Newly identified antibacterial leads are topoisomerase poisons in African trypanosomes

30 Pages·2009·1.57 MB·English

AAC Accepts, published online ahead of print on 14 December 2009
Antimicrob. Agents Chemother. doi:10.1128/AAC.01025-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Newly identified antibacterial leads are topoisomerase
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poisons in African trypanosomes
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3 Running Title: Anti-trypanosomal topoisomerase poisons
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5 Sonya C. Tang, Theresa A. Shapiro* n
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6 Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University d
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7 School of Medicine, Baltimore, MD 21205, USA fr
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9 *Corresponding Author :
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10 Theresa A. Shapiro c
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11 Johns Hopkins University School of Medicine m
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12 301 Hunterian Building, 725 North Wolfe Street, g
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13 Baltimore, MD 21205 USA

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14 Tel.: [phone]
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16 e-mail: [email protected]
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Version of 11Nov09
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17 Abstract
18 Human African trypanosomiasis, caused by the Trypanosoma brucei protozoan parasite, is fatal
19 when left untreated. Current therapies are antiquated and there is a need for new pharmacologic
20 agents against T. brucei targets that have no human ortholog. Trypanosomes have a single
21 mitochondrion with a unique mitochondrial DNA, known as kinetoplast DNA (kDNA), a D
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22 topologically complex network that contains thousands of interlocking circular DNAs, termed n
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23 minicircles (~1 kb) and maxicircles (~23 kb). Replication of kDNA depends on topoisomerases, d
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24 enzymes that catalyze reactions that change DNA topology. T. brucei has an unusual type IA f
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25 topoisomerase that is dedicated to kDNA metabolism. This enzyme has no ortholog in humans

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26 and RNAi studies have shown that it is essential for parasite survival, making it an ideal drug p
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27 target. In a large chemical library screen, two compounds were recently identified as poisons of a
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28 bacterial topoisomerase IA. We found that these compounds are trypanocidal in the low m
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29 micromolar range, and that they promote the formation of linearized minicircles covalently r
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30 bound to protein on the 5’-end, consistent with the poisoning of mitochondrial topoisomerase IA. n

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31 Surprisingly, however, band depletion studies showed that it is topoisomerase II , and not n
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32 topoisomerase IAmt, that is trapped. Both compounds are planar aromatic polycyclic structures ry

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33 that intercalate into and unwind DNA. These findings reinforce the utility of topoisomerase II 2
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34 as a target for development of new drugs for African sleeping sickness. 9

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35 Introduction
36 Human African trypanosomiasis, also known as African sleeping sickness, is a protozoal
37 infection caused by Trypanosoma brucei that is fatal without treatment (3). Parasites are
38 transmitted to humans via the tsetse fly, which is found exclusively in sub-Saharan Africa and
39 thus limits the geographic distribution of the disease. Currently, there is an estimated 50,000 to D
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40 70,000 cases (25). Despite the lethality of sleeping sickness, the drugs currently available for its n
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41 treatment are toxic and they require parenteral administration (10). In addition to these d
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42 obstacles, resistance has been a growing concern, and availability and cost are major problems f
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43 for the population affected. In light of these issues, new drugs for African trypanosomiasis are in

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44 demand. The recently completed T. brucei genome sequence has made possible the p
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45 identification of potential drug targets which are essential to trypanosomes and have no human a
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46 ortholog (2). m
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47 r
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48 Trypanosomes have a single mitochondrion with an unusual mitochondrial DNA, known n

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49 as kinetoplast DNA (kDNA). The kinetoplast is a massive and topologically complex structure, n
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50 comprised of thousands of interlocked ~1 kb DNA minicircles and a few dozen ~23 kb ry

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51 maxicircles (structure and replication of kDNA reviewed in Ref. 16). This intricate network is in 2
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52 striking contrast with the monomeric circular DNA molecule found in several copies in each 9

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53 human mitochondrion. Replication of kDNA requires the release of individual covalently closed g
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54 minicircles from the network into a pool of “free” minicircles, DNA synthesis via theta structure s
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55 intermediates, and reattachment to the network of progeny minicircles, which retain nicks or
56 gaps until all circles have been replicated. Every step of kDNA replication depends on the
57 function of topoisomerases. These enzymes catalyze reactions that alter the topological state of
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58 DNA by cleaving the phosphodiester backbone through the formation of a covalent
59 phosphotyrosine link, transferring segments of DNA through the break, and then ligating the
60 DNA together (topoisomerases reviewed in Refs. 7, 9, 28). They are classified into two types,
61 based on the number of DNA strands cleaved: type I enzymes cleave one strand while type II
62 enzymes cleave both strands in reactions requiring ATP. Type I enzymes are further classified D
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63 into A and B subfamilies based on structural and mechanistic differences. Topoisomerase IA n
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64 enzymes catalyze the relaxation of negatively supercoiled DNA through an “enzyme bridging d
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65 mechanism”. After cleavage of a single strand, topoisomerase IA remains covalently bound to f
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66 the 5’-end as it processively unwinds the DNA. If the enzyme acts across from a pre-existing

