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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/11/07">
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<meta name="citation_author" content="Leigh C. Carmody">
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<meta name="citation_author" content="Andrew R. Germain">
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<meta name="citation_author" content="Juan C. Engel">
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<meta name="citation_author" content="Jiri Gut">
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<meta name="citation_author" content="Marcel Kaiser">
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<meta name="citation_author" content="Ivan Jewett">
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<meta name="citation_author" content="Sebastian LeQuemen">
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<meta name="citation_author" content="Jean-Charles Marie">
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<meta name="citation_author" content="Sivaraman Dandapani">
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<meta name="citation_author" content="Ana Rodriguez">
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<meta name="citation_author" content="Jose R. Perez">
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<meta name="citation_author" content="James H. McKerrow">
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<meta name="citation_author" content="Michelle A. J. Palmer">
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<meta name="citation_author" content="Benito Munoz">
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<meta name="citation_author" content="Stuart L. Schrieber">
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<meta name="citation_pmid" content="24479197">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK179828/">
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<meta name="DC.Title" content="Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi">
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<meta name="DC.Contributor" content="Leigh C. Carmody">
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<meta name="DC.Contributor" content="Andrew R. Germain">
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<meta name="DC.Contributor" content="Juan C. Engel">
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<meta name="DC.Contributor" content="Jiri Gut">
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<meta name="DC.Contributor" content="Marcel Kaiser">
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<meta name="DC.Contributor" content="Ivan Jewett">
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<meta name="DC.Contributor" content="Sebastian LeQuemen">
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<meta name="DC.Contributor" content="Jean-Charles Marie">
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<meta name="DC.Contributor" content="Sivaraman Dandapani">
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<meta name="DC.Contributor" content="Ana Rodriguez">
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<meta name="DC.Contributor" content="Jose R. Perez">
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<meta name="DC.Contributor" content="James H. McKerrow">
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<meta name="DC.Contributor" content="Michelle A. J. Palmer">
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<meta name="DC.Contributor" content="Benito Munoz">
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<meta name="DC.Contributor" content="Stuart L. Schrieber">
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<meta name="DC.Date" content="2013/11/07">
