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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects a
b
Junhui Zhao & Chuixian Zhou a
Department of Anesthesiology, Weifang Medical University, 7166 Baotong Street (West), Weifang 261053, P.R. China b
Click for updates
Department of Neurosurgery, Weifang People's Hospital, Weifang 261041, P.R. China Published online: 05 Sep 2014.
To cite this article: Junhui Zhao & Chuixian Zhou (2014): Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.955491 To link to this article: http://dx.doi.org/10.1080/14786419.2014.955491
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.955491
SHORT COMMUNICATION Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects Junhui Zhaoa1* and Chuixian Zhoub1 a
Downloaded by [Florida State University] at 06:39 21 December 2014
Department of Anesthesiology, Weifang Medical University, 7166 Baotong Street (West), Weifang 261053, P.R. China; bDepartment of Neurosurgery, Weifang People’s Hospital, Weifang 261041, P.R. China (Received 2 July 2014; final version received 13 August 2014) Protein kinase C (PKC) plays a key role in neurotransmission in the central nervous system, and targeting PKC domain is considered as a strategy to modulate the anaesthetic effects. In this study, we described a synthetic pipeline to perform high-throughput virtual screening against a large library of 3D structural natural products released recently in order to discover those potential PKC modulators. A total of 100 natural products with top scores were raised, from which 12 promising candidates were tested to determine their inhibitory potencies against PKC. As might be expected, the promiscuous kinase inhibitor staurosporine showed a high PKC inhibitory activity (IC50 ¼ 64 nM), and other two tested compounds, i.e. fisetin and tetrahydropapaverine, were also highly potent with their activities at nanomolar level (IC50 ¼ 370 and 190, respectively). Keywords: anaesthetic; protein kinase C; natural product; rational drug discovery
1. Introduction Phospholipid-regulated protein kinase C (PKC) signalling is known to be involved in cellular functions relevant to a variety of neurobiological events, including ion channel modulation, receptor regulation, neurotransmitter release, synaptic plasticity and survival (Battaini & Pascale 2005). Strong supports for the protein theory of anaesthesia showed that the regulatory region of PKC contains a hydrophobic binding site for alcohols and anaesthetics, and inhibition of its kinase activity by small-molecule agents can result in significant modulation of general anaesthetic effects, supporting a role for the enzyme in the anaesthetic process (Firestone et al. 1993; Slater et al. 1997). Among this multigene family of serine/threonine kinases, the PKCd is a pivotal signalling step in ischaemic preconditioning and in transferring the anaesthetic preconditioning stimulus to mitochondrial K channels (Uecker et al. 2003). In this study, we performed a highthroughput virtual screening against a large library of 3D structural natural products in order to discover potential PKCd modulators. To solidify this scheme, we performed kinase assay to measure the inhibitory activity of several highly promising candidates arising from the virtual screening against PKCd. The molecular mechanism and structural basis of the intermolecular interactions between several potent modulators and PKCd domain were also characterised on the basis of computationally modelled complex structure data (Zhou et al. 2013). 2. Results, discussion and conclusions Consensus scoring function was first evaluated using a number of structure-solved, affinityknown kinase –inhibitor complexes, which was further employed to evaluate the binding
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Florida State University] at 06:39 21 December 2014
2
J. Zhao and C. Zhou
strength of natural product ligands extracted from the CamMedNP library (Ntie-Kang et al. 2013) to the PKCd domain. Before scoring, the structure of PKCd domain was constructed by homology modelling and its complexes with the 2147 natural products in the library were predicted using the automatic molecular docking scheme. The resulting consensus score values for these samples are shown in Figure 1. Evidently, the score bars roughly obey a normal distribution of centre 0 and standard deviation 1; this is expected if considering that the original score values were normalised before being used to derive the consensus score. Since all the original scores correlate negatively with ligand binding capability, ligands with negative values of consensus score are always stronger