Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Cherry JD. 2012. Why do pertussis vaccines fail? Pediatrics 129:968–970. 10.1542/peds.2011-2594 PubMed DOI

Khelef N, Zychlinsky A, Guiso N. 1993. Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect. Immun. 60:4064–4071 PubMed PMC

Bachelet M, Richard MJ, Francois D, Polla BS. 2002. Mitochondrial alterations precede Bordetella pertussis-induced apoptosis. FEMS Immunol. Med. Microbiol. 32:125–131. 10.1111/j.1574-695X.2002.tb00544.x PubMed DOI

Vojtova J, Kamanova J, Sebo P. 2006. Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr. Opin. Microbiol. 9:69–75. 10.1016/j.mib.2005.12.011 PubMed DOI

Masure HR. 1993. The adenylate cyclase toxin contributes to the survival of Bordetella pertussis within human macrophages. Microb. Pathog. 14:253–260. 10.1006/mpat.1993.1025 PubMed DOI

Martín C, Gómez-Bilbao G, Ostolaza H. 2010. Bordetella adenylate cyclase toxin promotes calcium entry into both CD11b+ and CD11b− cells through cAMP-dependent L-type-like calcium channels. J. Biol. Chem. 285:357–364. 10.1074/jbc.M109.003491 PubMed DOI PMC

Fiser R, Masin J, Bumba L, Pospisilova E, Fayolle C, Basler M, Sadilkova L, Adkins I, Kamanova J, Cerny J, Konopasek I, Osicka R, Leclerc C, Sebo P. 2012. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores. PLoS Pathog. 8:e1002580. 10.1371/journal.ppat.1002580 PubMed DOI PMC

Holy A. 2003. Phosphonomethoxyalkyl analogs of nucleotides. Curr. Pharmaceut. Des. 9:2567–2592. 10.2174/1381612033453668 PubMed DOI

Marcellin P, Chang TT, Lim SG, Tong MJ, Sievert W, Shiffman ML, Jeffers L, Goodman Z, Wulfsohn MS, Xiong S, Fry J, Brosgart CL, Adefovir Dipivoxil 437 Study Group 2003. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N. Engl. J. Med. 348:808–816. 10.1056/NEJMoa020681 PubMed DOI

Shen YQ, Zhukovskaya NL, Zimmer MI, Soelaiman S, Wang CR, Gibbs CS, Tang WJ. 2004. Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection. Proc. Natl. Acad. Sci. U. S. A. 101:3242–3247. 10.1073/pnas.0306552101 PubMed DOI PMC

Göttle M, Dove S, Steindel P, Shen Y, Tang WJ, Geduhn J, König B, Seifert R. 2007. Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent nucleotides. Mol. Pharmacol. 72:526–535. 10.1124/mol.107.034413 PubMed DOI

Guo Q, Shen Y, Lee YS, Gibbs CS, Mrksich M, Tang WJ. 2005. Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin. EMBO J. 24:3190–3201. 10.1038/sj.emboj.7600800 PubMed DOI PMC

Palu G, Stefanelli S, Rassu M, Parolin C, Balzarini J, De Clercq E. 1991. Cellular uptake of phosphonylmethoxyalkylpurine derivatives. Antivir. Res. 16:115–119. 10.1016/0166-3542(91)90063-W PubMed DOI

Naesens L, Balzarini J, Bischofberger N, De Clercq E. 1996. Antiretroviral activity and pharmacokinetics in mice of oral bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine, the bis(pivaloyloxymethyl) ester prodrug of 9-(2-phosphonylmethoxyethyl)adenine. Antimicrob. Agents Chemother. 40:22–28 PubMed PMC

Balestra E, Perno C, Aquaro S, Lazzarino G, Tavazzi B, Di Pierro D, Calio R, Balzarini J, De Clercq E. 1995. Phosphorylation of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is greater in macrophages than in replicating lymphocytes and fibroblasts. Antivir. Res. 26:275–276

Cundy KC, Barditch-Crovo P, Walker RE, Collier AC, Ebeling D, Toole J, Jaffe HS. 1995. Clinical pharmacokinetics of adefovir in human immunodeficiency virus type 1-infected patients. Antimicrob. Agents Chemother. 39:2401–2405. 10.1128/AAC.39.11.2401 PubMed DOI PMC

Barditch-Crovo PA, Cundy KC, Wachsman M, Toole J, Burgee H, Ebeling D. 1995. Pharmacokinetic profile of 9-[2-(bispivaloyloxy-methyl)phosphonylmethoxy] adenine (bis-POM PMEA), an orally bioavailable prodrug of the antiviral nucleotide, PMEA, abstr p 488 The Second National Conference on Human Retroviruses, Washington, DC

Shaw JP, Cundy KC. 1993. Biological screens of PMEA prodrugs. Pharm. Res. 10(Suppl):S294

Law ST, Li KK, Ho YY. 2012. Nephrotoxicity, including acquired Fanconi's syndrome, caused by adefovir dipivoxil-is there a safe dose? J. Clin. Pharm. Ther. 37:128–131. 10.1111/j.1365-2710.2011.01278.x PubMed DOI

Jansa P, Baszczyňski O, Dračínský M, Votruba I, Zídek Z, Bahador G, Stepan G, Cihlar T, Mackman R, Holý A, Janeba Z. 2011. A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities. Eur. J. Med. Chem. 46:3748–3754. 10.1016/j.ejmech.2011.05.040 PubMed DOI

Jansa P, Holý A, Dračínský M, Baszczyňski O, Ccaronesnek M, Janeba Z. 2011. Efficient and ‘green' microwave-assisted synthesis of haloalkylphosphonates via the Michaelis-Arbuzov reaction. Green Chem. 13:882–888. 10.1039/c0gc00509f DOI