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<meta name="description" content="Chagas disease, which is endemic to 18 Latin American countries, and is the major cause of heart failure in the region affecting 8 million people world-wide [1]. Chagas disease is caused by the parasite Trypanosoma cruzi which is transmitted to humans in 3 ways: 1) From fecal matter from blood-feeding insect vectors that belong to the Reduviidae family; 2) Through transfusion with infected blood; 3) Congenitally, from infected mother to fetus. The acute effects of the T. cruzi infection appear soon after infection and symptoms can include fever, swelling at the site of the insect bite and of the lymph glands, and enlargement of the liver and spleen. The chronic phase primarily affects cardiac and enteric cells and patients will suffer from irreversible damage to the heart and digestive tract which can occur even 20 years after infection [1-3]. This project aims to identify novel trypanocidal agents for the treatment of Chagas disease with little to no toxicity in mammalian host cells. An initial pilot high throughput screen of 22,378 compounds derived from the Broad Institute's Diveristy-Oriented Synthesis (DOS) collection (AID 651903) were tested against recombinant Tulahuen strain of T. cruzi stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH3T3 [4-7]. 1,092 ‘actives’ inhibited T. cruzi replication by 55% of control (at 5 μM). 800 compounds were retested at dose and tested for host cell toxicity. A SnAr 8-ortho (CID 44492448) scaffold compound exhibited greater than 1000-fold selectivity towards T. cruzi and was effective against amastigotes, the replicating form of the parasite inside the host cell. Activity against amastogotes is critical for the development of drugs targeting the chronic phase of Chagas disease. Preliminary SAR studies on the hit resulted in the probe ML341 which inhibited T. cruzi replication (IC50=0.9 nM). Potency of ML341 was confirmed against 2 strains of T. cruzi amastigotes (Tulahuen and CA-I/72). Moreover, ML341 is cidal at 750 nM after 20 days of compound treatment and cidal at the lowest dose tested (4 nM) after 29 days of compound treatment, and exhibited minimal toxicity to any mammalian cells tested (NIH3T3, L6, BESM, HepG2). Therefore, we have identified a potent and cidal inhibitor of T. cruzi that is non-toxic to mammalian host cell. Future studies to determine the efficacy in mouse models are currently being undertaken.">
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<meta name="og:description" content="Chagas disease, which is endemic to 18 Latin American countries, and is the major cause of heart failure in the region affecting 8 million people world-wide [1]. Chagas disease is caused by the parasite Trypanosoma cruzi which is transmitted to humans in 3 ways: 1) From fecal matter from blood-feeding insect vectors that belong to the Reduviidae family; 2) Through transfusion with infected blood; 3) Congenitally, from infected mother to fetus. The acute effects of the T. cruzi infection appear soon after infection and symptoms can include fever, swelling at the site of the insect bite and of the lymph glands, and enlargement of the liver and spleen. The chronic phase primarily affects cardiac and enteric cells and patients will suffer from irreversible damage to the heart and digestive tract which can occur even 20 years after infection [1-3]. This project aims to identify novel trypanocidal agents for the treatment of Chagas disease with little to no toxicity in mammalian host cells. An initial pilot high throughput screen of 22,378 compounds derived from the Broad Institute's Diveristy-Oriented Synthesis (DOS) collection (AID 651903) were tested against recombinant Tulahuen strain of T. cruzi stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH3T3 [4-7]. 1,092 ‘actives’ inhibited T. cruzi replication by 55% of control (at 5 μM). 800 compounds were retested at dose and tested for host cell toxicity. A SnAr 8-ortho (CID 44492448) scaffold compound exhibited greater than 1000-fold selectivity towards T. cruzi and was effective against amastigotes, the replicating form of the parasite inside the host cell. Activity against amastogotes is critical for the development of drugs targeting the chronic phase of Chagas disease. Preliminary SAR studies on the hit resulted in the probe ML341 which inhibited T. cruzi replication (IC50=0.9 nM). Potency of ML341 was confirmed against 2 strains of T. cruzi amastigotes (Tulahuen and CA-I/72). Moreover, ML341 is cidal at 750 nM after 20 days of compound treatment and cidal at the lowest dose tested (4 nM) after 29 days of compound treatment, and exhibited minimal toxicity to any mammalian cells tested (NIH3T3, L6, BESM, HepG2). Therefore, we have identified a potent and cidal inhibitor of T. cruzi that is non-toxic to mammalian host cell. Future studies to determine the efficacy in mouse models are currently being undertaken.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK179828_"><span class="title" itemprop="name">Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against <i>Trypanosoma cruzi</i></span></h1><p class="contribs">Carmody LC, Germain AR, Engel JC, et al.</p><p class="fm-aai"><a href="#_NBK179828_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Chagas disease, which is endemic to 18 Latin American countries, and is the major cause of heart failure in the region affecting 8 million people world-wide [<a class="bibr" href="#ml341.r1" rid="ml341.r1">1</a>]. Chagas disease is caused by the parasite <i>Trypanosoma cruzi</i> which is transmitted to humans in 3 ways: 1) From fecal matter from blood-feeding insect vectors that belong to the Reduviidae family; 2) Through transfusion with infected blood; 3) Congenitally, from infected mother to fetus. The acute effects of the <i>T. cruzi</i> infection appear soon after infection and symptoms can include fever, swelling at the site of the insect bite and of the lymph glands, and enlargement of the liver and spleen. The chronic phase primarily affects cardiac and enteric cells and patients will suffer from irreversible damage to the heart and digestive tract which can occur even 20 years after infection [<a class="bibr" href="#ml341.r1" rid="ml341.r1">1</a>-<a class="bibr" href="#ml341.r3" rid="ml341.r3">3</a>]. This project aims to identify novel trypanocidal agents for the treatment of Chagas disease with little to no toxicity in mammalian host cells. An initial pilot high throughput screen of 22,378 compounds derived from the Broad Institute's Diveristy-Oriented Synthesis (DOS) collection (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651903" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651903</a>) were tested against recombinant Tulahuen strain of <i>T. cruzi</i> stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH3T3 [<a class="bibr" href="#ml341.r4" rid="ml341.r4">4</a>-<a class="bibr" href="#ml341.r7" rid="ml341.r7">7</a>]. 1,092 ‘actives’ inhibited <i>T. cruzi</i> replication by 55% of control (at 5 μM). 800 compounds were retested at dose and tested for host cell toxicity. A SnAr 8-ortho (CID 44492448) scaffold compound exhibited greater than 1000-fold selectivity towards <i>T. cruzi</i> and was effective against amastigotes, the replicating form of the parasite inside the host cell. Activity against amastogotes is critical for the development of drugs targeting the chronic phase of Chagas disease. Preliminary SAR studies on the hit resulted in the probe <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> which inhibited <i>T. cruzi</i> replication (IC<sub>50</sub>=0.9 nM). Potency of <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> was confirmed against 2 strains of <i>T. cruzi</i> amastigotes (Tulahuen and CA-I/72). Moreover, <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> is cidal at 750 nM after 20 days of compound treatment and cidal at the lowest dose tested (4 nM) after 29 days of compound treatment, and exhibited minimal toxicity to any mammalian cells tested (NIH3T3, L6, BESM, HepG2). Therefore, we have identified a potent and cidal inhibitor of <i>T. cruzi</i> that is non-toxic to mammalian host cell. Future studies to determine the efficacy in mouse models are currently being undertaken.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03-MH-085673-01</p><p><b>Screening Center Name & PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Chemistry Center Name & PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Assay Submitter & Institution:</b> Ana Rodriguez, PhD., New York University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">624280</a></p><div id="ml341.s1"><h2 id="_ml341_s1_">Probe Structure & Characteristics</h2><div id="ml341.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml341f1&p=BOOKS&id=179828_ml341f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK179828/bin/ml341f1.jpg" alt="Image ml341f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml341t1"><a href="/books/NBK179828/table/ml341.t1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml341t1" rid-ob="figobml341t1"><img class="small-thumb" src="/books/NBK179828/table/ml341.t1/?report=thumb" src-large="/books/NBK179828/table/ml341.t1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml341.t1"><a href="/books/NBK179828/table/ml341.t1/?report=objectonly" target="object" rid-ob="figobml341t1">Table</a></h4></div></div></div><div id="ml341.s2"><h2 id="_ml341_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>The goal of this project is to identify a cidal small-molecule inhibitor of the amastigote life stage of protist <i>T. cruzi</i>, the causative parasite of Chagas disease. Although a few treatments for Chagas disease are available, these treatments are limited because they are only effective against the early (acute) stages of disease and have significant toxicity to the patient [<a class="bibr" href="#ml341.r1" rid="ml341.r1">1</a>-<a class="bibr" href="#ml341.r3" rid="ml341.r3">3</a>]. Anti-trypanosomal agents will aid in developing a drug treatment for both the acute and chronic stages of Chagas disease. This probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) is cidal towards <i>T. cruzi</i> within the mammalian host cell without being toxic to the host cell.</p><p>The probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) described in this report inhibits the replication of the Tulahuen strain <i>T. cruzi</i> with an IC<sub>50</sub> of 0.9 nM. The compound is toxic towards amastigotes (Tulahuen strain and CA-I/72) Moreover, <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> is cidal after 20 days of compound treatment at 750 nM and is cidal at the lowest dose tested 4 nM after 29 of compound treatment. Furthermore, <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> exhibits minimal toxicity to any mammalian cells tested (NIH3T3, L6, BESM, HepG2). This probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) provides a valuable tool that will allow microbiologists investigating Chagas disease to identify new targets in <i>T. cruzi</i>. In addition, <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> can also be advanced along the drug development pathway targeting both the acute and chronic phases of Chagas Disease. In that regard, we are currently evaluating the efficacy of <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> in a mouse model of Chagas disease.</p></div><div id="ml341.s3"><h2 id="_ml341_s3_">2. Materials and Methods</h2><div id="ml341.s4"><h3>2.1. Assays</h3><div id="ml341.s5"><h4>2.1.1. <i>T. cruzi</i> Inhibition Assay (AID 651903, AID 651817, AID 651845)</h4><div id="ml341.s6"><h5>Materials and Methods</h5><p>T175 culture flasks with vented caps were obtained from BD Falcon, and hyperflasks were obtained from Corning (Corning, NY; Catalog no.10024). Disposable sterile filter units (500 ml or 1 L; pore size, 0.20 μM were obtained from Nalgene (Catalog no. 566-0020). Dulbecco's modified Eagle's medium (DMEM) with Phenol Red, high glucose, with L-glutamine and sodium pyruvate was obtained from Cellgro (Mediatech Inc, Manassas, VA; Catalog no. 10-013-CM). Penicillin-streptomycin-L-glutamine (PSG, Catalog no. 10378-016), FBS-heat inactivated fetal bovine serum (FBS, Catalog no.16140-089), and 0.25% Trypsin-EDTA 1X (Catalog no. 25200-072) were purchased from Gibco-Invitrogen. Sterile horse serum, from donor herd (if appearance of epimastigotes) was obtained from Sigma (Catalog no. H1270). Sterile, Ca<sup>2+</sup>/ Mg<sup>2+</sup> free Phosphate Buffered Saline (PBS) 1X was prepared in house. Nonidet P-40 (NP40, now called Igepal CA 360) was obtained from Fluka (Sigma-Aldrich, St. Louis, MO; Catalog no. 56741) and Gal-Screen Buffer B was obtained from AB Biosciences (CA; Catalog no.T1031).</p><div id="ml341.s30"><h5>Cell Lines</h5><ul><li class="half_rhythm"><div>The following cell lines were used in this study: LLC-MK2 cells (rhesus monkey kidney epithelial cell line) and NIH/3T3 cells (mouse embryonic fibroblastic cell line) were initially obtained from the Assay Provider, then from ATCC. <i>T. cruzi</i> expressing β-galactosidase (<i>T. cruzi</i> -β-gal: Tulahuen strain, clone C4; as described in Buckner et al., [<a class="bibr" href="#ml341.r13" rid="ml341.r13">13</a>].</div></li></ul><p><b>For cell propagation:</b> 90% DMEM, Phenol Red, 10% FBS, and 1% Pen/Strep/Glutamine (PSG) were mixed and filtered through a 0.2 micron membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.</p><p><b>For <i>T. cruzi</i> culture and assays:</b> 98% DMEM, Phenol Red, 2% FBS, and 1% PSG were mixed and filtered through a 0.2 micron membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.