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30RESEARCH RESULT
H A y M H bl i l P E 3 y JÏ b T A I
Kravchenko D.V., Beskhmelnitsyna E.A., Korokin M.V., Avtina T.V., SernovL.N., Tishin AN., Kostina D.A. Molecular screening of prospective candidates for TRPA1 ion channel selective antagonists. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, №1 (2): 63-66.
Pyc. Eng.
UDC: 616-01/09
DOI: 10.18413/2313-8971-2016-2-1-63-66
Kravchenko D.V.
Beskhmelnitsyna E.A.
Korokin M.V. Avtina T.V. Sernov L.N. MOLECULAR SCREENING OF PROSPECTIVE CANDIDATES FOR
TRPA1 ION CHANNEL SELECTIVE ANTAGONISTS
Tishin A.N.
Kostina D.A.
1) Chief manager. Closed corporation "Chemical Diversity Research Institute" 2a, Rabochaya street, Khimki, Moscow region, 141400, Russia. e-mail: [email protected] 2) Postgraduate student of pharmacological department. Belgorod State National Research University 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] 3) Doctor of medicine, professor of the pharmacological department. Belgorod State National Research University. 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected] 4) PhD in Pharmaceutical Sciences, Associate Professor. Belgorod State National Research University 85, Pobedy St., Belgorod, 308005, Russia. e-mail: [email protected] 5) Doctor of medicine, Professor, General Director of LLC "Farmkonsalting" 19, Kirov street, Moscow region, Staraya Kupavna, 142450, Russia. e-mail: [email protected] 6) Teaching assistant of pharmacological department. Belgorod State National Research University 85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]
7) Postgraduate student of pharmacological department. Belgorod State National Research University
85, Pobedy St., Belgorod, 308015, Russia. e-mail: [email protected]
8) Postgraduate student of pharmacological department/ Belgorod State National Research University
85, Pobedy St., Belgorod, 308015, Russia/ e-mail:[email protected]
Abstract: TRPAj is an ankyrin receptor of TRP family. It is mostly expressed in the small-diameter nociceptors with cell bodies located in the dorsal roots, as well as in trigeminal, nodose and jugular ganglia. Recently, more and more information has been found in the literature on the role of TRPAi in the realization of cold and pain sensitivity, and also in the formation and maintenance of inflammation. Subject to these data there is an increasing interest in finding and studying pharmacological agents able to selectively block the TRPA1 receptors and thereby reduce the severity of pain and inflammation.
Using the molecular modeling techniques, we analyzed spectra of biological activity of a series of promising candidates for selective antagonists of TRPA1 ion channel in order to find potentially active compounds against pain and inflammation.
Substances that showed in high throughput screening the percentage of the cellular calcium response inhibition above 50% - 3*SD maximum and its own agonistic activity less than 10% + 3*SD minimum were identified as hits. The value IC50 for hits was determined immediately after re-testing at 10 ^M concentration and repeatedly after the determination of the leading chemical series. As the result of the studies, the biologically active molecules of leading chemical series have been confirmed. The research was partially supported by the grant of the President of the Russian Federation №MD-4711.2015.7 and MK-6135.2016.4.
Key words: TRPion channel; TRPAj; pain; nociception; inflammation; molecular screening; hits.
Over the past 20 years there has been a revival of interest in the fundamental mechanisms of nociception [1, 2]. Recent advances in immunohistochemistry, neuropharmacology and neurophysiology contributed to new discoveries in the field of physiology and pathophysiology of pain. The conducted studies resulted in the discovery of new receptors and mechanisms of
pain perception. The obtained information ensured the opportunity to create and apply new tools and methods for more effective and safe control over pain [3]. One of the modern methods of pain treatment is the use of various substances that can selectively block the receptors that directly perceive both pain stimuli and
RESEARCH RESULT
H A y M H bl i l P E 3 y JÏ b T A I
Kravchenko D.V., Beskhmelnitsyna E.A., Korokin M.V., Avtina T.V., SernovL.N., Tishin AN., Kostina D.A. Molecular screening of prospective candidates for TRPAi ion channel selective antagonists. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, №1 (2): 63-66.
inflammatory mediators. One of these receptors is an ankyrin receptor TRPAi.
The TRPA1 is mostly expressed in the small-diameter nociceptors with cell bodies located in the dorsal roots, as well as in trigeminal, nodose and jugular ganglia [1, 2, 4]. Some authors argue, according to the results of their research, for the increasing TRPA1 expression in pathological conditions such as inflammation, neuropathy [5, 6, 7], by virtue whereof there has been an increase in interest in the TRPA1 as the receptor of pain stimuli [8, 9].
Objective of the study was to confirm the activity of hits of the leading chemical series, obtained from high-throughput screening of a focused library.
