LED fluorescence microscopy offers potential benefits to the diagnosis of human African trypanosomiasis, as well as to other aspects of diseases management, such as detection of drug resistant strains. To advance such approaches reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we report a series of novel green fluorescent diamidines and their suitability as probes to stain trypanosomes

Arvind Kumar

Bailey

Baker

Barrett

Bieler

Bridges

Carter

Chappuis

David W. Boykin

de Koning

Federica Giordani

Hanscheid

Jones

Keiser

Lanteri

Manoj Munde

Mathis

Matovu

Michael P. Barrett

Minion

Mohamed A. Ismail

Paine

Steingart

Stewart

Vincent

W. David Wilson

Wilson

English

PubMed

ABSTRACT
          Light-emitting diode (LED) fluorescence microscopy offers potential benefits in the diagnosis of human African trypanosomiasis and in other aspects of diseases management, such as detection of drug-resistant strains. To advance such approaches, reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we describe a series of novel green fluorescent diamidines and their suitability as probes with which to stain trypanosomes.</jats:p

Crossref

Antimicrobial Agents and Chemotherapy

Green Fluorescent Diamidines as Diagnostic Probes for Trypanosomes

Giordani, Federica

Munde, Manoj

Wilson, W. David

Ismail, Mohamed A.

Kumar, Arvind

Boykin, David W.

Barrett, Michael P.

