the fourth from the 3′ꢀtRNA) is more the original antibiotic. Substances of this type are quite
preferable for strong binding than Trp. This is confirmed promising for the study of the interaction between regulaꢀ
by the fact that the Arg7 residue of regulatory peptide tory peptides and the ribosomal tunnel.
ErmBL, being at the fourth position from the 3′ꢀtRNA
end, is crucial for translation arrest [20]. Judging by cryoꢀ
The authors are grateful to M. N. Zhmak for tripepꢀ
electron microscopy data, it apparently interacts with tide synthesis, to A. L. Konevega for ribosomes given for
U2586 nucleotide, and it is also observed for the arginine investigation, to M. V. Serebryakova for massꢀspectromeꢀ
residue of peptide chloramphenicol analog II based on try analyses, and to Yu. K. Grishin for NMR spectra.
molecular docking (Fig. 5b). Moreover, the arginine side
This research was supported by the Russian Science
chain in the complex of II with the E. coli ribosome is Foundation (project No. 14ꢀ50ꢀ00029, molecular modelꢀ
located approximately in the same environment as ing) and by the Russian Foundation for Basic Research
Arg163 of SecM peptide [26]. This suggests that residues (projects Nos. 13ꢀ04ꢀ00986ꢀa, synthesis of chloramꢀ
Arg7 in ErmBL and Arg163 in SecM form with rRNA phenicol analogs; 13ꢀ04ꢀ40211ꢀH, study of analogs bindꢀ
nucleotide moieties an ensemble of hydrogen bonds simꢀ ing to bacterial ribosomes). The work was carried out
ilar to that formed by the arginine residue in compound using equipment purchased with funds of Lomonosov
II.
Moscow State University Program of Development.
BIOCHEMISTRY (Moscow) Vol. 81 No. 4 2016

PEPTIDE CHLORAMPHENICOL ANALOGS
399
peptidyl transferase center in eubacteria, Nature, 413, 814ꢀ
821.
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