Natural compounds as angiogenic enzyme thymidine phosphorylase inhibitors: In vitro biochemical inhibition, mechanistic, and in silico modeling studies

Autoři: Sumaira Javaid aff001;  Muniza Shaikh aff001;  Narjis Fatima aff001;  M. Iqbal Choudhary aff001
Působiště autorů: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center of Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan aff001;  H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan aff002;  Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225056


Natural flora is the richest source of novel therapeutic agents due to their immense chemical diversity and novel biological properties. In this regard, eighteen natural products belonging to different chemical classes were evaluated for their thymidine phosphorylase (TP) inhibitory activity. TP shares identity with an angiogenic protein platelet derived endothelial cell growth factor (PD-ECGF). It assists tumor angiogenesis and is a key player in cancer progression, thus an ideal target to develop anti-angiogenic drugs. Eleven compounds 12, 510, 11, 15, and 18 showed a good to weak TP inhibitory activity (IC50 values between 44.0 to 420.3 μM), as compared to standards i.e. tipiracil (IC50 = 0.014 ± 0.002 μM) and 7-deazaxanthine (IC50 = 41.0 ± 1.63 μM). Kinetic studies were also performed on active compounds, in order to deduce the mechanism of ligand binding to enzyme. To get further insight into receptor protein (enzyme) and ligand interaction at atomic level, in- sillico studies were also performed. Active compounds were finally evaluated for cytotoxicity test against mouse fibroblast (3T3) cell line. Compound 18 (Masoprocol) showed a significant TP inhibitory activity (IC50 = 44.0 ± 0.5 μM). Kinetic studies showed that it inhibits the enzyme in a competitive manner (Ki = 25.6 ± 0.008 μM), while it adopts a binding pose different than the substrate thymidine. It is further found to be non-toxic in MTT cytotoxicity assay. This is the first report on TP inhibitory activity of several natural compounds, some of which may serve as leads for further research towards drug the development.

Klíčová slova:

Angiogenesis – Cytotoxicity assay – Enzyme inhibitors – Hydrogen bonding – MTT assay – Phosphorylases – Thymidines – Coumarins


1. de Moura Sperotto ND, Deves Roth C, Rodrigues-Junior VS, Ev Neves C, Reisdorfer Paula F, da Silva Dadda et al., 2019. Design of novel inhibitors of human thymidine phosphorylase: Synthesis, enzyme inhibition, in vitro toxicity, and impact on human glioblastoma cancer. J Med Chem. 2019; 62:1231–1245. doi: 10.1021/acs.jmedchem.8b01305 30615449

2. Brown NS, Bicknell R. Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis. Biochem J. 1998; 334: 1–8. doi: 10.1042/bj3340001 9693094

3. Bronckaers A, Gago F, Balzarini J, Liekens S. The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med Res Rev. 2009; 29: 903–953. doi: 10.1002/med.20159 19434693

4. Fox SB, Westwood M, Moghaddam A, Comley M, Turley H, Whitehouse RM, et al., The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase is up-regulated in breast cancer epithelium and endothelium, Br J Cancer. 1996; 73: 275–280. doi: 10.1038/bjc.1996.49 8562330

5. Takebayashi Y, Akiyama S, Akiba S, Yamada K, Miyadera K, Sumizawa T, et al., Clinicopathologic and prognostic factor significance of an angiogenic factor thymidine phosphorylase in human colorectal carcinoma, J. Natl. Cancer Inst. 1996; 88:1110–1117. doi: 10.1093/jnci/88.16.1110 8757190

6. O' Brien TS, Fox SB, Dickinson AJ, Turley H, Westwood M, et al. Expression of the angiogenic factor thymidine phosphorylase/platelet-derived endothelial cell growth factor in primary bladder cancers. Cancer Res. 1996; 56: 4799–4804. 8841001

7. Igarashi M, Dhar DK, Kubota H, Yamamoto A, El-Assal O, Nagasue N. The prognostic significance of microvessel density and thymidine phosphorylase expression in squamous cell carcinoma of the esophagus. Cancer. 1998; 82:1225–1232. doi: 10.1002/(sici)1097-0142(19980401)82:7<1225::aid-cncr3>;2-e 9529012

8. de-Bruin M, Temmink OH, Hoekman K, Pinedo HM, Peters GJ. Role of platelet derived endothelial cell growth factor / thymidine phosphorylase in health and disease. Cancer Ther. 2006;4:954–967.

9. Miyadera K, Emura T, Suzuki N, Akiyama S, Fukushima M, Yamada Y. Novel functional antitumor nucleoside TAS-102, combined form of F3dThd and its modulator (2): Inhibitory effect of TPI on tumor-derived angiogenesis and metastasis. Proc Natl Assoc Cancer Res. 1998;39: 609.

10. Matsushita S, Nitanda T, Furukawa T, Sumizawa T, Tani A, Nishimoto K, et al. The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors. Cancer Res. 1999; 59:1911–1916. 10213500

11. Pomeisl K, Votruba I, Holy A, Pohl R. Syntheses of pyrimidine acyclic nucleoside phosphonates as potent inhibitors of thymidine phosphorylase (PD-ECGF) from SD-lymphoma. Nucleos Nucleot Nucl. 2007; 26:1025–1028.

12. Gbaj A, Edwards PN, Reigan P, Freeman S, Jaffar M, Douglas KT. Thymidine phosphorylase from Escherichia coli: Tight-binding inhibitors as enzyme active-site titrants. J Enzyme Inhib Med Chem. 2006; 21:69–73. doi: 10.1080/14756360500424010 16570508

13. Nencka R, Votruba I, Hrebabecky H, Jansa P, Tloustova E, Horska K, et al., Discovery of 5-substituted-6-chlorouracils as efficient inhibitors of human thymidine phosphorylase. J Med Chem. 2007; 50:6016–6023. doi: 10.1021/jm070644i 17963370

14. McNally VA, Rajabi M, Gbaj A, Stratford IJ, Edwards PN, Douglas KT, et al., Design, synthesis and enzymatic evaluation of 6-bridged imidazolyluracil derivatives as inhibitors of human thymidine phosphorylase. J Pharm Pharmacol. 2007;59:537–547. doi: 10.1211/jpp.59.4.0008 17430637

15. Liekens S, Balzarini J, Hernández AI, De Clercq E, Priego EM, Camarasa MJ, et al., Thymidine phosphorylase is noncompetitively inhibited by 5'-O-trityl-inosine (KIN59) and related compounds. Nucleos Nucleot Nucl. 2006;25: 975–980.

16. Casanova E, Hernandez AI, Priego EM, Liekens S, Camarasa MJ, Balzarini J, et al.,5‘-O-Tritylinosine and analogues as allosteric inhibitors of human thymidine phosphorylase. J Med Chem. 2006; 49:5562–5570. doi: 10.1021/jm0605379 16942029

17. Mayer RJ, Cutsem EV, Falcone A, Yoshino T, Garcia-Carbonero R, Mizunuma N, et al., Randomized trial of TAS-102 for refractory metastatic colorectal cancer. New Engl J Med. 2015; 372:1909–1919. doi: 10.1056/NEJMoa1414325 25970050

18. Balzarini J, Gamboa AE, Esnouf R, Liekens S, Neyts J, De Clercq E, et al., 7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase. FEBS Lett. 1998; 438:91–95 doi: 10.1016/s0014-5793(98)01271-x 9821965

19. Liekens S, De Clercq E, Neyts J. Angiogenesis: Regulators and clinical applications. Biochem Pharmacol. 2001; 61: 253–270. doi: 10.1016/s0006-2952(00)00529-3 11172729

20. Liekens S, Bilsen F, De Clercq E, Priego EM, Camarasa MJ, Pérez-Pérez MJ, et al., Anti-angiogenic activity of a novel multi-substrate analogue inhibitor of thymidine phosphorylase. FEBS Lett. 2002; 510: 83–88. doi: 10.1016/s0014-5793(01)03233-1 11755536

21. Khan KM, Ambreen N, Hussain S, Perveen S, Choudhary MI. Schiff bases of 3-formylchromone as thymidine phosphorylase inhibitors. Bioorg Med Chem. 2009; 17:2983–2988. doi: 10.1016/j.bmc.2009.03.020 19329330

22. Khan KM, Rani M, Ambreen N, Ali M, Hussain S, Perveen S, et al., 2,5-Disubstituted-1,3,4-oxadiazoles: Thymidine phosphorylase inhibitors. Med Chem Res. 2013;22: 6022–6028.