Enlighten

     Giordani, Federica, Munde, Manoj, Wilson, W. David, Ismail, Mohamed A., Kumar, Arvind, Boykin, David W., and Barrett, Michael P. (2014) Green fluorescent diamidines as diagnostic probes for trypanosomes. Antimicrobial Agents and Chemotherapy, 58 (3). pp. 1793-1796. ISSN 0066-4804   Copyright © 2014 The Authors    http://eprints.gla.ac.uk/90388    Deposited on:  02 April 2014                         Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk   Published Ahead of Print 23 December 2013. 10.1128/AAC.02024-13. 2014, 58(3):1793. DOI:Antimicrob. Agents Chemother. Michael P. BarrettMohamed A. Ismail, Arvind Kumar, David W. Boykin and Federica Giordani, Manoj Munde, W. David Wilson, Diagnostic Probes for TrypanosomesGreen Fluorescent Diamidines ashttp://aac.asm.org/content/58/3/1793Updated information and services can be found at: These include:SUPPLEMENTAL MATERIAL Supplemental materialREFERENCEShttp://aac.asm.org/content/58/3/1793#ref-list-1at: This article cites 21 articles, 11 of which can be accessed freeCONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from  on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from Green Fluorescent Diamidines as Diagnostic Probes for TrypanosomesFederica Giordani,c Manoj Munde,a W. David Wilson,a Mohamed A. Ismail,a,b Arvind Kumar,a David W. Boykin,a Michael P. BarrettcDepartment of Chemistry, Georgia State University, Atlanta, Georgia, USAa; Department of Chemistry, College of Science, King Faisal University, Hofuf, Saudi Arabiab;Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University ofGlasgow, Glasgow, United KingdomcLight-emitting diode (LED) fluorescence microscopy offers potential benefits in the diagnosis of human African trypanosomiasisand in other aspects of diseases management, such as detection of drug-resistant strains. To advance such approaches, reliableand specific fluorescent markers to stain parasites in human fluids are needed. Here we describe a series of novel green fluores-cent diamidines and their suitability as probes with which to stain trypanosomes.Early diagnosis of human African trypanosomiasis (HAT) (1) isparamount for favorable prognosis, but it is difficult to achievein the field (2). Conventional light microscopy, in particular, isinsensitive and time-consuming. Deployment of better diagnos-tics, together with more effective drugs, will be essential to elimi-nate the disease as a public health problem.The development of portable and efficient light-emitting diode(LED)-illuminated microscopes (3–5) allows fluorescence mi-croscopy to offer improvements in detection. Its use in the diag-nosis of other tropical diseases has already been shown to increasethe sensitivity and rapidity of sample preparation and screening(6, 7). A similar approach would also be useful in diagnosis ofanimal trypanosomiasis, which suffers limitations similar to thoseof diagnosis of HAT. The diagnostic fluorescence tests developedfor HAT (8, 9) use acridine orange to stain trypanosomes. This isa cheap but aspecific fluorophore, which can label the nucleicacids of any cell in the specimen, and as a DNA intercalator, itposes potential risks to laboratory technicians (although, based onavailable data, the IARC inserts it in group 3, as not classifiable asto its carcinogenicity for humans).The aim of this work was to synthesize and evaluate a series offluorescent diamidines as targeted diagnostic probes for trypano-somes. Diamidines (10, 11) appeared promising since they arerapidly internalized by Trypanosoma brucei via specific surfacetransporters, including the aminopurine P2/TbAT1 transporter(12–14), and several of them are fluorescent (11, 15, 16). The UVemission of DB99 was previously exploited in our laboratory todevelop a simple assay for detection of drug-resistant strains lack-ing P2 (17). Unfortunately, although LED fluorescence micro-scopes are being increasingly used, UV LED-illuminated instru-ments are not yet widely available, most fluorescence biologicalwork being carried out at longer wavelengths.(Biological aspects of this work were conducted by F. Giordaniin partial fulfillment of the requirements for a Ph.D. from theUniversity of Glasgow, Glasgow, Scotland, 2011.)By addition of a series of ultraconjugated ring structures in thelinker between the two amidine groups (containing the recogni-tion motif for the parasite’s P2 transporter), we generated a seriesof new diamidines with longer emission wavelengths than the pa-rental UV-emitting DB75 (chemistry data are presented in thesupplemental material). Spectral measurements confirmed thatall novel dications emitted in the green wavelength range (Table1). As expected for diamidines (11), the newmolecules had a highaffinity for DNA. Values of DNA binding by thermal melting(Tm, measured as described in reference 15) revealed the bindingaffinity to be greater than that of pentamidine (12.6°C) but lowerthan that of DB75 (25°C) (Table 1).The dependence of the compounds on P2 transport for uptakeinto trypanosomes was confirmed by an alamarBlue assay (18)performed on threeTrypanosoma brucei brucei strains: a wild-typeline (bloodstream Lister 427), a line not expressing P2 (tbat1/)(19), and a derived clone, in which high-affinity pentamidinetransporter (HAPT1, now identified as an aquaglyceroporin [20])activity is also lost (B48) (21). Compound DB1692 showed thegreatest dependency on P2 for transport (50% inhibitory concen-tration [IC50] 25-fold higher for the tbat1/ line than for thewildtype), followed by DB1645 (16-fold higher), DB1465 (10-fold),and DB1464 (8-fold) (Table 2). Data using the B48 line showedthat HAPT1 activity also contributed to the accumulation ofDB1645 and DB1465. In contrast, the addition of a single furanring in the linker of DB1680 highly affected the rate of uptake ofthis compound through the P2 transporter, and further loss ofHAPT1 did not affect its toxicity, suggesting other routes of inter-nalization (e.g., other carriers or endocytosis). Equally notewor-thy is the potent in vitro activity shown by these new compoundsagainst the wild-type line, especially by DB1464, which had anIC50 close to that of pentamidine. This indicates that these mole-culesmay be attractive candidates to pursue as possible leads againstHAT, provided that ADME (absorption, distribution, metabolism,and excretion) characterizationdemonstrates goodpharmacokineticproperties and rules out potential toxicity issues previously observedfor somemolecules belonging to this class.All five diamidines stained live T. b. brucei in infected rat bloodfilms using a standard fluorescein isothiocyanate (FITC) filter set(excitation, band-pass, 450 to 490 nm; beam splitter, 510 nm; andemission, band-pass, 515 to 565 nm) and a Zeiss Axioplan fluo-Received 16 September 2013 Returned for modification 6 December 2013Accepted 17 December 2013Published ahead of print 23 December 2013Address correspondence to Michael P. Barrett, michael.barrett@glasgow.ac.uk.Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02024-13.Copyright © 2014 Giordani et al. This is an open-access article distributed underthe terms of the Creative Commons Attribution 3.0 Unported license.doi:10.1128/AAC.02024-13March 2014 Volume 58 Number 3 Antimicrobial Agents and Chemotherapy p. 1793–1796 aac.asm.org 1793 on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from rescence microscope (Fig. 1A). Under our experimental condi-tions (ex vivo incubation for 15 min at 37°C, with 50 M [each]fluorophore), all five diamidines provided specific staining of try-panosomes without labeling erythrocytes or white blood cells.Staining of the parasite nucleus was not observed, but all com-pounds selectively accumulated inside the kinetoplast (the para-site’smitochondrial genome, at the posterior of the cell) and othercytoplasmic organelles, spread throughout the cell body and alsovisualized at longer wavelengths (Fig. 1B). These were likely to beacidocalcisomes, previously observed to be stained by other di-amidines (15). For DB1692, a prominent red fluorescent spot,putatively identified as the lysosome, also appeared in a regionbetween the nucleus and the kinetoplast after 1 h of treatment.Under the FITC filter, parasites were readily detectable amongblood cells based on their movement and fluorescent dots (espe-cially the bright kinetoplast). Cell labeling was achieved only byincubating at room temperature or 37°C but not at 4°C. Thistemperature sensitivity could lead to variability in field tests usingambient temperature in different sites; hence, the use of temper-ature-controlled devices (e.g., water baths or heating blocks)might be required in such settings. Although the fluorescence in-tensity increased with the time of incubation for all probes, theoverall yields remained below those identified with DB75 andcould not be improved by increasing the fluorophore concentra-tion. The low emission intensity of the new compounds could beTABLE 1 Structures and physical properties of the fluorescent diamidinesCompound StructureEX(nm)aEM(nm)bFluorescence intensityat maxcTm valuesd withpoly(dA) · poly(dT)DB1464 405 542 73 9.4DB1465 417 513 123 18.5DB1645 424 576 36 14.6DB1680 418 546 8 15.0DB1692 400 546 40 17.0a Excitation wavelength.b Emission wavelength.c max, maximum.d Melting temperature of the compound-DNA complexes as compared to values for free DNA. Tm values are in degrees Celsius and have an error of0.5°C.TABLE 2 In vitro trypanocidal activities of the five diamidines against T.b. brucei S427 wild-type and derived cell linesaCompoundIC50 [nM] ( SEM) for:RFbIC50 [nM] ( SEM)for B48 RFWild typetbat1/lineDB1464 0.9 0.3 6.9 2.1 8 7.3 0.7 8DB1465 6.9 1.9 71.4 4.4 10 334 11 48DB1645 14.5 2.8 233 43 16 610 60 42DB1680 266 21 591 43 2 341 11 1DB1692 17.6 4.4 436 41 25 344 14 20Pentamidine 0.6 0.3 0.9 0.2 1.5 167 10 278a n 3.b RF, resistance factor (ratio of the IC50 measured against the tbat1/ and B48 celllines to its value obtained for the wild type).Giordani et al.1794 aac.asm.org Antimicrobial Agents and Chemotherapy on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from due to their intrinsically low fluorescence yields (Table 1) but alsoto the discrepancy between their excitation maxima, all close to400 nm, and the standard FITC filter wavelength utilized for mi-croscopy. Hence, the use of these specific fluorophores with theseparticular filters may not be directly applicable to HAT diagnosisdue to the risk of false negatives. However, these same compoundswith customized filters or further derivatives with higher fluores-cence yields could be considered as probes for the specific stainingof trypanosomes using existing LED-based fluorescence micro-scopes.Despite the compounds’ partial dependence on the P2 trans-porter for uptake, they did not distinguish wild-type and tbat1/knockout line by fluorescencemicroscopy (50Mfor incubationsranging from 15 min to up to 2 h), indicating that uptake routesindependent of P2 play important roles in their internalizationwhen used under these conditions.Implementation of fluorescence tests for trypanosome detec-tion in human fluids must rely on affordable, efficient and prefer-ably specificmarkers. This work shows the feasibility of generatingcheap fluorophores, emitting at differential wavelengths, by sim-ple addition of cyclic groups within the chemical structure of di-amidines, the precursor of which, DB75, is fluorescent only in theUV region. The exploitation of surface transporters peculiar totrypanosomes is a straightforward way to specifically target fluo-rophores to these parasites, and improved compounds may bedeveloped following this approach to target both human and an-imal infective trypanosomes.ACKNOWLEDGMENTSThisworkwas supported by an award from theBill andMelindaGates Foun-dation through theConsortiumforParasiticDrugDevelopment (CPDD) (toM.P.B., W.D.W., and D.W.B.), an NIH grant (AI064200) (to W.D.W. andFIG 1 (A) Fluorescence images of infected rat blood incubated ex vivo with the five green fluorescent diamidines (note that the parasite position can changebetween the bright-field and corresponding fluorescence micrographs), 40 objective. (B) In vitro trypanosomes treated with DB1680 and DB1692 (50 M, 1h, 37°C), 100 objective; 4=,6-diamidino-2-phenylindole (DAPI) (20M)was used as aDNA counterstain. Asterisk, kinetoplast; arrow, cytoplasmic corpuscles,possibly acidocalcisomes; arrowhead, nucleus; hash mark, possibly lysosome. Bar, 10 m.Fluorescent Diamidines as Probes for TrypanosomesMarch 2014 Volume 58 Number 3 aac.asm.org 1795 on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from D.W.B.), the SirHalley StewartTrust (toM.P.B. andF.G.), and theWellcomeTrust.TheWellcomeTrustCentre forMolecularParasitology is supportedbycore funding from theWellcome Trust (085349).REFERENCES1. Barrett MP, Burchmore RJ, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ,Krishna S. 2003. The trypanosomiases. Lancet 362:1469–1480. http://dx.doi.org/10.1016/S0140-6736(03)14694-6.2. Chappuis F, Loutan L, Simarro P, Lejon V, Buscher P. 2005. Options forfield diagnosis of human African trypanosomiasis. Clin. Microbiol. Rev.18:133–146. http://dx.doi.org/10.1128/CMR.18.1.133-146.2005.3. Hanscheid T. 2008. The future looks bright: low-cost fluorescent micro-scopes for detection of Mycobacterium tuberculosis and Coccidiae. Trans.R. Soc. Trop. Med. Hyg. 102:520–521. http://dx.doi.org/10.1016/j.trstmh.2008.02.020.4. Jones D, Nyalwidhe J, Tetley L, Barrett MP. 2007. McArthur revisited:fluorescence microscopes for field diagnostics. Trends Parasitol. 23:468–469. http://dx.doi.org/10.1016/j.pt.2007.08.003.5. Minion J, Sohn H, Pai M. 2009. Light-emitting diode technologies for TBdiagnosis: what is on the market? Expert. Rev. Med. Devices 6:341–345.http://dx.doi.org/10.1586/erd.09.26.6. Keiser J, Utzinger J, Premji Z, Yamagata Y, Singer BH. 2002. Acridineorange for malaria diagnosis: its diagnostic performance, its promotionand implementation in Tanzania, and the implications for malaria con-trol. Ann. Trop. Med. Parasitol. 96:643–654. http://dx.doi.org/10.1179/000349802125001834.7. Steingart KR, Henry M, Ng V, Hopewell PC, Ramsay A, Cunning-ham J, Urbanczik R, Perkins M, Aziz MA, Pai M. 2006. Fluorescenceversus conventional sputum smear microscopy for tuberculosis: a sys-tematic review. Lancet Infect. Dis. 6:570–581. http://dx.doi.org/10.1016/S1473-3099(06)70578-3.8. Bailey JW, Smith DH. 1992. The use of the acridine orange QBC tech-nique in the diagnosis of African trypanosomiasis. Trans. R. Soc. Trop.Med. Hyg. 86:630. http://dx.doi.org/10.1016/0035-9203(92)90160-E.9. Bieler S, Matovu E, Mitashi P, Ssewannyana E, Bi Shamamba SK,Bessell PR, Ndung’u JM. 2012. Improved detection of Trypanosomabrucei by lysis of red blood cells, concentration and LED fluorescencemicroscopy. Acta Trop. 121:135–140. http://dx.doi.org/10.1016/j.actatropica.2011.10.016.10. Paine MF, Wang MZ, Generaux CN, Boykin DW, Wilson WD, deKoning HP, Olson CA, Pohlig G, Burri C, Brun R, Murilla GA, ThuitaJK, Barrett MP, Tidwell RR. 2010. Diamidines for human Africantrypanosomiasis. Curr. Opin. Investig. Drugs 11:876–883.11. Wilson WD, Nguyen B, Tanious FA, Mathis A, Hall JE, Stephens CE,Boykin DW. 2005. Dications that target the DNA minor groove: com-pound design and preparation, DNA interactions, cellular distributionand biological activity. Curr. Med. Chem. Anticancer Agents 5:389–408.http://dx.doi.org/10.2174/1568011054222319.12. Carter NS, Berger BJ, Fairlamb AH. 1995. Uptake of diamidine drugs bythe P2 nucleoside transporter in melarsen-sensitive and -resistantTrypanosoma brucei brucei. J. Biol. Chem. 270:28153–28157. http://dx.doi.org/10.1074/jbc.270.47.28153.13. de Koning HP, Anderson LF, Stewart M, Burchmore RJ, Wallace LJ,Barrett MP. 2004. The trypanocide diminazene aceturate is accumu-lated predominantly through the TbAT1 purine transporter: addi-tional insights on diamidine resistance in African trypanosomes. An-timicrob. Agents Chemother. 48:1515–1519. http://dx.doi.org/10.1128/AAC.48.5.1515-1519.2004.14. Lanteri CA, Stewart ML, Brock JM, Alibu VP, Meshnick SR, TidwellRR, Barrett MP. 2006. Roles for the Trypanosoma brucei P2 transporter inDB75 uptake and resistance. Mol. Pharmacol. 70:1585–1592. http://dx.doi.org/10.1124/mol.106.024653.15. Mathis AM, Bridges AS, Ismail MA, Kumar A, Francesconi I, An-bazhagan M, Hu Q, Tanious FA, Wenzler T, Saulter J, Wilson WD,Brun R, Boykin DW, Tidwell RR, Hall JE. 2007. Diphenyl furans andaza analogs: effects of structural modification on in vitro activity, DNAbinding, and accumulation and distribution in trypanosomes. Antimi-crob. Agents Chemother. 51:2801–2810. http://dx.doi.org/10.1128/AAC.00005-07.16. Mathis AM, Holman JL, Sturk LM, Ismail MA, Boykin DW, TidwellRR, Hall JE. 2006. Accumulation and intracellular distribution of antit-rypanosomal diamidine compounds DB75 and DB820 in African try-panosomes. Antimicrob. Agents Chemother. 50:2185–2191. http://dx.doi.org/10.1128/AAC.00192-06.17. Stewart ML, Krishna S, Burchmore RJ, Brun R, de Koning HP, BoykinDW, Tidwell RR, Hall JE, Barrett MP. 2005. Detection of arsenical drugresistance in Trypanosoma brucei with a simple fluorescence test. Lancet366:486–487. http://dx.doi.org/10.1016/S0140-6736(05)66793-1.18. Vincent IM, Creek D, Watson DG, Kamleh MA, Woods DJ, Wong PE,Burchmore RJ, Barrett MP. 2010. A molecular mechanism for eflorni-thine resistance in African trypanosomes. PLoS Pathog. 6:e1001204. http://dx.doi.org/10.1371/journal.ppat.1001204.19. Matovu E, Stewart ML, Geiser F, Brun R, Maser P, Wallace LJ, Burch-more RJ, Enyaru JC, Barrett MP, Kaminsky R, Seebeck T, de KoningHP. 2003. Mechanisms of arsenical and diamidine uptake and resistancein Trypanosoma brucei. Eukaryot. Cell 2:1003–1008. http://dx.doi.org/10.1128/EC.2.5.1003-1008.2003.20. Baker N, Glover L, Munday JC, Aguinaga AD, Barrett MP, de KoningHP, Horn D. 2012. Aquaglyceroporin 2 controls susceptibility to melar-soprol and pentamidine in African trypanosomes. Proc. Natl. Acad. Sci.U. S. A. 109:10996–11001. http://dx.doi.org/10.1073/pnas.1202885109.21. Bridges DJ, Gould MK, Nerima B, Maser P, Burchmore RJ, de KoningHP. 2007. Loss of the high-affinity pentamidine transporter is responsiblefor high levels of cross-resistance between arsenical and diamidine drugsin African trypanosomes. Mol. Pharmacol. 71:1098–1108. http://dx.doi.org/10.1124/mol.106.031351.Giordani et al.1796 aac.asm.org Antimicrobial Agents and Chemotherapy on April 2, 2014 by Glasgow University Libraryhttp://aac.asm.org/Downloaded from 

Green fluorescent diamidines as diagnostic probes for trypanosomes

Enlighten: Publications

  
 
 
 
Giordani, Federica, Munde, Manoj, Wilson, W. David, Ismail, Mohamed 
A., Kumar, Arvind, Boykin, David W., and Barrett, Michael P. (2014) 
Green fluorescent diamidines as diagnostic probes for trypanosomes. 
Antimicrobial Agents and Chemotherapy, 58 (3). pp. 1793-1796. ISSN 
0066-4804 
 
 
Copyright © 2014 The Authors 
 
 
 
http://eprints.gla.ac.uk/90388 
 
 
 
Deposited on:  02 April 2014 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
  Published Ahead of Print 23 December 2013. 
10.1128/AAC.02024-13. 
2014, 58(3):1793. DOI:Antimicrob. Agents Chemother. 
Michael P. Barrett
Mohamed A. Ismail, Arvind Kumar, David W. Boykin and 
Federica Giordani, Manoj Munde, W. David Wilson,
 
Diagnostic Probes for Trypanosomes
Green Fluorescent Diamidines as
http://aac.asm.org/content/58/3/1793
Updated information and services can be found at: 
These include:
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 Supplemental material
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Green Fluorescent Diamidines as Diagnostic Probes for Trypanosomes
Federica Giordani,c Manoj Munde,a W. David Wilson,a Mohamed A. Ismail,a,b Arvind Kumar,a David W. Boykin,a Michael P. Barrettc
Department of Chemistry, Georgia State University, Atlanta, Georgia, USAa; Department of Chemistry, College of Science, King Faisal University, Hofuf, Saudi Arabiab;
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of
Glasgow, Glasgow, United Kingdomc
Light-emitting diode (LED) fluorescence microscopy offers potential benefits in the diagnosis of human African trypanosomiasis
and in other aspects of diseases management, such as detection of drug-resistant strains. To advance such approaches, reliable
and specific fluorescent markers to stain parasites in human fluids are needed. Here we describe a series of novel green fluores-
cent diamidines and their suitability as probes with which to stain trypanosomes.
Early diagnosis of human African trypanosomiasis (HAT) (1) isparamount for favorable prognosis, but it is difficult to achieve
in the field (2). Conventional light microscopy, in particular, is
insensitive and time-consuming. Deployment of better diagnos-
tics, together with more effective drugs, will be essential to elimi-
nate the disease as a public health problem.
The development of portable and efficient light-emitting diode
(LED)-illuminated microscopes (3–5) allows fluorescence mi-
croscopy to offer improvements in detection. Its use in the diag-
nosis of other tropical diseases has already been shown to increase
the sensitivity and rapidity of sample preparation and screening
(6, 7). A similar approach would also be useful in diagnosis of
animal trypanosomiasis, which suffers limitations similar to those
of diagnosis of HAT. The diagnostic fluorescence tests developed
for HAT (8, 9) use acridine orange to stain trypanosomes. This is
a cheap but aspecific fluorophore, which can label the nucleic
acids of any cell in the specimen, and as a DNA intercalator, it
poses potential risks to laboratory technicians (although, based on
available data, the IARC inserts it in group 3, as not classifiable as
to its carcinogenicity for humans).
The aim of this work was to synthesize and evaluate a series of
fluorescent diamidines as targeted diagnostic probes for trypano-
somes. Diamidines (10, 11) appeared promising since they are
rapidly internalized by Trypanosoma brucei via specific surface
transporters, including the aminopurine P2/TbAT1 transporter
(12–14), and several of them are fluorescent (11, 15, 16). The UV
emission of DB99 was previously exploited in our laboratory to
develop a simple assay for detection of drug-resistant strains lack-
ing P2 (17). Unfortunately, although LED fluorescence micro-
scopes are being increasingly used, UV LED-illuminated instru-
ments are not yet widely available, most fluorescence biological
work being carried out at longer wavelengths.
(Biological aspects of this work were conducted by F. Giordani
in partial fulfillment of the requirements for a Ph.D. from the
University of Glasgow, Glasgow, Scotland, 2011.)
By addition of a series of ultraconjugated ring structures in the
linker between the two amidine groups (containing the recogni-
tion motif for the parasite’s P2 transporter), we generated a series
of new diamidines with longer emission wavelengths than the pa-
rental UV-emitting DB75 (chemistry data are presented in the
supplemental material). Spectral measurements confirmed that
all novel dications emitted in the green wavelength range (Table
1). As expected for diamidines (11), the newmolecules had a high
affinity for DNA. Values of DNA binding by thermal melting
(Tm, measured as described in reference 15) revealed the binding
affinity to be greater than that of pentamidine (12.6°C) but lower
than that of DB75 (25°C) (Table 1).
The dependence of the compounds on P2 transport for uptake
into trypanosomes was confirmed by an alamarBlue assay (18)
performed on threeTrypanosoma brucei brucei strains: a wild-type
line (bloodstream Lister 427), a line not expressing P2 (tbat1/)
(19), and a derived clone, in which high-affinity pentamidine
transporter (HAPT1, now identified as an aquaglyceroporin [20])
activity is also lost (B48) (21). Compound DB1692 showed the
greatest dependency on P2 for transport (50% inhibitory concen-
tration [IC50] 25-fold higher for the tbat1
/ line than for thewild
type), followed by DB1645 (16-fold higher), DB1465 (10-fold),
and DB1464 (8-fold) (Table 2). Data using the B48 line showed
that HAPT1 activity also contributed to the accumulation of
DB1645 and DB1465. In contrast, the addition of a single furan
ring in the linker of DB1680 highly affected the rate of uptake of
this compound through the P2 transporter, and further loss of
HAPT1 did not affect its toxicity, suggesting other routes of inter-
nalization (e.g., other carriers or endocytosis). Equally notewor-
thy is the potent in vitro activity shown by these new compounds
against the wild-type line, especially by DB1464, which had an
IC50 close to that of pentamidine. This indicates that these mole-
culesmay be attractive candidates to pursue as possible leads against
HAT, provided that ADME (absorption, distribution, metabolism,
and excretion) characterizationdemonstrates goodpharmacokinetic
properties and rules out potential toxicity issues previously observed
for somemolecules belonging to this class.
All five diamidines stained live T. b. brucei in infected rat blood
films using a standard fluorescein isothiocyanate (FITC) filter set
(excitation, band-pass, 450 to 490 nm; beam splitter, 510 nm; and
emission, band-pass, 515 to 565 nm) and a Zeiss Axioplan fluo-
Received 16 September 2013 Returned for modification 6 December 2013
Accepted 17 December 2013
Published ahead of print 23 December 2013
Address correspondence to Michael P. Barrett, michael.barrett@glasgow.ac.uk.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.02024-13.
Copyright © 2014 Giordani et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution 3.0 Unported license.
doi:10.1128/AAC.02024-13
March 2014 Volume 58 Number 3 Antimicrobial Agents and Chemotherapy p. 1793–1796 aac.asm.org 1793
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n
 April 2, 2014 by G
lasgow University Library
http://aac.asm
.org/
D
ow
nloaded from
 
rescence microscope (Fig. 1A). Under our experimental condi-
tions (ex vivo incubation for 15 min at 37°C, with 50 M [each]
fluorophore), all five diamidines provided specific staining of try-
panosomes without labeling erythrocytes or white blood cells.
Staining of the parasite nucleus was not observed, but all com-
pounds selectively accumulated inside the kinetoplast (the para-
site’smitochondrial genome, at the posterior of the cell) and other
cytoplasmic organelles, spread throughout the cell body and also
visualized at longer wavelengths (Fig. 1B). These were likely to be
acidocalcisomes, previously observed to be stained by other di-
amidines (15). For DB1692, a prominent red fluorescent spot,
putatively identified as the lysosome, also appeared in a region
between the nucleus and the kinetoplast after 1 h of treatment.
Under the FITC filter, parasites were readily detectable among
blood cells based on their movement and fluorescent dots (espe-
cially the bright kinetoplast). Cell labeling was achieved only by
incubating at room temperature or 37°C but not at 4°C. This
temperature sensitivity could lead to variability in field tests using
ambient temperature in different sites; hence, the use of temper-
ature-controlled devices (e.g., water baths or heating blocks)
might be required in such settings. Although the fluorescence in-
tensity increased with the time of incubation for all probes, the
overall yields remained below those identified with DB75 and
could not be improved by increasing the fluorophore concentra-
tion. The low emission intensity of the new compounds could be
TABLE 1 Structures and physical properties of the fluorescent diamidines
Compound Structure
EX
(nm)a
EM
(nm)b
Fluorescence intensity
at maxc
Tm values
d with
poly(dA) · poly(dT)
DB1464 405 542 73 9.4
DB1465 417 513 123 18.5
DB1645 424 576 36 14.6
DB1680 418 546 8 15.0
DB1692 400 546 40 17.0
a Excitation wavelength.
b Emission wavelength.
c max, maximum.
d Melting temperature of the compound-DNA complexes as compared to values for free DNA. Tm values are in degrees Celsius and have an error of0.5°C.
TABLE 2 In vitro trypanocidal activities of the five diamidines against T.
b. brucei S427 wild-type and derived cell linesa
Compound
IC50 [nM] ( SEM) for:
RFb
IC50 [nM] ( SEM)
for B48 RFWild type
tbat1/
line
DB1464 0.9 0.3 6.9 2.1 8 7.3 0.7 8
DB1465 6.9 1.9 71.4 4.4 10 334 11 48
DB1645 14.5 2.8 233 43 16 610 60 42
DB1680 266 21 591 43 2 341 11 1
DB1692 17.6 4.4 436 41 25 344 14 20
Pentamidine 0.6 0.3 0.9 0.2 1.5 167 10 278
a n 3.
b RF, resistance factor (ratio of the IC50 measured against the tbat1
/ and B48 cell
lines to its value obtained for the wild type).
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due to their intrinsically low fluorescence yields (Table 1) but also
to the discrepancy between their excitation maxima, all close to
400 nm, and the standard FITC filter wavelength utilized for mi-
croscopy. Hence, the use of these specific fluorophores with these
particular filters may not be directly applicable to HAT diagnosis
due to the risk of false negatives. However, these same compounds
with customized filters or further derivatives with higher fluores-
cence yields could be considered as probes for the specific staining
of trypanosomes using existing LED-based fluorescence micro-
scopes.
Despite the compounds’ partial dependence on the P2 trans-
porter for uptake, they did not distinguish wild-type and tbat1/
knockout line by fluorescencemicroscopy (50Mfor incubations
ranging from 15 min to up to 2 h), indicating that uptake routes
independent of P2 play important roles in their internalization
when used under these conditions.
Implementation of fluorescence tests for trypanosome detec-
tion in human fluids must rely on affordable, efficient and prefer-
ably specificmarkers. This work shows the feasibility of generating
cheap fluorophores, emitting at differential wavelengths, by sim-
ple addition of cyclic groups within the chemical structure of di-
amidines, the precursor of which, DB75, is fluorescent only in the
UV region. The exploitation of surface transporters peculiar to
trypanosomes is a straightforward way to specifically target fluo-
rophores to these parasites, and improved compounds may be
developed following this approach to target both human and an-
imal infective trypanosomes.
ACKNOWLEDGMENTS
Thisworkwas supported by an award from theBill andMelindaGates Foun-
dation through theConsortiumforParasiticDrugDevelopment (CPDD) (to
M.P.B., W.D.W., and D.W.B.), an NIH grant (AI064200) (to W.D.W. and
FIG 1 (A) Fluorescence images of infected rat blood incubated ex vivo with the five green fluorescent diamidines (note that the parasite position can change
between the bright-field and corresponding fluorescence micrographs), 40 objective. (B) In vitro trypanosomes treated with DB1680 and DB1692 (50 M, 1
h, 37°C), 100 objective; 4=,6-diamidino-2-phenylindole (DAPI) (20M)was used as aDNA counterstain. Asterisk, kinetoplast; arrow, cytoplasmic corpuscles,
possibly acidocalcisomes; arrowhead, nucleus; hash mark, possibly lysosome. Bar, 10 m.
Fluorescent Diamidines as Probes for Trypanosomes
March 2014 Volume 58 Number 3 aac.asm.org 1795
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D.W.B.), the SirHalley StewartTrust (toM.P.B. andF.G.), and theWellcome
Trust.TheWellcomeTrustCentre forMolecularParasitology is supportedby
core funding from theWellcome Trust (085349).
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Green fluorescent diamidines as diagnostic probes for trypanosomes

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