binders than those with positive values. Therefore, we considered the 100 natural products with the most negative scores as promising candidates, and they are structurally diverse, including classic kinase inhibitor structures such as quinoline and indole derivatives, as well as some non-classic types. Some of these high-score candidates have been previously found to target an array of kinases involved in diverse cellular events, such as signalling pathway (i.e. STAT), cell cycle (i.e. GSK), immune response (i.e. Akt) and molecular trafficking (i.e. mTOR). Specifically, the promiscuous kinase inhibitor staurosporine, which has previously been reported to have high inhibitory potency against several PKC isoforms (Firestone et al. 1993), was properly identified by the consensus score as a top PKCd binder. A number of flavonoids such as quercetin, chrysin and baicalein were suggested to be potent PKCd inhibitors. In addition, several natural products under clinical trial or preclinical investigation such as emodin and radicicol are also listed here. In order to test the theoretical results arising from virtual screening, we further performed assay to determine the inhibitory activity of 12 promising compounds from the 100 candidates against PKCd, including bergenin, honokiol, fisetin, myricitrin, rutaecarpine, tetrahydropapaverine, theobromine, aloin, indirubin, puerarin and chrysophanic acid as well as the promiscuous staurosporine (Table S2). As might be expected, 8 out of the 12 tested candidates showed high or moderate inhibitory activity against PKCd, with IC50 values ranging from 0.064 (staurosporine) to 560 (theobromine) mM. In particular, the biological activities of fisetin, tetrahydropapaverine and staurosporine were determined at nanomolar level (IC50 ¼ 370, 190 and 64 nM), and five tested compounds honokiol, myricitrin, theobromine, aloin and puerarin also show relatively high or moderate inhibitory potencies (IC50 ¼ 110, 12, 860, 320 and 57 mM, respectively). However, the natural products bergenin and chrysophanic acid, as well as the rutaecarpine and indirubin that are currently under clinical trials, have not been detected to possess observable efficacy (IC50 . 1000 mM), suggesting that these compounds may not be the good lead compounds for further PKCd modulator development, although they were predicted in silico to
Figure 1. Diagrammatic representation of consensus scores for 2147 natural products bound to PKCd.
Natural Product Research
3
have strong binding capability towards PKCd. Further structural examination of PKCd interactions with two high-activity compounds fisetin and tetrahydropapaverine and complicated networks of non-bonded interactions (Zhou et al. 2012) were found at the complex interfaces of PKCd with these small-molecule binders. Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 and S2. Note
Downloaded by [Florida State University] at 06:39 21 December 2014
1. These two authors contributed equally to this work.
References Battaini F, Pascale A. 2005. Protein kinase C signal transduction regulation in physiological and pathological aging. Ann N Y Acad Sci. 1057:177–192. Firestone S, Firestone LL, Ferguson C, Blanck D. 1993. Staurosporine, a protein kinase C inhibitor, decreases the general anesthetic requirement in Rana pipiens tadpoles. Anesth Analg. 77:1026–1030. Ntie-Kang F, Mbah JA, Mbaze LM, Lifongo LL, Scharfe M, Hanna JN, Cho-Ngwa F, Ongue´ne´ PA, Owono Owono LC, Megnassan E, et al. 2013. CamMedNP: building the Cameroonian 3D structural natural products database for virtual screening. BMC Complement Altern Med. 13:88. Slater SJ, Kelly MB, Larkin JD, Ho C, Mazurek A, Taddeo FJ, Yeager MD, Stubbs CD. 1997. Interaction of alcohols and anesthetics with protein kinase C alpha. J Biol Chem. 272:6167 –6173. Uecker M, Da Silva R, Grampp T, Pasch T, Schaub MC, Zaugg M. 2003. Translocation of protein kinase C isoforms to subcellular targets in ischemic and anesthetic preconditioning. Anesthesiology. 99:138–147. Zhou P, Huang J, Tian F. 2012. Specific noncovalent interactions at protein-ligand interface: implications for rational drug design. Curr Med Chem. 19:226–238. Zhou P, Wang C, Ren Y, Yang C, Tian F. 2013. Computational peptidology: a new and promising approach to therapeutic peptide design. Curr Med Chem. 20:1985– 1996.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects a
b
Junhui Zhao & Chuixian Zhou a
Department of Anesthesiology, Weifang Medical University, 7166 Baotong Street (West), Weifang 261053, P.R. China b
Click for updates
Department of Neurosurgery, Weifang People's Hospital, Weifang 261041, P.R. China Published online: 05 Sep 2014.
