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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
Evaluation of flavonols and derivatives as human cathepsin B inhibitor a
a
Suelem D. Ramalho , Lorena R. F. de Sousa , Marcela C. M. a
b
a
Burger , Maria Inês S. Lima , M. Fátima das G. F. da Silva , João B. a
Fernandes & Paulo C. Vieira
a
a
Department of Chemistry, Federal University of São Carlos, 13565-905 São Carlos, SP, Brazil b
Click for updates
Department de Botany, Federal University of São Carlos, 13565-905 São Carlos, SP, Brazil Published online: 27 Jan 2015.
To cite this article: Suelem D. Ramalho, Lorena R. F. de Sousa, Marcela C. M. Burger, Maria Inês S. Lima, M. Fátima das G. F. da Silva, João B. Fernandes & Paulo C. Vieira (2015): Evaluation of flavonols and derivatives as human cathepsin B inhibitor, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.1002404 To link to this article: http://dx.doi.org/10.1080/14786419.2014.1002404
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 [Pennsylvania State University] at 05:47 14 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1002404
SHORT COMMUNICATION Evaluation of flavonols and derivatives as human cathepsin B inhibitor Suelem D. Ramalhoa, Lorena R. F. de Sousaa, Marcela C. M. Burgera, Maria Ineˆs S. Limab, M. Fa´tima das G. F. da Silvaa, Joa˜o B. Fernandesa and Paulo C. Vieiraa* a
Department of Chemistry, Federal University of Sa˜o Carlos, 13565-905 Sa˜o Carlos, SP, Brazil; Department de Botany, Federal University of Sa˜o Carlos, 13565-905 Sa˜o Carlos, SP, Brazil
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
b
(Received 21 August 2014; final version received 18 December 2014)
OH OH O
HO
OH OH
OH
O
Ki = 11.3 µM
IC50 = 4.9 ± 0.5 µM
Cathepsin B (catB) is a cysteine protease involved in tumour progression and represents a potential therapeutic target in cancer. Among the 15 evaluated extracts from cerrado biome, Myrcia lingua Berg. (Myrtaceae) extract demonstrated to be a source of compounds with potential to inhibit catB. Using bioactivity-guided fractionation, we have found flavonols as inhibitors and also some other derivatives were obtained. From the evaluated compounds, myricetin (5) and quercetin (6) showed the most promising results with IC50 of 4.9 and 8.2 mM, respectively, and mode of inhibition as uncompetitive on catB. The results demonstrated polyhydroxylated flavonols as promising inhibitors of catB. Keywords: flavonoids; cathepsin B; uncompetitive inhibitor
1. Introduction Cancer is one of the leading causes for death worldwide and is characterised by uncontrolled growth and spread of cells (WHO 2014). Several studies showed that increased expression of cathepsin B (catB) is strongly related with malignant types of tumours and can play an important role in tumour progression (Withana et al. 2012). Low pH is requested to activate these cysteine proteases, which degrade extracellular matrix proteins and lead to tumour cell growth, invasion and metastasis (Skrzydlewska et al. 2005). The majority of cysteine proteases act as endopeptidases, although catB differs from those cathepsins in some aspects. The occluding loop is a structural feature unique to catB and has two His residues (His-110 and His-111) (Tomoo 2010). Plant diversity is an important source of natural products, which continues to play a significant role in drug discovery (Cragg et al. 2009). Besides being devastated, cerrado biome
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
S.D. Ramalho et al.
covers about 23% of Brazil land surface and is rich in non-studied medicinal plants (Pinheiro & Monteiro 2010). We have selected seven different cerrado plants for the initial screening. In the course of the screening of ethanolic extracts for catB inhibitors, we used bioactivity-guided fractionation and found high activity on ethyl acetate fraction from Myrcia lingua Berg. (Myrtaceae) leaves. To the best of our knowledge, there are no reports in the literature on the phytochemical study of Myrcia lingua Berg.