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67 nick, it can pass a double helix through the resulting double stranded break. The type II enzymes p
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68 also utilize a 5’-end linkage during catalysis, in contrast to topoisomerases IB, which create a a
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69 covalent link at the 3’-end of the cleavage site. m
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70 r
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71 T. brucei has at least six catalytically active topoisomerases and two of these appear to n

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72 function exclusively in the mitochondrion: topoisomerase IA and topoisomerase II (15, 21). n
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73 These mitochondrial enzymes are especially attractive drug targets since they are essential ry

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74 according to RNAi and drug studies (21), and their depletion leads to akinetoplasty (the loss of 2
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75 kDNA), which in turn results in death (13). 9

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76 g
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77 Compounds that target topoisomerases can be classified as poisons or non-poisoning s
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78 inhibitors (17). Poisons bind to the Michaelis complex comprised of DNA and enzyme, forming
79 a ternary DNA-protein-inhibitor “cleavable complex”. In situ, the presence of a poison prevents
80 strand ligation and thus the topoisomerase remains in an inert complex that is fixed on the DNA
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81 helix. Covalent adducts of topoisomerase and DNA can be forged by rapid denaturation of the
82 topoisomerase with detergents such as sodium dodecyl sulfate (SDS). Non-specific inhibitors
83 also decrease topoisomerase activity, but do not promote the formation of cleavable complexes.
84 They may include agents that deform the DNA substrate, bind proteins with high affinity, or
85 compete for binding at active sites, such as DNA and ATP analogs. In the cell, stationary D
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86 cleavable complexes prove lethal when replication machinery collides with them, producing n
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87 breaks in the DNA (11, 17). Therapeutically useful topoisomerase-targeting agents are all d
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88 poisons: fluoroquinolones and etoposide are topoisomerase II poisons for antibacterial and anti- f
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89 tumor therapy, respectively, while camptothecin is an anti-tumor agent that poisons

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90 topoisomerase IB. For T. brucei, poisons against topoisomerase IA would be ideal since this p
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91 enzyme has no human ortholog and is exclusively found in the mitochondrion with kDNA (21). a
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92 m
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93 Recently, a luciferase reporter system that detects the accumulation of covalent DNA r
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94 complexes with recombinant Yersinia pestis topoisomerase IA in Escherichia coli was developed n

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95 (8). The system was used in a high throughput assay to screen for compounds that poisoned Y. n
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96 pestis topoisomerase IA. Three compounds were found to promote intracellular cleavable ry

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97 complex formation by the recombinant topoisomerase IA and to inhibit relaxation of DNA by the 2
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98 isolated enzyme. These compounds also inhibited the growth of bacterial cells. 9