</p><p>Solutions: Gal-Screen. Using a Gal-Screen base kit, Buffer B (Catalog no. T2361) was mixed with 1:25 substrate (Catalog no. T2359). For <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651903" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651903</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651817" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem"> AID 651817</a> a 1:400 dilution of 20% NP40 was added.</p><p><b><u>NIH/3T3 Cell Culture.</u></b> NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.</p><p><b><u>LLC-MK2 Cell Culture.</u></b> LLC-MK2 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 flasks in 50 ml total of medium. Cells were usually passaged twice a week at 1:4 to 1:8 ratios.</p><p><b><u>Parasite Culture: <i>T. cruzi</i> β-gal (Tc).</u></b> [<a class="bibr" href="#ml341.r4" rid="ml341.r4">4</a>] <i>T. cruzi</i> -β-gal were cultivated in DMEM supplemented with 2% FBS and 1% PSG in T175 flasks with vented caps in 50 ml total of medium.</p></div></div></div><div id="ml341.s7"><h4>2.1.2. Growth Inhibition of <i>Trypanosoma cruzi</i> Assay for HTS (384-well plates)</h4><p>DMEM was supplemented with FBS to a final concentration of 2% and warmed to 37°C prior to use. The parasites were harvested in 50-ml tubes, and spun for 10 minutes at 2200 rpm. Approximately 15 ml of media was aspirated, and the samples were incubated for 3-5 hours. The NIH/3T3 cells were trypsinized (refer to cell culture protocol). When the NIH/3T3 cells were detached, the cells were harvested in DMEM, 2% FBS, and 1% PSG, then counted using the Nexcelom Cellometer. The cells were diluted to 166,667 cells/ml, and then added to a flask and plated 5,000 cells/ 30 μL per well using a standard cassette multidrop Combi (Thermo Scientific). The cells were incubated for 3 hours, and then <i>T. cruzi</i> cells were counted, diluted to 0.250 million cells/ml, and transferred to a 2-liter flask. Then, 50 nL compounds/DMSO were pinned to each well with NIH/3T3 cells. Next, 20 μL per well of parasites (5000 <i>T. cruzi</i>) were added with a standard cassette multiwell drop Combi on slow speed, and incubated for 4 days (or a minimum of 90 hours). Gal-Screen was prepared with 0.05% NP40, 30 μLper well were dispensed in a 384-well plate, incubated for 60 minutes, and the luminescence was read using EnVision (Perkin-Elmer) at 0.1 sec/well.</p></div><div id="ml341.s8"><h4>2.1.3. Cell Toxicity Assay: NIH/3T3 Cells (AID 651818, AID 651844)</h4><p>For the cell toxicity assay with NIH/3T3 cells, the same materials as for <i>T. cruzi</i> co-culture assay were used. NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.</p></div><div id="ml341.s9"><h4>2.1.4. <i>T. cruzi</i> Tulahuen strain amastigote study (Swiss Tropical Public Health Institute, AID 651881 and AID 651890)</h4><p>Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 μL RPMI 1640 medium with 10% FBS and 2 mM l-glutamine. After 24 h the medium was removed and replaced by 100 μL per well containing 5000 trypomastigote forms of <i>T. cruzi</i> Tulahuen strain C2C4 containing the β-galactosidase (Lac Z) gene [<a class="bibr" href="#ml341.r13" rid="ml341.r13">13</a>]. After 48 hours the medium was removed from the wells and replaced by 100 μl fresh medium with or without a serial compound dilution of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/ml. After 96 hours of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 μl) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic program Softmax Pro (Molecular Devices), which calculated IC<sub>50</sub> values by linear regression [<a class="bibr" href="#ml341.r14" rid="ml341.r14">14</a>] from the sigmoidal dose inhibition curves. Benznidazole is used as control.</p></div><div id="ml341.s10"><h4>2.1.5. <i>Leishmania donovani</i> axenic amastigote study (Swiss Tropical Public Health Institute, AID 651891 and AID 651892)</h4><p>Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37 °C in SM medium (Cunnigham et al. 1977) at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO<sub>2</sub> in air. One hundred microliters of culture medium with 10<sup>5</sup> amastigotes from axenic culture with or without a serial compound dilution were seeded in 96-well microtitre plates. Serial compound dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg /ml were prepared. After 70 hours of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 μL of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) [<a class="bibr" href="#ml341.r15" rid="ml341.r15">15</a>] were then added to each well and the plates incubated for another 2 hours. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. Data were analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease of fluorescence (= inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the compound concentrations. From the sigmoidal inhibition curves the IC<sub>50</sub> values were calculated by linear regression [<a class="bibr" href="#ml341.r14" rid="ml341.r14">14</a>]. Miltefosine is used as control</p></div><div id="ml341.s11"><h4>2.1.6. <i>Trypanosoma brucei rhodesiense</i> (Swiss Tropical Public Health Institute, AID 651893, AID 651894)</h4><p><i>Trypanosoma brucei rhodesiense</i> STIB900 stock was isolated in 1982 from a human patient in Tanzania and after several mouse passages cloned and adapted to axenic culture conditions [<a class="bibr" href="#ml341.r16" rid="ml341.r16">16</a>] Minimum Essential Medium (50 μL) supplemented with 25 mM HEPES, 1g/l additional glucose, 1% MEM non-essential amino acids (100x), 0.2 mM 2-mercaptoethanol, 1mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial compound dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/ml were prepared. Then 4×10<sup>3</sup> bloodstream forms of T. b. rhodesiense STIB 900 in 50 μL was added to each well and the plate incubated at 37 °C under a 5 % CO<sub>2</sub> atmosphere for 70 h. 10 μL Alamar Blue (resazurin, 12.5 mg in 100 ml double-distilled water) was then added to each well and incubation continued for a further 2-4 h [<a class="bibr" href="#ml341.r17" rid="ml341.r17">17</a>]. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC<sub>50</sub> values were calculated by linear regression [<a class="bibr" href="#ml341.r14" rid="ml341.r14">14</a>] from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Melarsoprol is used as control.</p></div><div id="ml341.s12"><h4>2.1.7. <i>Plasmodium falciparum</i> NF54 study (Swiss Tropical Public Health Institute, AID 651844, AID 651889)</h4><p>In vitro activity against erythrocytic stages of P. falciparum was determined using a <sup>3</sup>H-hypoxanthine incorporation assay [<a class="bibr" href="#ml341.r18" rid="ml341.r18">18</a>], using the drug sensitive NF54 strain (Schipol Airport, The Netherlands, [<a class="bibr" href="#ml341.r19" rid="ml341.r19">19</a>]) and the standard drug chloroquine (Sigma C6628). Compounds were dissolved in DMSO at 10 mg/ml and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO<sub>3</sub> (2.1 g/l), neomycin (100 U/ml), Albumax<sup>R</sup> (5 g/L) and washed human red cells A<sup>+</sup> at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg /ml were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 °C; 4% CO<sub>2</sub>, 3% O<sub>2</sub>, 93% N<sub>2</sub>. After 48 h 50 μl of <sup>3</sup>H-hypoxanthine (=0.5 uCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate™ cell harvester (Wallac, Zurich, Switzerland), and the red blood cells transferred onto a glass fibre filter then washed with distilled water. The dried filters were inserted into a plastic foil with 10 ml of scintillation fluid, and counted in a Betaplate™ liquid scintillation counter (Wallac, Zurich, Switzerland). IC<sub>50</sub> values were calculated from sigmoidal inhibition curves by linear regression [<a class="bibr" href="#ml341.r14" rid="ml341.r14">14</a>] using Microsoft Excel. Chloroquine and artemisinin are used as control.</p></div><div id="ml341.s13"><h4>2.1.8. L-6 rat myocyte toxicity (Swiss Tropical Public Health Institute, AID 651896, AID 651897)</h4><p>Assays were performed in 96-well microtiter plates, each well containing 100 μL of RPMI 1640 medium supplemented with 1% L-glutamine (200mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts) [<a class="bibr" href="#ml341.r20" rid="ml341.r20">20</a>,<a class="bibr" href="#ml341.r21" rid="ml341.r21">21</a>]. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg /ml were prepared. After 70hours of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 μL of Alamar Blue was then added to each well and the plates incubated for another 2 hours. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC<sub>50</sub> values were calculated by linear regression [<a class="bibr" href="#ml341.r14" rid="ml341.r14">14</a>] from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxine is used as control.