23. Javaid S, Saad SM, Perveen S, Khan KM, Choudhary MI. 2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase inhibitors. Bioorg Chem. 2015;63:142–151. doi: 10.1016/j.bioorg.2015.10.006 26547232

24. Javaid S, Ishtiaq M, Shaikh M, Hameed A, Choudhary MI. Thymidine esters as substrate analogue inhibitors of angiogenic enzyme thymidine phosphorylase in vitro. Bioorg Chem. 2017; 70: 44–56. doi: 10.1016/j.bioorg.2016.11.007 27955923

25. Hussain S, Gaffney J, Ahmed N, Slevin M, Choudhary MI, Ahmad VU, et al., An investigation of the kinetic and anti-angiogenic properties of plant glycoside inhibitors of thymidine phosphorylase. J Asian Nat Prod Res. 2009; 11:159–167. doi: 10.1080/10286020802618860 19219729

26. Abbasi MA, Ahmad VU, Zubair M, Fatima N, Farooq U, Hussain S, et al., Phosphodiesterase and thymidine phosphorylase inhibiting Salirepin derivatives from Symplocos racemosa. Planta Med. 2004;70:1189–1194. doi: 10.1055/s-2004-835850 15643556

27. Vane JR, Botting RM. Anti-inflammatory drugs and their mechanism of action. Inflamm Res.1998; 47:S78–87. doi: 10.1007/s000110050284 9831328

28. Djerassi C, Gorman M, Nussbaum AL, Reynoso L. Alkaloid studies. IV. The isolation of reserpine, serpentine and ajmaline from Rauwolfia heterophylla Roem. and Schult. J Am Chem Soc.1954; 76:4463–4465

29. Dias DA, Urban S, Roessner U. A historical overview of natural products in drug discovery. Metabolites. 2012; 3: 303–336.

30. Lacy A, O’Kennedy R. Studies on coumarins and coumarin-related compounds to determine their therapeutic role in the treatment of cancer. Curr Pharm Des. 2004 10:3797–3811. doi: 10.2174/1381612043382693 15579072

31. Borges F, Roleira F, Milhazes N, Santana L, Uriarte E. Simple coumarins and analogues in medicinal chemistry: Occurrence, synthesis and biological activity. Curr Med Chem. 2005; 12: 887–916. doi: 10.2174/0929867053507315 15853704

32. Miadoková E. Isoflavonoids–An overview of their biological activities and potential health benefits. Interdiscip Toxicol. 2009; 2:211–218. doi: 10.2478/v10102-009-0021-3 21217857

33. Moss GP. Nomenclature of lignans and neolignans (IUPAC Recommendations 2000). Pure Appl Chem. 2000; 72:1493–1523.

34. Facchini PJ. Alkaloid biosynthesis in plants: Biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annu Rev Plant Physiol Plant Mol Biol. 2001; 52:29–66. doi: 10.1146/annurev.arplant.52.1.29 11337391

35. Ballatore C, Huryn DM, Smith AB III. Carboxylic acid (bio)isosteres in drug design. Chem Med Chem. 2013;8: 385–395. doi: 10.1002/cmdc.201200585 23361977

36. Dutta S, Ray S, Nagarajan K. Glutamic acid as anticancer agent: An overview. Saudi Pharm J. 2013; 21: 337–343. doi: 10.1016/j.jsps.2012.12.007 24227952

37. Saarinen N, Mäkelä S, Santti R. Anticancer effects of lignans. In: Yagasaki K., Miura Y., Hatori M., Nomura Y.(eds), Animal Cell Technology: Basic & Applied Aspects, 2003;13:55–58.

38. Mughal UR, Fatima I, Malik A, Tareen RB. Loasifolin, a new flavonoid from Eremostachys loasifolia. J Asian Nat Prod Res. 2010; 12: 328–330. doi: 10.1080/10286021003627379 20419544

39. Rauf A, Khan R, Muhammad N. Antioxidant studies of various solvent fractions and chemical constituents of Potentilla evestita Th. Wolf, Afr. J Pharm Pharmacol. 2013; 7: 2707–2710.

40. Faizi S, Dar A, Siddiqi H, Naqvi S, Naz A, Bano S, et al.,. Bioassay-guided isolation of antioxidant agents with analgesic properties from flowers of Tagetes patula. Pharm Biol. 2011;49: 516–525. doi: 10.3109/13880209.2010.523006 21284510

41. Miftakhova AF, Burasheva GSh, Abilov ZhA, Ahmad VU, Zahid M. Coumarins from the aerial part of Halocnemum strobilaceum. Fitoterapia, 2001; 72:319–321. doi: 10.1016/s0367-326x(00)00301-4 11295318

42. Abbaskhan A, Choudhary MI, Ghayur MN, Parween Z, Shaheen F, Gilani AUH, et al.,. Biological Activities of Indian Celery, Seseli diffusum (Roxb. ex Sm.) Sant. & Wagh. Phytother Res. 2012;26: 783–786. doi: 10.1002/ptr.3600 22095902

43. Tatuedom OK, Kouam SF, Yapna DB, Ngadjui BT, Green IR, Choudhary MI, et al.,. Spiroalkaloids and coumarins from the stem bark of Pauridiantha callicarpoides. Z Naturforsch. 2014; 69b: 747–752.

44. Shaheen F, Zeeshan M, Ahmad M, Anjum S, Ali S, Fun HK, et al., (2006). Norditerpenoid alkaloids from Delphinium nordhagenii. J Nat Prod. 2006; 69: 823–825. doi: 10.1021/np050478m 16724850

45. Bera H, Dolzhenko AV, Sun L, Gupta SD, Chui W. Synthesis and in vitro evaluation of 1,2,4-triazolo[1,5-a][1,3,5]triazine derivatives as thymidine phosphorylase inhibitors. Chemical Biol Drug Des. 2013; 82: 351–360.

46. LigPrep, version 3.6, Schrödinger, LLC, New York, NY, 2015.

47. Protein Preparation Wizard 2015–4; Epik version 2.4, Schrödinger, LLC, New York, NY, 2015; Impact version 5.9, Schrödinger, LLC, New York, NY, 2015; Prime version 3.2, Schrödinger, LLC, New York, NY, 2015.

48. Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman WJ. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments, Compute Aided Mol Des. 2013; 27: 221–234.

49. Halgren T. Identifying and characterizing binding sites and assessing druggability. J Chem Inf Model. 2009; 49:377–389. doi: 10.1021/ci800324m 19434839

50. Schrödinger Release 2015–4: SiteMap, version 3.7, Schrödinger, LLC, New York, NY, 2015.

51. Glide, version 6.9, Schrödinger, LLC, New York, NY, 2015.

52. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al., Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004; 47: 1739–1749. doi: 10.1021/jm0306430 15027865

53. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, et al., Glide: A new approach for rapid, accurate docking and scoring. 2. Chapter 1: Introduction 6 Schrödinger software release 2015–4 enrichment factors in database screening, J Med Chem. 2004;47:1750–1759. doi: 10.1021/jm030644s 15027866

54. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, et al.,. Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem. 2006;49: 6177–6196. doi: 10.1021/jm051256o 17034125

55. Prime, version 4.2, Schrödinger, LLC, New York, NY, 2015.

56. Dimas K, Demetzos C, Marsellos M, Stiriadou R, Malamas M, Kokkinopoulos D. Cytotoxic activity of labdane type diterpenes against human leukemia cell lines in vitro. Planta Med. 1997; 64: 208–211.

57. Khan JA, Javaid S, Maryam AG, Huwait E, Shaikh M, Shafqat A, et al., (2017). Studies on new urease inhibitors by using biochemical, STD-NMR spectroscopy, and molecular docking methods. Med Chem Res. 2017; 26: 2452–2467.

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2019 Číslo 11