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
2. Results and discussion 2.1. Bioactivity-guided fractionation and biochemical evaluation Figure S1 (Supplementary Figure S1 – online only) shows the inhibitory effect of 15 crude extracts from 7 different cerrado plants against catB and L. at 125 mg/mL. From the evaluated extracts, we selected MLFE (Myrcia lingua leaves ethanolic extract) to continue the bioactivity-guided fractionation. The inhibition of MLFE was higher than 90% on both cysteine proteases. In the first moment, all compounds (Supplementary Figure S2 – online only) were tested at the concentration of 100 mM and the active compounds had their IC50 (Supplementary Table S1 – online only) determined by kinetic assays using seven different inhibitor concentrations. The obtained results revealed that the presence of hydroxyl groups in the ring B is important for catB inhibition. Flavonol aglycones 5 and 6 showed to be most potent inhibitors of catB, with IC50 of 4.9 and 8.2 mM, respectively. However, compound 7 with only one hydroxyl in ring B showed a slight decrease in the activity with IC50 of 15.0 mM on catB. From the biological activities, it is known that the presence of hydroxyl group(s) in phenolic compounds, including flavonoids, might affect their biological activities by binding to the active site of enzymes, forming hydrogen bonds with certain enzymes or biomembranes resulting in alteration of their metabolism (Harborne & Williams 2000). Comparison of the activity of flavonoids 6 (IC50 8.2 mM) and 11 (IC50 36.2 mM) showed that the replacement of hydroxyl group by hydrogen substituent at position C-3 causes a decrease in the potency on inhibition of catB. This result indicated that the hydroxyl at C-3 might contribute to the activity. The analysis of the enzyme inhibition of flavan-3-ols derivatives (15 –18) demonstrated the importance of both double bond between C-2 and C-3 and the ketone group at C-4, once these compounds cannot inhibit catB and L. We also observed that enzyme inhibition of catB was completely lost with the replacement of hydroxyl groups by methoxyl substituents in ring B. Such relationship was observed for compounds 1, 9 and 6, 12 –14. All these insights demonstrated the importance of hydroxyl groups on ring B of flavonols for enzyme inhibition. It is now well documented that not all natural products are free of risk, and the compound origin is not a determinant of its physicochemical properties and its side effects on mammals or experimental animals (Nenaah, 2014). Nevertheless, natural flavonoids are classified as one of the most safe phytochemicals included in the human diets, since, nutritionists estimated the average intake of flavonoids by human, in normal diet, to be 1– 2 g/day (de Vries et al. 1997). They also have very low toxicity against rats (Havsteen 2002). After experiments required to validate various limitations about their mammalian safety, such extracts and phytochemicals could be play a role in the management of certain cancer diseases. 2.2. Mechanism of inhibition Flavonoids 5 –7 were selected to determine the type of inhibition of catB, although due to their low activity the kinetics on catL were not investigated. The evaluated compounds showed uncompetitive inhibition (Supplementary Figure S3 – online only). Uncompetitive inhibitors bind only to the enzyme– substrate complex. Also, the apparent value of Vmax and Km decreases
Natural Product Research
3
with increasing concentration of uncompetitive inhibitors and as it is demonstrated in Supplementary Figure S3 – online only, double reciprocal plots are composed of parallel lines. (Copeland 2005; Bisswanger 2008). The Ki values (Supplementary Table S2 – online only) were obtained using Dixon plots analyses, which consist of the secondary plot of the intercepts versus the inhibitor concentration.
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
3. Conclusion It is necessary to discover more effective and safer compounds that can be applied in tumour treatment and metastasis control. Our results indicate that flavonoids need to be further investigated on enzyme inhibition. Some flavonoids and derivatives were evaluated, and especially the polyhydroxylated flavonols (5 – 7) showed to be uncompetitive inhibitors with moderate activity on catB. These compounds may be used for the design of more potent catB inhibitors. Supplementary material Experimental details related to this article are available online, alongside Tables S1– S2 and Figures S1 –S30. Funding This work was supported by the Sa˜o Paulo Research Foundation-FAPESP [2010/52326-9]; National Council for Scientific and Technological Development-CNPq, Brazil.
