Structure-based drug design, synthesis and biological assays of P. falciparum Atg3–Atg8 protein–protein interaction inhibitors

Abstract

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein–protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3–Atg8 reciprocal protein–protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

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Abbreviations

PE:

Phosphatidylethanolamine

PPI:

Protein–protein interactions

MD:

Molecular dynamics

SPR:

Surface plasmon resonance

References

  1. 1.

    World Health Organization (2017) http://www.who.int/mediacentre/factsheets/fs094/en/. Accessed 16 may 2017

  2. 2.

    Ashley E, McGready R, Proux S, Nosten F (2006) Malaria. Travel Med Infect Dis 4:159–173. https://doi.org/10.1016/j.tmaid.2005.06.009

    Article  Google Scholar 

  3. 3.

    Maeno Y, Culleton R, Quang NT, Kawai S, Marchand RP, Nakazawa S (2016) Plasmodium knowlesi and human malaria parasites in Khan Phu, Vietnam: gametocyte production in humans and frequent co-infection of mosquitoes. Parasitology 144:527–535. https://doi.org/10.1017/S0031182016002110

    Article  Google Scholar 

  4. 4.

    Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NPJ, Lindegardh N, Socheat D, White NJ (2009) Artemisinin resistance in Plasmodium falciparum malaria. New Engl J Med 361:455–467. https://doi.org/10.1056/NEJMoa0808859

    CAS  Article  Google Scholar 

  5. 5.

    Dondorp AM, Yeung S, White L, Nguon C, Day NPJ, Socheat D, von Seidlein L (2010) Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol 8:530–530. https://doi.org/10.1038/nrmicro2385

    CAS  Article  Google Scholar 

  6. 6.

    Beesley T, Gascoyne N, Knott-Hunziker V, Petursson S, Waley SG, Jaurin B, Grundström T (1983) The inhibition of class C β-lactamases by boronic acids. Biochem J 209:229–233. https://doi.org/10.1042/bj2090229

    CAS  Article  Google Scholar 

  7. 7.

    World Health Organization (2017) Malaria vaccine: WHO position paper, January 2016—Recommendations. Vaccine. https://doi.org/10.1016/j.vaccine.2016.10.047

    Google Scholar 

  8. 8.

    Jana S, Paliwal J (2007) Novel molecular targets for antimalarial chemotherapy. Int J Antimicrob Agents 30:4–10. https://doi.org/10.1016/j.ijantimicag.2007.01.002

    CAS  Article  Google Scholar 

  9. 9.

    Ettari R, Micale N, Grazioso G, Bova F, Schirmeister T, Grasso S, Zappalà M (2012) Synthesis and molecular modeling studies of derivatives of a highly potent peptidomimetic vinyl ester as falcipain-2 inhibitors. ChemMedChem 7:1594–1600. https://doi.org/10.1002/cmdc.201200274

    CAS  Article  Google Scholar 

  10. 10.

    Marco M, Coteron JM (2012) Falcipain inhibition as a promising antimalarial target. Curr Top Med Chem 12:408–444

    CAS  Article  Google Scholar 

  11. 11.

    Alvarez VE, Kosec G, Sant’Anna C, Turk V, Cazzulo JJ, Turk B (2008) Autophagy is involved in nutritional stress response and differentiation in Trypanosoma cruzi. J Biol Chem 283:3454–3464. https://doi.org/10.1074/jbc.M708474200

    CAS  Article  Google Scholar 

  12. 12.

    Sinai AP, Roepe PD (2012) Autophagy in Apicomplexa: A life sustaining death mechanism?. Trends Parasitol 28:358–364. https://doi.org/10.1016/j.pt.2012.06.006

    Article  Google Scholar 

  13. 13.

    Hain AUP, Weltzer RR, Hammond H, Jayabalasingham B, Dinglasan RR, Graham DRM, Colquhoun DR, Coppens I, Bosch J (2012) Structural characterization and inhibition of the Plasmodium Atg8–Atg3 interaction. J Struct Biol 180:551–562. https://doi.org/10.1016/j.jsb.2012.09.001

    CAS  Article  Google Scholar 

  14. 14.

    Besteiro S (2012) Which roles for autophagy in Toxoplasma gondii and related apicomplexan parasites? Mol Biochem Parasitol 184:1–8. https://doi.org/10.1016/j.molbiopara.2012.04.001

    CAS  Article  Google Scholar 

  15. 15.

    Chen D, Lin J, Liu Y, Li X, Chen G, Hua Q, Nie Q, Hu X, Tan F (2016) Identification of TgAtg8–TgAtg3 interaction in Toxoplasma gondii. Acta Trop 153:79–85. https://doi.org/10.1016/j.actatropica.2015.09.013

    CAS  Article  Google Scholar 

  16. 16.

    Geng J, Klionsky DJ (2008) The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. Embo Rep 9:859

    CAS  Article  Google Scholar 

  17. 17.

    Abada A, Elazar Z (2014) Getting ready for building: signaling and autophagosome biogenesis. Embo Rep 15:839

    CAS  Article  Google Scholar 

  18. 18.

    Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA (2003) Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301:1503–1508. https://doi.org/10.1126/science.1087025

    Article  Google Scholar 

  19. 19.

    Duszenko M, Ginger ML, Brennand A, Gualdrón-López M, Colombo MI, Coombs GH, Coppens I, Jayabalasingham B, Langsley G, Lisboa de Castro S, Menna-Barreto R, Mottram JC, Navarro M, Rigden DJ, Romano PS, Stoka V, Turk B, Michels PAM (2011) Autophagy in protists. Autophagy 7:127–158. https://doi.org/10.4161/auto.7.2.13310

    CAS  Article  Google Scholar 

  20. 20.

    Hain AU, Miller AS, Levitskaya J, Bosch J (2016) Virtual screening and experimental validation identify novel inhibitors of the Plasmodium falciparum Atg8–Atg3 protein–protein interaction. ChemMedChem 11:900–910. https://doi.org/10.1002/cmdc.201500515

    CAS  Article  Google Scholar 

  21. 21.

    Hain AUP, Bartee D, Sanders NG, Miller AS, Sullivan DJ, Levitskaya J, Meyers CF, Bosch J (2014) Identification of an Atg8–Atg3 Protein–Protein interaction inhibitor from the medicines for malaria venture malaria box active in blood and liver stage Plasmodium falciparum parasites. J Med Chem 57:4521–4531. https://doi.org/10.1021/jm401675a

    CAS  Article  Google Scholar 

  22. 22.

    Scott DE, Bayly AR, Abell C, Skidmore J (2016) Small molecules, big targets: drug discovery faces the protein–protein interaction challenge. Nat Rev Drug Discov 15:533–550. https://doi.org/10.1038/nrd.2016.29

    CAS  Article  Google Scholar 

  23. 23.

    Valverde IE, Mindt TL (2013) 1,2,3-Triazoles as amide-bond surrogates in peptidomimetics. Chimia 67:262–266

    CAS  Article  Google Scholar 

  24. 24.

    Stucchi M, Grazioso G, Lammi C, Manara S, Zanoni C, Arnoldi A, Lesma G, Silvani A (2016) Disrupting the PCSK9/LDLR protein–protein interaction by an imidazole-based minimalist peptidomimetic. Org Biomol Chem 14:9736–9740. https://doi.org/10.1039/C6OB01642A

    CAS  Article  Google Scholar 

  25. 25.

    Ni Z, Giordano L, Tenaglia A (2014) Cyclobutene formation in PtCl2-catalyzed cycloisomerizations of heteroatom-tethered 1,6-enynes. Chem-Eur J 20:11703–11706. https://doi.org/10.1002/chem.201403643

    CAS  Article  Google Scholar 

  26. 26.

    Masciocchi D, Gelain A, Porta F, Meneghetti F, Pedretti A, Celentano G, Barlocco D, Legnani L, Toma L, Kwon B-M, Asaid A, Villa S (2013) Synthesis, structure–activity relationships and stereochemical investigations of new tricyclic pyridazinone derivatives as potential STAT3 inhibitors. MedChemComm 4:1181–1188

    CAS  Article  Google Scholar 

  27. 27.

    Toma L, Legnani L, Rencurosi A, Poletti L, Lay L, Russo G (2009) Modeling of synthetic phosphono and carba analogues of N-acetyl-alpha-D-mannosamine 1-phosphate, the repeating unit of the capsular polysaccharide from Neisseria meningitidis serovar A. Org Biomol Chem 7:3734–3740. https://doi.org/10.1039/b907000a

    CAS  Article  Google Scholar 

  28. 28.

    Luparia M, Legnani L, Porta A, Zanoni G, Toma L, Vidari G (2009) Enantioselective synthesis and olfactory evaluation of bicyclic alpha- and gamma-ionone derivatives: the 3D arrangement of key molecular features relevant to the violet odor of ionones. J Org Chem 74:7100–7110. https://doi.org/10.1021/jo9014936

    CAS  Article  Google Scholar 

  29. 29.

    Legnani L, Colombo D, Venuti A, Pastori C, Lopalco L, Toma L, Mori M, Grazioso G, Villa S (2017) Diazabicyclo analogues of maraviroc: synthesis, modeling, NMR studies and antiviral activity. MedChemComm 8:422–433. https://doi.org/10.1039/C6MD00575F

    CAS  Article  Google Scholar 

  30. 30.

    Lee C, Yang W, Parr RG (1988) Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys Rev B 37:785–789

    CAS  Article  Google Scholar 

  31. 31.

    Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J Chem Phys 98:5648–5652. https://doi.org/10.1063/1.464913

    CAS  Article  Google Scholar 

  32. 32.

    Wolinski K, Hinton JF, Pulay P (1990) Efficient implementation of the gauge-independent atomic orbital method for NMR chemical shift calculations. J Am Chem Soc 112:8251–8260. https://doi.org/10.1021/ja00179a005

    CAS  Article  Google Scholar 

  33. 33.

    Ditchfield R (1974) Self-consistent perturbation theory of diamagnetism. Mol Phys 27:789–807. https://doi.org/10.1080/00268977400100711

    CAS  Article  Google Scholar 

  34. 34.

    Case DA, Darden TA, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Walker C, Zhang W, Merz KM, Roberts B, Hayik S, Roitberg A, Seabra G, Swails J, Goetz AW, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wolf RM, Liu J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh MJ, Cui G, Roe DR, Mathews DH, Seetin MG, Salomon-Ferrer R, Sagui C, Babin V, Luchko T, Gusarov S, Kovalenko A, Kollman PA (2012) AMBER 12. University of California, San Francisco

  35. 35.

    Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comp Chem 25:1157–1174. https://doi.org/10.1002/jcc.20035

    CAS  Article  Google Scholar 

  36. 36.

    Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935. https://doi.org/10.1063/1.445869

    CAS  Article  Google Scholar 

  37. 37.

    Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593

    CAS  Article  Google Scholar 

  38. 38.

    Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690. https://doi.org/10.1063/1.448118

    CAS  Article  Google Scholar 

  39. 39.

    Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–748. https://doi.org/10.1006/jmbi.1996.0897

    CAS  Article  Google Scholar 

  40. 40.

    Korb O, Stutzle T, Exner TE (2009) Empirical scoring functions for advanced protein-ligand docking with PLANTS. J Chem Inf Model 49:84–96. https://doi.org/10.1021/ci800298z

    CAS  Article  Google Scholar 

  41. 41.

    Dallanoce C, Magrone P, Bazza P, Grazioso G, Rizzi L, Riganti L, Gotti C, Clementi F, Frydenvang K, De Amici M (2009) New analogues of epiboxidine incorporating the 4,5-dihydroisoxazole nucleus: synthesis, binding affinity at neuronal nicotinic acetylcholine receptors, and molecular modeling investigations. Chem Biodivers 6:244–259. https://doi.org/10.1002/cbdv.200800077

    CAS  Article  Google Scholar 

  42. 42.

    Nilsson BM, Vargas HM, Ringdahl B, Hacksell U (1992) Phenyl-substituted analogues of oxotremorine as muscarinic antagonists. J Med Chem 35:285–294

    CAS  Article  Google Scholar 

  43. 43.

    Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JJA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross BJ, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann, ER, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski WJ, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian09. Revision A.02

  44. 44.

    Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193:673–675

    CAS  Article  Google Scholar 

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Acknowledgements

We acknowledge the CINECA and the Regione Lombardia award under the LISA initiative, for the availability of high performance computing resources and support. We thank the Johns Hopkins Malaria Research Institute parasite and insectary facility for assistance with our experiments. This work was partially funded through The Bloomberg Family Foundation (J.B.). We thank Professors D. Taramelli and M. De Amici for helpful discussion. L. L. thanks the University of Pavia for partial financial support.

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Correspondence to Giovanni Grazioso.

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Villa, S., Legnani, L., Colombo, D. et al. Structure-based drug design, synthesis and biological assays of P. falciparum Atg3–Atg8 protein–protein interaction inhibitors. J Comput Aided Mol Des 32, 473–486 (2018). https://doi.org/10.1007/s10822-018-0102-5

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Keywords

  • Malaria
  • Autophagy
  • Atg8 inhibitors
  • Docking
  • PPI inhibitors
  • Peptidomimetics
  • 1,2,3-Triazole