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99 g
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100 Lead Compounds 1 and 2 identified in the Y. pestis model (Fig. 1), hereafter referred to s
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101 as #1895 and #0020, respectively, were tested against T. brucei to assess cytotoxic activity and
102 ability to poison topoisomerase IA . These compounds were found to be trypanocidal at
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103 micromolar concentrations, they appear to intercalate into DNA, and, unexpectedly, they
104 promote linearization of free minicircles by poisoning intracellular topoisomerase II .
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106 Materials and Methods
107 All experiments were performed with bloodstream form Trypanosoma brucei brucei (MiTat 1.2 D
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108 strain 427; doubling time 6-8 h), containing a c-Myc-tagged topoisomerase IA allele (21), grown n
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109 at 37 °C in HMI-9 containing 10% fetal bovine serum, 10% Serum Plus and 0.8 µg/mL G418 d
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110 (Gibco BRL Life Technologies, Inc.). Compounds #1895 and #0020 were purchased from f
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111 ChemDiv (San Diego, CA; catalog 4333-1895 and 4711-0020, respectively), dissolved in
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112 DMSO, aliquoted, and stored at -20 °C. :
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113 c
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114 Cytotoxicity assay. Compounds #1895 and #0020 were assayed for anti-trypanosomal activity m
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115 using an acid phosphatase-based 96-well plate method (4). Briefly, 100 µL of cells (2 x 105/mL) rg
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116 were incubated with 100 µL of medium containing solvent or 2x serially diluted compound (20-
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117 24 h, 37 °C). Each concentration was assayed in quadruplicate. Final DMSO concentrations did u
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118 not exceed 1%. Lethality was confirmed by microscopic examination for motility. Acid
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119 phosphatase activity from surviving cells was measured by adding 20 µL of buffer containing 20 0
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120 mg/mL p-nitrophenyl phosphate in 1M sodium acetate pH 5.5, 1% Triton X-100 (5 h, 37 °C). b
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121 Absorbance was measured at 405 nm. Each compound was assayed three times. Curve fitting
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122 and EC determinations were obtained with the E model (12) and DeltaGraph Pro 3.5. Based
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123 on Chauvenet’s criterion (26), values from three (of 576 total) wells were identified as outliers
124 and were dropped from analysis.
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126 Free minicircle profiles. For each concentration, 12 µL of 10x compound was added to 1.2 x
127 107 cells in 108 µL of medium (30 min, 37 °C). Just before lysis, a portion was taken for
128 western blot analysis (below). The remaining cells were lysed with an equal volume of 10 mM
129 Tris HCl pH 8.0, 75 mM Na EDTA, 1% SDS, with or without 2 mg/mL Proteinase K
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130 (2 h, 55 °C), then treated with 100 µg/mL RNase A (30 min, 37 °C). Samples were separated by D
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131 electrophoresis in 1.5% agarose gels containing 89 mM Tris base, 89 mM boric acid, 2 mM n
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132 EDTA with 1 µg/mL ethidium bromide (35 V, 24 h). DNA was transferred to Hybond XL
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133 membrane (GE Healthcare); washed in 750 mM NaCl and 75 mM sodium citrate; UV r
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134 crosslinked (Stratagene UV Crosslinker 1800) to the membranes; and hybridized in Church- h
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135 Gilbert Solution with a 32P-labeled probe that recognizes the 124 bp conserved minicircle :/
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136 sequence (overnight, 45 °C) (21). Membranes were exposed to a PhosphoImager plate that was c
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137 scanned (Fujix BAS 1000 PhosphorImager) and visualized with Image Gauge version 3.45 (Fuji
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138 Photo Films Company). Unless indicated otherwise, Adobe Photoshop was used to adjust /

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139 contrast and brightness only.
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140 u
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141 Exonuclease treatment. Trypanosomes were treated with 25.5 µM #1895 or 63 µM #0020 (1 h, 4
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142 37 °C), then lysed and digested with Proteinase K and RNase A, as described above. DNA was 0
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143 extracted with buffer-saturated phenol, then with phenol/chloroform/isoamyl alcohol (25:24:1), b
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144 followed by back extraction and ethanol precipitation. Samples were redissolved in 100 µL of e
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145 10 mM Tris HCl pH 8.0, 1 mM Na EDTA; applied to QiaAmp DNA micro kit (Qiagen) for
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146 cleanup; eluted in 30 µL distilled water; and treated with 10 U λ exonuclease in buffer
147 containing 67 mM Glycine-KOH, pH 9.4, 2.5 mM MgCl and 50 µg/mL BSA or with 10 U
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148 exonuclease III in buffer containing 10 mM Bis-Tris-Propane-HCl, pH 7.0, 10 mM MgCl , 1
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149 mM dithiothreitol in a final volume of 40 µL (30 min, 37 °C). Hind III-linearized pBluescript
150 (80 ng) was added to each treatment as an internal control. Minicircle DNA was separated and
151 visualized as described above.
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153 Western blot band depletion. The 8 x 106 cells reserved above were lysed with an equal D
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154 volume of 117 mM Tris HCl pH 6.8, 3.4% SDS, 10% glycerol, 0.2 M dithiothreitol, 0.004% n
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155 bromophenol blue. Proteins were separated by SDS PAGE and transferred to polyvinyl d
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156 difluoride membranes (Millipore). Blots were blocked with 3% bovine serum albumin in 25 mM f
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157 Tris HCl pH 7.4, 150 mM NaCl, 0.05% Tween 20, and probed with the following primary and
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158 then secondary antibodies: anti-c-Myc (1:1000; Santa Cruz Biotechnologies)/anti-rabbit-HRP :
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159 (1:20,000; Santa Cruz Biotechnologies); anti-T. brucei-topoisomerase IImt (1:5000; Ref. 15)/anti- c
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160 guinea pig-HRP (1:40,000; Sigma). Loading control was provided by anti-human enolase m
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161 (1:200; Santa Cruz Biotechnologies)/anti-rabbit-HRP (1:30,000). Quantitation was done with rg
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162 Image J (1). n

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163 n
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164 Precipitation of topoisomerase-DNA complexes. The ability of compounds #1895 and #0020 ry