References Bisswanger H. 2008. Enzyme kinetics: principles and methods. 2nd ed. Weinheim: Wiley. Copeland RA. 2005. Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2nd ed. New Jersey: Wiley. Cragg GM, Grothaus PG, Newman DJ. 2009. Impact of natural products on developing new anti-cancer agents. Chem Rev. 109:3012– 3043. de Vries J, Janseen P, Hollman P, Staveren W, Katan M. 1997. Consumption of quercetin and kaempferol in free living subjects eating a variety of diets. Cancer Lett. 114:141–144. Harborne JB, Williams C. 2000. Advances in flavonoid research since 1992. Phytochemistry. 55:481–504. Havsteen BH. 2002. The biochemistry and medical significance of flavonoids. Pharmacol Ther. 96:66–202. Nenaah G. 2014. Toxic and antifeedant activities of prenylated flavonoids isolated from Tephrosia apollinea L. against three major coleopteran pests of stored grains with reference to their structure-activity relationship. Nat Prod Res. 28:2245–2252. Pinheiro MHO, Monteiro R. 2010. Contribution to the discussions on the origin of the cerrado biome: brazilian savanna. Braz J Biol. 70:95–102. Skrzydlewska E, Sulkowska M, Wincewicz A, Koda M, Sulkowski S. 2005. Evaluation of serum cathepsin B and D in relation to clinicopathological staging of colorectal cancer. World J Gastroenterol. 11:4225–4229. Tomoo K. 2010. Development of cathepsin inhibitors and structure-based design of cathepsin B-Specific Inhibitor. Curr Top Med Chem. 10:696– 707. WHO: World Health Organization. [Internet]. [cited 2014 June 15]. Available from: http://www.who.int/features/qa/15/ en/index.html Withana NP, Blum G, Sameni M, Slaney C, Anbalagan A, Olive MB, Bidwell BN, Edgington L, Wang L, Moin K, et al. 2012. Cathepsin B inhibition limits bone metastasis in breast cancer. Cancer Res. 72:1199– 1209.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Evaluation of flavonols and derivatives as human cathepsin B inhibitor a
a
Suelem D. Ramalho , Lorena R. F. de Sousa , Marcela C. M. a
b
a
Burger , Maria Inês S. Lima , M. Fátima das G. F. da Silva , João B. a
Fernandes & Paulo C. Vieira
a
a
Department of Chemistry, Federal University of São Carlos, 13565-905 São Carlos, SP, Brazil b
Click for updates
Department de Botany, Federal University of São Carlos, 13565-905 São Carlos, SP, Brazil Published online: 27 Jan 2015.
To cite this article: Suelem D. Ramalho, Lorena R. F. de Sousa, Marcela C. M. Burger, Maria Inês S. Lima, M. Fátima das G. F. da Silva, João B. Fernandes & Paulo C. Vieira (2015): Evaluation of flavonols and derivatives as human cathepsin B inhibitor, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.1002404 To link to this article: http://dx.doi.org/10.1080/14786419.2014.1002404
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 [Pennsylvania State University] at 05:47 14 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1002404
SHORT COMMUNICATION Evaluation of flavonols and derivatives as human cathepsin B inhibitor Suelem D. Ramalhoa, Lorena R. F. de Sousaa, Marcela C. M. Burgera, Maria Ineˆs S. Limab, M. Fa´tima das G. F. da Silvaa, Joa˜o B. Fernandesa and Paulo C. Vieiraa* a
Department of Chemistry, Federal University of Sa˜o Carlos, 13565-905 Sa˜o Carlos, SP, Brazil; Department de Botany, Federal University of Sa˜o Carlos, 13565-905 Sa˜o Carlos, SP, Brazil
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
b
(Received 21 August 2014; final version received 18 December 2014)
OH OH O
HO
OH OH
OH
O
Ki = 11.3 µM
IC50 = 4.9 ± 0.5 µM
Cathepsin B (catB) is a cysteine protease involved in tumour progression and represents a potential therapeutic target in cancer. Among the 15 evaluated extracts from cerrado biome, Myrcia lingua Berg. (Myrtaceae) extract demonstrated to be a source of compounds with potential to inhibit catB. Using bioactivity-guided fractionation, we have found flavonols as inhibitors and also some other derivatives were obtained. From the evaluated compounds, myricetin (5) and quercetin (6) showed the most promising results with IC50 of 4.9 and 8.2 mM, respectively, and mode of inhibition as uncompetitive on catB. The results demonstrated polyhydroxylated flavonols as promising inhibitors of catB. Keywords: flavonoids; cathepsin B; uncompetitive inhibitor