To cite this article: Junhui Zhao & Chuixian Zhou (2014): Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.955491 To link to this article: http://dx.doi.org/10.1080/14786419.2014.955491
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Florida State University] at 06:39 21 December 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.955491
SHORT COMMUNICATION Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects Junhui Zhaoa1* and Chuixian Zhoub1 a
Downloaded by [Florida State University] at 06:39 21 December 2014
Department of Anesthesiology, Weifang Medical University, 7166 Baotong Street (West), Weifang 261053, P.R. China; bDepartment of Neurosurgery, Weifang People’s Hospital, Weifang 261041, P.R. China (Received 2 July 2014; final version received 13 August 2014) Protein kinase C (PKC) plays a key role in neurotransmission in the central nervous system, and targeting PKC domain is considered as a strategy to modulate the anaesthetic effects. In this study, we described a synthetic pipeline to perform high-throughput virtual screening against a large library of 3D structural natural products released recently in order to discover those potential PKC modulators. A total of 100 natural products with top scores were raised, from which 12 promising candidates were tested to determine their inhibitory potencies against PKC. As might be expected, the promiscuous kinase inhibitor staurosporine showed a high PKC inhibitory activity (IC50 ¼ 64 nM), and other two tested compounds, i.e. fisetin and tetrahydropapaverine, were also highly potent with their activities at nanomolar level (IC50 ¼ 370 and 190, respectively). Keywords: anaesthetic; protein kinase C; natural product; rational drug discovery
1. Introduction Phospholipid-regulated protein kinase C (PKC) signalling is known to be involved in cellular functions relevant to a variety of neurobiological events, including ion channel modulation, receptor regulation, neurotransmitter release, synaptic plasticity and survival (Battaini & Pascale 2005). Strong supports for the protein theory of anaesthesia showed that the regulatory region of PKC contains a hydrophobic binding site for alcohols and anaesthetics, and inhibition of its kinase activity by small-molecule agents can result in significant modulation of general anaesthetic effects, supporting a role for the enzyme in the anaesthetic process (Firestone et al. 1993; Slater et al. 1997). Among this multigene family of serine/threonine kinases, the PKCd is a pivotal signalling step in ischaemic preconditioning and in transferring the anaesthetic preconditioning stimulus to mitochondrial K channels (Uecker et al. 2003). In this study, we performed a highthroughput virtual screening against a large library of 3D structural natural products in order to discover potential PKCd modulators. To solidify this scheme, we performed kinase assay to measure the inhibitory activity of several highly promising candidates arising from the virtual screening against PKCd. The molecular mechanism and structural basis of the intermolecular interactions between several potent modulators and PKCd domain were also characterised on the basis of computationally modelled complex structure data (Zhou et al. 2013). 2. Results, discussion and conclusions Consensus scoring function was first evaluated using a number of structure-solved, affinityknown kinase –inhibitor complexes, which was further employed to evaluate the binding
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Florida State University] at 06:39 21 December 2014
2
J. Zhao and C. Zhou
strength of natural product ligands extracted from the CamMedNP library (Ntie-Kang et al. 2013) to the PKCd domain. Before scoring, the structure of PKCd domain was constructed by homology modelling and its complexes with the 2147 natural products in the library were predicted using the automatic molecular docking scheme. The resulting consensus score values for these samples are shown in Figure 1. Evidently, the score bars roughly obey a normal distribution of centre 0 and standard deviation 1; this is expected if considering that the original score values were normalised before being used to derive the consensus score. Since all the original scores correlate negatively with ligand binding capability, ligands with negative values of consensus score