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165 to promote intracellular cleavable complex formation with nuclear DNA was determined by a 2
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166 KSDS assay (5, 27). Trypanosomes (107 cells per mL) in thymidine-free medium were 9
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167 radiolabeled with 333 µCi/mL [3H]thymidine (20 Ci/mmol; Perkin Elmer; 1 h, 37 °C), washed g
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168 three times, resuspended at 107 cells/mL and treated with DMSO, 500 µM etoposide, 50 µM t
169 #1895 or 50 µM #0020 (30 min, 37 °C). Samples were assayed in triplicate. An equal volume
170 of lysis buffer (1.25% SDS, 5 mM EDTA, 0.4 mg/mL calf thymus DNA) was added and lysates
171 were incubated (10 min, 65 °C). Covalent DNA-protein complexes were precipitated by the
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172 addition of ice-cold 65 mM KCl; washed three times with 10 mM Tris HCl, pH 8.0, 100 mM
173 KCl, 1 mM EDTA, 0.1 mg/mL calf thymus DNA; and radioactivity was counted by liquid
174 scintillation. Total incorporation of radioactivity by control cells was measured in triplicate by
175 aliquoting 5 x 105of untreated labeled cells onto glass filter paper and precipitating DNA with

176 5% ice-cold trichloroacetic acid. To calculate the percent of DNA trapped cleavable complexes, D
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177 the averaged counts per minute detected in KSDS precipitates was divided by the averaged n
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178 counts per minute in trichloroacetic acid precipitates. d
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179 f
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180 DNA unwinding by #1895 and #0020 was assayed as described previously (24). In brief,

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181 250 ng of supercoiled pBluescript or of pBluescript relaxed by Escherichia coli topoisomerase p
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182 IA was treated with various concentrations of #1895 and #0020 in 50 mM Tris HCl, pH 7.5, 50 a
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183 mM NaCl and 5 mM MgCl2, after which 1 pM vaccinia topoisomerase IB (gift from Dr. James m
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184 Stivers) was added (1 h, 37 °C). Reactions were quenched by addition of 10x DNA loading dye rg
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185 containing 0.625% SDS, 0.125% xylene cyanol, 0.125% bromophenol blue, 62.5% glycerol. n

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186 DNA was separated in 1% agarose in 89 mM Tris base, 89 mM boric acid, 2 mM EDTA and n
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187 visualized by ethidium bromide fluorescence. y

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188 2
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189 Results
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190 Trypanocidal activity in vitro. Increasing concentrations of #1895 or #0020 were tested against g
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191 bloodstream T. brucei to assess their killing activity (Fig. 2). In three determinations, the EC50 t
192 for #1895 is 6.3 ± 4.3 µM and for #0020 is 16.9 ± 8.8 µM. For perspective, these potencies
193 against trypanosomes are comparable to those of known topoisomerase poisons: camptothecin,
194 1.5 µM (4), ciprofloxacin, 52 µM (18); and are low when compared to some clinically used
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195 agents for trypanosomiasis: pentamidine, 14 nM (4), berenil, 94 nM (4), but not
196 difluoromethylornithine, >147 µM (29).
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198 Compounds #1895 and #0020 promote minicircle linearization. To determine whether
199 antitrypanosomal activity is related to topoisomerase IA , free minicircle replication D
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200 intermediates from treated cells were analyzed by Southern hybridization (Fig. 3). When n
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201 electrophoresed in the presence of ethidium bromide, free minicircles are separated into d
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202 nicked/gapped daughter circles, linears, and covalently closed template circles. As expected, f
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203 etoposide, a known poison of T. brucei topoisomerase IImt (23), increased linearized minicircles h
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204 in a concentration-dependent fashion (Fig. 3A, left panel). These linear forms are substantially :
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205 reduced in samples not treated with Proteinase K, consistent with their covalent linkage to c
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206 protein (Fig. 3A, right panel). m
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207 rg
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208 Compounds #1895 and #0020 also give rise to an increase in protease-sensitive linear n

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209 forms (Fig. 3B and 3C). For #1895, at concentrations between 0 and 40 µM, there is a steady n
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210 increase in linears, but above 60 µM, the population decreases (Fig. 3B). Based on Proteinase K y

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211 sensitivity, at 80 and 100 µM, the linear forms are not protein bound. For #0020, the pattern of 0
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212 protein-bound linears is more complicated (Fig. 3C). In the presence of Proteinase K, linears b
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213 increase steadily between 0 and 40 µM and level off at 80 µM. At 100 µM there appears to be a u
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214 decrease in linears, which then rebound at 120 µM. This wavering pattern has been observed
215 reproducibly in several experiments.
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