1. Introduction Cancer is one of the leading causes for death worldwide and is characterised by uncontrolled growth and spread of cells (WHO 2014). Several studies showed that increased expression of cathepsin B (catB) is strongly related with malignant types of tumours and can play an important role in tumour progression (Withana et al. 2012). Low pH is requested to activate these cysteine proteases, which degrade extracellular matrix proteins and lead to tumour cell growth, invasion and metastasis (Skrzydlewska et al. 2005). The majority of cysteine proteases act as endopeptidases, although catB differs from those cathepsins in some aspects. The occluding loop is a structural feature unique to catB and has two His residues (His-110 and His-111) (Tomoo 2010). Plant diversity is an important source of natural products, which continues to play a significant role in drug discovery (Cragg et al. 2009). Besides being devastated, cerrado biome
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
S.D. Ramalho et al.
covers about 23% of Brazil land surface and is rich in non-studied medicinal plants (Pinheiro & Monteiro 2010). We have selected seven different cerrado plants for the initial screening. In the course of the screening of ethanolic extracts for catB inhibitors, we used bioactivity-guided fractionation and found high activity on ethyl acetate fraction from Myrcia lingua Berg. (Myrtaceae) leaves. To the best of our knowledge, there are no reports in the literature on the phytochemical study of Myrcia lingua Berg.
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
2. Results and discussion 2.1. Bioactivity-guided fractionation and biochemical evaluation Figure S1 (Supplementary Figure S1 – online only) shows the inhibitory effect of 15 crude extracts from 7 different cerrado plants against catB and L. at 125 mg/mL. From the evaluated extracts, we selected MLFE (Myrcia lingua leaves ethanolic extract) to continue the bioactivity-guided fractionation. The inhibition of MLFE was higher than 90% on both cysteine proteases. In the first moment, all compounds (Supplementary Figure S2 – online only) were tested at the concentration of 100 mM and the active compounds had their IC50 (Supplementary Table S1 – online only) determined by kinetic assays using seven different inhibitor concentrations. The obtained results revealed that the presence of hydroxyl groups in the ring B is important for catB inhibition. Flavonol aglycones 5 and 6 showed to be most potent inhibitors of catB, with IC50 of 4.9 and 8.2 mM, respectively. However, compound 7 with only one hydroxyl in ring B showed a slight decrease in the activity with IC50 of 15.0 mM on catB. From the biological activities, it is known that the presence of hydroxyl group(s) in phenolic compounds, including flavonoids, might affect their biological activities by binding to the active site of enzymes, forming hydrogen bonds with certain enzymes or biomembranes resulting in alteration of their metabolism (Harborne & Williams 2000). Comparison of the activity of flavonoids 6 (IC50 8.2 mM) and 11 (IC50 36.2 mM) showed that the replacement of hydroxyl group by hydrogen substituent at position C-3 causes a decrease in the potency on inhibition of catB. This result indicated that the hydroxyl at C-3 might contribute to the activity. The analysis of the enzyme inhibition of flavan-3-ols derivatives (15 –18) demonstrated the importance of both double bond between C-2 and C-3 and the ketone group at C-4, once these compounds cannot inhibit catB and L. We also observed that enzyme inhibition of catB was completely lost with the replacement of hydroxyl groups by methoxyl substituents in ring B. Such relationship was observed for compounds 1, 9 and 6, 12 –14. All these insights demonstrated the importance of hydroxyl groups on ring B of flavonols for enzyme inhibition. It is now well documented that not all natural products are free of risk, and the compound origin