are always stronger binders than those with positive values. Therefore, we considered the 100 natural products with the most negative scores as promising candidates, and they are structurally diverse, including classic kinase inhibitor structures such as quinoline and indole derivatives, as well as some non-classic types. Some of these high-score candidates have been previously found to target an array of kinases involved in diverse cellular events, such as signalling pathway (i.e. STAT), cell cycle (i.e. GSK), immune response (i.e. Akt) and molecular trafficking (i.e. mTOR). Specifically, the promiscuous kinase inhibitor staurosporine, which has previously been reported to have high inhibitory potency against several PKC isoforms (Firestone et al. 1993), was properly identified by the consensus score as a top PKCd binder. A number of flavonoids such as quercetin, chrysin and baicalein were suggested to be potent PKCd inhibitors. In addition, several natural products under clinical trial or preclinical investigation such as emodin and radicicol are also listed here. In order to test the theoretical results arising from virtual screening, we further performed assay to determine the inhibitory activity of 12 promising compounds from the 100 candidates against PKCd, including bergenin, honokiol, fisetin, myricitrin, rutaecarpine, tetrahydropapaverine, theobromine, aloin, indirubin, puerarin and chrysophanic acid as well as the promiscuous staurosporine (Table S2). As might be expected, 8 out of the 12 tested candidates showed high or moderate inhibitory activity against PKCd, with IC50 values ranging from 0.064 (staurosporine) to 560 (theobromine) mM. In particular, the biological activities of fisetin, tetrahydropapaverine and staurosporine were determined at nanomolar level (IC50 ¼ 370, 190 and 64 nM), and five tested compounds honokiol, myricitrin, theobromine, aloin and puerarin also show relatively high or moderate inhibitory potencies (IC50 ¼ 110, 12, 860, 320 and 57 mM, respectively). However, the natural products bergenin and chrysophanic acid, as well as the rutaecarpine and indirubin that are currently under clinical trials, have not been detected to possess observable efficacy (IC50 . 1000 mM), suggesting that these compounds may not be the good lead compounds for further PKCd modulator development, although they were predicted in silico to
Figure 1. Diagrammatic representation of consensus scores for 2147 natural products bound to PKCd.
Natural Product Research
3
have strong binding capability towards PKCd. Further structural examination of PKCd interactions with two high-activity compounds fisetin and tetrahydropapaverine and complicated networks of non-bonded interactions (Zhou et al. 2012) were found at the complex interfaces of PKCd with these small-molecule binders. Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 and S2. Note
Downloaded by [Florida State University] at 06:39 21 December 2014
1. These two authors contributed equally to this work.
References Battaini F, Pascale A. 2005. Protein kinase C signal transduction regulation in physiological and pathological aging. Ann N Y Acad Sci. 1057:177–192. Firestone S, Firestone LL, Ferguson C, Blanck D. 1993. Staurosporine, a protein kinase C inhibitor, decreases the general anesthetic requirement in Rana pipiens tadpoles. Anesth Analg. 77:1026–1030. Ntie-Kang F, Mbah JA, Mbaze LM, Lifongo LL, Scharfe M, Hanna JN, Cho-Ngwa F, Ongue´ne´ PA, Owono Owono LC, Megnassan E, et al. 2013. CamMedNP: building the Cameroonian 3D structural natural products database for virtual screening. BMC Complement Altern Med. 13:88. Slater SJ, Kelly MB, Larkin JD, Ho C, Mazurek A, Taddeo FJ, Yeager MD, Stubbs CD. 1997. Interaction of alcohols and anesthetics with protein kinase C alpha. J Biol Chem. 272:6167 –6173. Uecker M, Da Silva R, Grampp T, Pasch T, Schaub MC, Zaugg M. 2003. Translocation of protein kinase C isoforms to subcellular targets in ischemic and anesthetic preconditioning. Anesthesiology. 99:138–147. Zhou P, Huang J, Tian F. 2012. Specific noncovalent interactions at protein-ligand interface: implications for rational drug design. Curr Med Chem. 19:226–238. Zhou P, Wang C, Ren Y, Yang C, Tian F. 2013. Computational peptidology: a new and promising approach to therapeutic peptide design. Curr Med Chem. 20:1985– 1996.