Materials and research methods. Embryonic human liver epithelial cells exogenously expressing the IPTG (isopropyl-P-D-1-thiogalactopyranoside)-induced TRPA1 ion channel, were suspended in culture medium with 1 mM IPTG so as to obtain a concentration 8x105 cells/ml, which would amount 20,000 cells per well. The resulting cell suspension was placed in a 384-well culture experimental dies with optical bottom with the use of automatic robotized stations Biomek FX or Biomek NX, 25 ^g suspension in each well. 384-well dies were centrifuged at 200xg for 5 minutes. Further experimental dies with the cell suspension were transferred to an incubator at 37°C, 5% CO2. On the day of the experiment cells were immersed in Ca2+-sensitive dye, prepared by the manufacturer's instructions. Experimental dies filled with Ca2+-sensitive dye were incubated for 1 hour at a room temperature in a dark place. Further step was
screening with FLIPR (Robotic High-Throughput Cellular Calcium Screening System).
The TRPA1 ion channel was stimulated with agonist at EC80 concentration, which was 30 ^M. For the inhibition of stimulated calcium response, we used the maximum inhibitory concentration of a control antagonist IC100, equal to 6 ^M. FLIPR was turned on 30 minutes prior to the experiment to preheat its laser. We performed a signal test and adjusted the laser so as the base signal level was equal to 10,000 relative fluorescence units.
To detect agonist activity of test substances 12.5 ^l substance was added to each well of 384-well die according to the die scheme. Further, the experimental dies were screened with FLIPR. After incubation, 12.5 ^l TRPA1 ion channel agonist and allyl isothiocyanate (AITC) were added for 11 minutes to each well of 384-well experimental die, and the second FLIPR screening immediately started to determine the antagonists.
The dependence of antagonist activity on concentration was analyzed for substances. IC50 value was determined from the obtained relation. The experimental data were analyzed with GraphPad Prizm (GraphPad Software, Inc., San Diego, CA). For the construction of concentration dependence the following equation was chosen: Y=Bottom curve plateau + (Upper plateau-Lower Plateau)/(1+10A((LogIC50-X)*Curve slope)).
Results of the study. The obtained results show that all previously discovered hits of leading chemical series have confirmed their activity towards the TRPA1 ion channel (Table 1, Figure 1-3).
Table 1
Summary table with IC50 data for verifiable hits
Substance IC50, ^M Comments
3455-3246 36.76
3455-3273 11.82
3455-3281 22.51
3455-3320 13.22 partial antagonist
4606-1800 13.56
5628-0670 12.81
6040-4753 7.439 partial antagonist
E002-2494 0.9008
E002-2495 3.747
E002-2498 3.039
G857-0153 7.6
G721-0484 9.623 partial antagonist
C276-1460 46.74
RESEARCH RESULT
H A y M H bl i 'l P E 3 y ji b T A I
Kravchenko D.V., Beskhmelnitsyna E.A., Korokin M.V., Avtina T.V., SernovL.N., Tishin AN., Kostina D.A. Molecular screening of prospective candidates for TRPAi ion channel selective antagonists. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, №1 (2): 63-66.
Figure 1. The concentration dependence of antagonist activity on the concentration for verifiable hits and control antagonist RR. Each concentration point is the average of two repetitions. Partial antagonists
Full TRPA-1
c o
OJ
c c ra
c o
100-
t 50--
1 m X S 4 n T ... T X
^ ■ -
\ i
iL ■
I i -1-1-
-10 -8 -6
Test substances
■ ruthenium red
a 3455-3281
T 3455-3246
• 3455-3273
□ C276-14G0
7 5628-0670
O 4606-1800
ruthenium red
3455-3346 3465-3273 3455J2S1 C276-1460 562S-0670 4606-1S00
50
2.339e-007
- 3.6760-005 1.1 S2e-005 2.251 e-005
- 4.6740-005 1.2S1 e-005
1.3566-005
Figure 2. The concentration dependence of antagonist activity on the concentration for verifiable hits and control antagonist RR. Each concentration point is the average of two repetitions. Full antagonists
RESEARCH RESULT
НАУЧНЫ Й Р Е 3 У Л Ь Т А I
Kravchenko D.V., Beskhmelnitsyna E.A., Korokin M.V., Avtina T.V., SernovL.N., Tishin AN., Kostina D.A. Molecular screening of prospective candidates for TRPA1 ion channel selective antagonists. Research result: pharmacology and clinical pharmacology. 2016. Vol. 2, №1 (2): 63-66.
Figure 3. The concentration dependence of antagonist activity on the concentration for verifiable hits and control antagonist RR. Each concentration point is the average of two repetitions. Full antagonists
Summary. As a result of molecular screening of biologically active molecules- candidates for selective antagonists of TRPA1 ion channel, it was found that all investigated hits of the leading chemical series confirmed their activity. Thus, these molecules can be recommended for further studies of their activity in the tests in vivo in laboratory animals.
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