is not a determinant of its physicochemical properties and its side effects on mammals or experimental animals (Nenaah, 2014). Nevertheless, natural flavonoids are classified as one of the most safe phytochemicals included in the human diets, since, nutritionists estimated the average intake of flavonoids by human, in normal diet, to be 1– 2 g/day (de Vries et al. 1997). They also have very low toxicity against rats (Havsteen 2002). After experiments required to validate various limitations about their mammalian safety, such extracts and phytochemicals could be play a role in the management of certain cancer diseases. 2.2. Mechanism of inhibition Flavonoids 5 –7 were selected to determine the type of inhibition of catB, although due to their low activity the kinetics on catL were not investigated. The evaluated compounds showed uncompetitive inhibition (Supplementary Figure S3 – online only). Uncompetitive inhibitors bind only to the enzyme– substrate complex. Also, the apparent value of Vmax and Km decreases
Natural Product Research
3
with increasing concentration of uncompetitive inhibitors and as it is demonstrated in Supplementary Figure S3 – online only, double reciprocal plots are composed of parallel lines. (Copeland 2005; Bisswanger 2008). The Ki values (Supplementary Table S2 – online only) were obtained using Dixon plots analyses, which consist of the secondary plot of the intercepts versus the inhibitor concentration.
Downloaded by [Pennsylvania State University] at 05:47 14 March 2015
3. Conclusion It is necessary to discover more effective and safer compounds that can be applied in tumour treatment and metastasis control. Our results indicate that flavonoids need to be further investigated on enzyme inhibition. Some flavonoids and derivatives were evaluated, and especially the polyhydroxylated flavonols (5 – 7) showed to be uncompetitive inhibitors with moderate activity on catB. These compounds may be used for the design of more potent catB inhibitors. Supplementary material Experimental details related to this article are available online, alongside Tables S1– S2 and Figures S1 –S30. Funding This work was supported by the Sa˜o Paulo Research Foundation-FAPESP [2010/52326-9]; National Council for Scientific and Technological Development-CNPq, Brazil.
References Bisswanger H. 2008. Enzyme kinetics: principles and methods. 2nd ed. Weinheim: Wiley. Copeland RA. 2005. Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2nd ed. New Jersey: Wiley. Cragg GM, Grothaus PG, Newman DJ. 2009. Impact of natural products on developing new anti-cancer agents. Chem Rev. 109:3012– 3043. de Vries J, Janseen P, Hollman P, Staveren W, Katan M. 1997. Consumption of quercetin and kaempferol in free living subjects eating a variety of diets. Cancer Lett. 114:141–144. Harborne JB, Williams C. 2000. Advances in flavonoid research since 1992. Phytochemistry. 55:481–504. Havsteen BH. 2002. The biochemistry and medical significance of flavonoids. Pharmacol Ther. 96:66–202. Nenaah G. 2014. Toxic and antifeedant activities of prenylated flavonoids isolated from Tephrosia apollinea L. against three major coleopteran pests of stored grains with reference to their structure-activity relationship. Nat Prod Res. 28:2245–2252. Pinheiro MHO, Monteiro R. 2010. Contribution to the discussions on the origin of the cerrado biome: brazilian savanna. Braz J Biol. 70:95–102. Skrzydlewska E, Sulkowska M, Wincewicz A, Koda M, Sulkowski S. 2005. Evaluation of serum cathepsin B and D in relation to clinicopathological staging of colorectal cancer. World J Gastroenterol. 11:4225–4229. Tomoo K. 2010. Development of cathepsin inhibitors and structure-based design of cathepsin B-Specific Inhibitor. Curr Top Med Chem. 10:696– 707. WHO: World Health Organization. [Internet]. [cited 2014 June 15]. Available from: http://www.who.int/features/qa/15/ en/index.html Withana NP, Blum G, Sameni M, Slaney C, Anbalagan A, Olive MB, Bidwell BN, Edgington L, Wang L, Moin K, et al. 2012. Cathepsin B inhibition limits bone metastasis in breast cancer. Cancer Res. 72:1199– 1209.