Jansa P, Baszczyňski O, Procházková E, Dračínský M, Janeba Z. 2012. Microwave-assisted hydrolysis of phosphonate diesters: an efficient protocol for the preparation of phosphonic acids. Green Chem. 14:2282–2288. 10.1039/c2gc35547g DOI

Fiser R, Masín J, Basler M, Krusek J, Spuláková V, Konopásek I, Sebo P. 2007. Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin: membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities. J. Biol. Chem. 282:2808–2820 PubMed

Grynkiewicz G, Poenie M, Tsien RY. 1985. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450 PubMed

Artursson P, Palm K, Luthman K. 2001. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46:27–43. 10.1016/S0169-409X(00)00128-9 PubMed DOI

Annaert P, Kinget R, Naesens L, de Clercq E, Augustijns P. 1997. Transport, uptake, and metabolism of the bis(pivaloyloxymethyl)-ester prodrug of 9-(2-phosphonylmethoxyethyl)adenine in an in vitro cell culture system of the intestinal mucosa (Caco-2). Pharm. Res. 14:492–496. 10.1023/A:1012155717819 PubMed DOI

Annaert P, Van Gelder J, Naesens L., De Clercq E, Van den Mooter G, Kinget R, Augustijns P. 1998. Carrier mechanisms involved in the transepithelial transport of bis(POM)-PMEA and its metabolites across Caco-2 monolayers. Pharm. Res. 15:1168–1173 PubMed

Sampath J, Adachi M, Hatse S, Naesens L, Balzarini J, Flatley R, Matherly L, Schuetz J. 2002. Role of MRP4 and MRP5 in biology and chemotherapy. AAPS PharmSci. 4:1–9. 10.1208/ps040314 PubMed DOI PMC

Srinivas RV, Robbins BL, Connelly MC, Gong YF, Bischofberger N, Fridland A. 1993. Metabolism and in vitro antiretroviral activities of bis(pivaloyloxymethyl) prodrugs of acyclic nucleoside phosphonates. Antimicrob. Agents Chemother. 37:2247–2250. 10.1128/AAC.37.10.2247 PubMed DOI PMC

Birkus G, Wang R, Liu X, Kutty N, MacArthur H, Cihlar T, Gibbs C, Swaminathan S, Lee W, McDermott M. 2007. Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131. Antimicrob. Agents Chemother. 51:543–550. 10.1128/AAC.00968-06 PubMed DOI PMC

Birkus G, Kutty N, Frey CR, Shribata R, Chou T, Wagner C, McDermott M, Cihlar T. 2011. Role of Cathepsin A and lysosomes in the intracellular activation of novel antipapillomavirus agent GS-9191. Antimicrob. Agents Chemother. 55:2166–2173. 10.1128/AAC.01603-10 PubMed DOI PMC

Luedtke CC, Andonian S, Igdoura S, Hermo L. 2000. Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors. J. Histochem. Cytochem. 48:1131–1146. 10.1177/002215540004800810 PubMed DOI

Rottier RJ, Hahn CN, Mann LW, del Pilar Martin M, Smeyne RJ, Suzuki K, d'Azzo A. 1998. Lack of PPCA expression only partially coincides with lysosomal storage in galactosialidosis mice: indirect evidence for spatial requirement of the catalytic rather than the protective function of PPCA. Hum. Mol. Genet. 7:1787–1794. 10.1093/hmg/7.11.1787 PubMed DOI

Annaert P, Van den Mooter G, Arimilli MN, Kim Ch De Clercq UE. 1998. Antiretroviral efficacy and pharmacokinetics of oral bis(isopropyloxycarbonyloxymethyl)9-(2-phosphonylmethoxypropyl)adenine in mice. Antimicrob. Agents Chemother. 42:1568–1573 PubMed PMC

Li F, Maag H, Alfredson T. 2008. Prodrugs of nucleoside analogues for improved oral absorption and tissue targeting. J. Pharm. Sci. 97:1109–1134. 10.1002/jps.21047 PubMed DOI

Basler M, Masin J, Osicka R, Sebo P. 2006. Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes. Infect. Immun. 74:2207–2214. 10.1128/IAI.74.4.2207-2214.2006 PubMed DOI PMC

Zídek Z, Kmoníčková E, Holý A. 2005. Cytotoxicity of pivoxil esters of antiviral acyclic nucleoside phosphonates: adefovir dipivoxil versus adefovir. Biomed. Papers Med. Fac. Univ. Palacky Olomouc Czech Republ. 149:315–319. 10.5507/bp.2005.049 PubMed DOI

Marcsek ZL, Kocsis Z, Szende B, Tompa A. 2007. Effect of formaldehyde and resveratrol on the viability of Vero, HepG2, and MCF-7 cells. Cel.l Biol. Int. 31:1214–1219. 10.1016/j.cellbi.2007.01.039 PubMed DOI

Wong K, Li X, Ma Y. 2006. Paraformaldehyde induces elevation of intracellular calcium and phosphatidylserine externalization in platelets. Thromb. Res. 117:537–542. 10.1016/j.thromres.2005.04.030 PubMed DOI

McConkey DJ. 1996. The role of calcium in the regulation of apoptosis. Scan. Microsc. 10:777–793 PubMed

Structure-Based Drug Design of ADRA2A Antagonists Derived from Yohimbine

Discovery of a potent and selective human AC2 inhibitor based on 7-deazapurine analogues of adefovir

Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors

Phosphonates and Phosphonate Prodrugs in Medicinal Chemistry: Past Successes and Future Prospects

Halogen-Dance-Based Synthesis of Phosphonomethoxyethyl (PME) Substituted 2-Aminothiazoles as Potent Inhibitors of Bacterial Adenylate Cyclases

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis

Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases