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Delivery of Antimicrobials Into Parasites

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Delivery of Antimicrobials Into Parasites Delivery of antimicrobials into parasites B. U. Samuel*†, B. Hearn†‡, D. Mack*†, P. Wender‡§¶, J. Rothbard‡§, M. J. Kirisits*, E. Mui*, S. Wernimont*, C. W. Robertsʈ, S. P. Muench**, D. W. Rice**, S. T. Prigge††,A.B.Law††, and R. McLeod*¶ *Department of Visual Sciences, University of Chicago, 5841 South Maryland, AMB S-208, Chicago, IL 60637; ‡Department of Chemistry, Stanford University, Stanford, CA 94305; §Cell Gate Inc., 552 Del Rey Avenue, Sunnyvale, CA 94086; ʈDepartment of Immunology, University of Strathclyde, G4 ONR Glasgow, Scotland; **Department of Molecular Biology, Sheffield University, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom; and ††Malaria Research Institute, The Johns Hopkins University School of Public Health, Baltimore, MD 21205 Contributed by P. Wender, September 25, 2003 To eliminate apicomplexan parasites, inhibitory compounds must tional single polypeptide (6–10). In apicomplexans, type II fatty cross host cell, parasitophorous vacuole, and parasite membranes acid synthesis enzymes are in a plastid organelle (6, 8). and cyst walls, making delivery challenging. Here, we show that Some monofunctional type II enzymes had been identified in short oligomers of arginine enter Toxoplasma gondii tachyzoites Plasmodia and T. gondii (6–10), but T. gondii ENR (TgENR) had and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP not been identified. The antimicrobial agent 5-chloro-2-[2,4- reductase (ENR), conjugated to arginine oligomers enters extracel- dichlorophenoxy]phenol (triclosan) had been found to inhibit lular tachyzoites, host cells, tachyzoites inside parasitophorous bacterial ENRs (11). We (6) and Surolia and Surolia (9) had vacuoles within host cells, extracellular bradyzoites, and brady- found that ␮M concentrations of triclosan dissolved in DMSO zoites within cysts. We identify, clone, and sequence T. gondii enr inhibit replication of apicomplexan parasites, presumably by and produce and characterize enzymatically active, recombinant binding to, and inhibiting, apicomplexan ENR. Triclosan was ENR. This enzyme has the requisite amino acids to bind triclosan. active only when solubilized in DMSO (6, 9). Structures of Triclosan released after conjugation to octaarginine via a readily triclosan, oligomers, and conjugates of oligoarginines are shown hydrolyzable ester linkage inhibits ENR activity, tachyzoites in in Fig. 1 (see also Fig. 5). vitro, and tachyzoites in mice. Delivery of an inhibitor to a micro- Materials and Methods organism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to seques- Short Arginine Oligomers, Conjugates, and Controls. Peptides were tered parasites, a model system to characterize transport across synthesized by using solid-phase techniques and commercially MEDICAL SCIENCES multiple membrane barriers and structures, a widely applicable available fluorenylmethoxycarbonyl amino acids, resins, and paradigm for treatment of active and encysted apicomplexan and reagents with an Applied Biosystems 433 peptide synthesizer. other infections, and a generic proof of principle for a mechanism Fastmoc cycles were used with O-(7-azabenzotriazol-1-yl)- of medicine delivery. 1,1,3,3-tetramethyluronium hexafluorophosphate (HATU). Peptides and conjugates were cleaved from resin by using 95% trifluoroacetic acid and 5% triisopropylsilane for 24 h. Peptides he vast majority of interesting small molecules will never be were subsequently filtered from resin, precipitated with diethyl Tused for therapeutics because they cannot traverse biologic ether, purified by using HPLC reverse-phase columns, and barriers to reach their targets. A special challenge for the characterized by using 1H NMR and electrospray MS. development of antimicrobials effective against apicomplexan For synthesis of triclosan-Orn(FITC)-Argn-CONH2, 4 and 5, parasites is delivery. Effective agents have to gain entry to host triclosan was reacted with ␣-bromoacetic acid to provide the cells, cross the parasitophorous vacuole, and enter parasites and desired ␣-substituted acetic acid product. Attachment of the their specialized organelles. This is potentially more difficult resin-bound carrier was accomplished by reaction of this acetic with Toxoplasma gondii bradyzoites, which reside in cysts com- acid product with the appropriate oligomer of L-argi- posed partly of host and partly of parasite constituents. The nine(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Pbf) studies described herein were performed to determine whether containing an N-terminal L-ornithine(methyltrityl). After cleav- short oligomers of arginine (Fig. 1A) could provide a solution to age of the side-chain methyltrityl protecting group with 1% these challenges. Arginine oligomers linked to immunomodula- trifluoroacetic acid in dichloromethane, the conjugates were tory and antitumor compounds can actively transport such labeled with FITC in the presence of diisopropylethylamine compounds across a variety of epithelial barriers, including skin, (DIPEA). The conjugates were then deprotected and cleaved lung, and eye (1–4). Oligoarginine-medicine conjugates have from the resin by using the previously described protocol. For the been administered safely to experimental animals and humans synthesis of triclosan-Orn(tetramethylrhodamine isothiocya- (ref. 1 and unpublished data). nate)-Arg8-CONH2, ent-6, tetramethylrhodamine isothiocya- Better approaches for treating apicomplexan parasite infec- nate was reacted in the solution phase with triclosan-D-Orn-D- tions (e.g., toxoplasmosis and malaria) are needed. For example, Arg8-CONH2 in the presence of DIPEA. Triclosan was reacted T. gondii bradyzoites infect 30–50% of people throughout the with glutaric anhydride (GA) in the presence of DIPEA to world, but no currently used medicines eradicate this slowly provide the desired glutaric acid product, i.e., triclosan-GA- growing, encysted, latent parasite that causes chronic, lifelong Argn-CONH2, 7 and 8. Attachment of the carrier was accom- infections in brain and eye with recrudescence causing severe plished by the solution-phase reaction with the appropriate illness (5). Available antimicrobial agents to eliminate rapidly oligomer of D-orL-arginine. For the synthesis of triclosan-acetic growing T. gondii tachyzoites, which cause tissue destruction during active infection, are limited by toxicity and allergy (5). Abbreviations: ENR, enoyl-ACP reductase; TgENR, Toxoplasma gondii ENR; Pbf, 2,2,4,6,7- Enoyl-ACP reductase (ENR) is a key enzyme active in fatty pentamethyldihydrobenzofuran-5-sulfonyl. acid synthesis (6). Bacterial and apicomplexan fatty acid synthe- Data deposition: The sequence reported in this paper has been deposited in the GenBank sis occurs by the type II pathway (6–10) (Fig. 5, which is database (accession no. AY372520). published as supporting information on the PNAS web site) and †B.U.S., B.H., and D.M. contributed equally to this work. depends on monofunctional polypeptides, including ENR. In ¶To whom correspondence should be addressed. E-mail: [email protected] or contrast, mammalian fatty acid synthesis occurs by the type I [email protected]. pathway in which the key enzymes are present on a polyfunc- © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2436169100 PNAS ͉ November 25, 2003 ͉ vol. 100 ͉ no. 24 ͉ 14281–14286 Downloaded by guest on September 26, 2021 Fig. 1. Schematic representations of oligomers and conjugates, uptake of short oligomers of arginine and lysine by T. gondii tachyzoites, encysted bradyzoites, and effect of azide on uptake of arginine triclosan conjugates by parasites. (A) Structures of short oligomers of arginine and conjugates used in these studies. Releasable conjugates use readily hydrolyzable ester linkages for the attachment of triclosan to the oligoarginine carrier (structures 7 and 8). Nonreleasable triclosan conjugates use nonhydrolyzable ether bonds for the attachment of triclosan (structures 4, 5, 6, and 9). Control compounds 10 and 11 are the hydrolysis products of releasable conjugates 7 and 8, respectively. Ent designates the dextro isomer. Synthetic details are in Materials and Methods.(B) Comparative flow cytometry to measure labeling of T. gondii tachyzoites incubated with FITC-conjugated polyamino acid compounds. Numbers in parentheses indicate percentage of positive cells. (Inset) Fluorescence and differential interference contrast (DIC) microscopy assays of uptake of r7 FITC, compound 1,byT. gondii tachyzoites. (C) Fluorescence images showing uptake of FITC-conjugated polyamino acid compounds by T. gondii tissue cysts. (Scale bar ϭ 10 ␮m.) (D–F) Fluorescein and DIC microscopy images showing inhibition of uptake of arginine triclosan conjugates by azide in T. gondii.(D) Tachyzoites. (E) Excysted bradyzoites. (F) Uptake of r8 triclosan (compound 7) and less inhibition in isolated brain cysts. The arginine carrier was conjugated to fluorophore and triclosan in a nonreleasable manner (4 and ent-6). (Scale bar ϭ 10 ␮m.) ␣ acid-Arg8-CONH2, 9, the previously described -substituted Expression and Purification of Recombinant TgENR. A construct of acetic acid product was attached to the carrier by reaction with TgENR containing residues 103–417 (and lacking the putative the resin-bound octa-L-arginine(Pbf). The conjugate was subse- signal and transit peptides) was designed for in vivo cleavage by quently cleaved from the resin, and the Pbf sulfonamides were tobacco
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