</p></div><div id="ml341.s14"><h4>2.1.9. <i>T. cruzi</i> CA-I/72 strain HCS Assay amastigote study (James McKerrow Lab, University of California, San Francisco (UCSF), AID 651885)</h4><p>The screen employs the IN Cell Analyzer 2000 (GE Healthcare Life Sciences), a flexible, modular, cellular and subcellular imaging system for fast, automated imaging of fixed and live cells built around an automated epifluorescence-based microscope and image acquisition software. The screening is performed in sterile and fluorescence transmissible 96 wells-cell culture plates. Each well is seeded with 1000 BESM cells (Bovine Embryo Skeletal Muscle Cells) allowed to attach for several hours. The culture medium (RPMI 1640 with 5 % FBS) is then replaced with fresh medium containing 1000 <i>T. cruzi</i> trypomastigotes (Wild type CA-I/72 <i>T. cruzi</i> was isolated from an Argentinean chronic chagasic patient ) per well. Sixteen hours post-infection, cells are washed once to remove extracellular parasites, and maintained in medium containing serial dilutions of compounds for 72 hours at 37°C. Cells are then washed once with PBS, fixed for 2 hours with 4% paraformaldehyde, and washed 1 time to remove the fixative. Then 50 μL of mounting medium containing a DNA fluorescent dye (i.e. DAPI or Hoechst) are added per well. The plates are kept in the dark at 4°C until scanned in the IN Cell Analyzer with a 10X objective, and the images are collected with appropriate fluorescent excitation/emission filters At least 100 infected cells are recorded per well. The ratio of <i>T. cruzi</i> amastigotes per infected is calculated in untreated controls and compared to those obtained for the different compounds and conditions tested. A significant reduction in the E/C ratio indicates inhibition of parasite growth rate. This information provides a quantitative measure of growth inhibition during the first 72 hours post-infection [<a class="bibr" href="#ml341.r22" rid="ml341.r22">22</a>].</p></div><div id="ml341.s15"><h4>2.1.10. <i>T. cruzi</i> CA-I/72 cidal (McKerrow Lab, UCSF, AID 651877, AID 651869)</h4><p>This assay assesses cidal activity of compounds in <i>T. cruzi</i> (CA-I/72)-infected Bovine Embryo Skeletal Muscle Cells (BESM). Twenty four hours after infection, infected cells are washed and compounds are added every other day for 20 days (protocol 1) or 29 days (protocol 2). After the initial period, compounds are removed and cells are observed for an additional 38 days (protocol 1) or 37 days (protocol 2) without compound. During which time cultures are monitored for reappearance of <i>T. cruzi</i> parasites. This procedure allows us to determine if host cells were effectively cured (i.e., compound is cidal) or if infection still persists (i.e, static activity) [<a class="bibr" href="#ml341.r23" rid="ml341.r23">23</a>].</p></div><div id="ml341.s16"><h4>2.1.11. Cruzain Inhibition (McKerrow Lab, UCSF, AID 651897)</h4><p>Methods were outlined previously [<a class="bibr" href="#ml341.r24" rid="ml341.r24">24</a>]. Briefly, recombinant cruzain was expressed and purified. For IC<sub>50</sub> determinations, compounds were serially diluted in DMSO in the range of 25 μM - 0.001 μM for cruzain. Protease inhibition assays for cruzain were carried out in 384 well plate format. Cruzain (4 nM) was incubated with test compound in 100 mM sodium acetate, pH 5.5, containing 5 mM DTT and 0.01% Triton X-100 (buffer A), for 5 min at room temperature (the higher concentration of TbCatB was required due to its lower intrinsic activity against the fluorogenic substrate). Then buffer A containing Z-Phe-Arg-AMC (Bachem) was added to enzyme-compound mixture to give 10 μM substrate in a final assay volume of 200 μL. The rate of free AMC release was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, with a microtiter plate spectrofluorimeter (SpectraMax M5, Molecular Devices) for 3 min. Percentage inhibition of test compound was calculated relative to the DMSO control (0% inhibition control). IC<sub>50</sub> values were determined with Prism 4 software (GraphPad, San Diego, CA) fitted by nonlinear regression to the variable-slope sigmoidal dose-response formula y=100/[1 + 10^(log IC<sub>50</sub> - X)^H, where H is the Hill coefficient (slope factor).</p></div></div><div id="ml341.s17"><h3>2.2. Probe Chemical Characterization</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml341t2"><a href="/books/NBK179828/table/ml341.t2/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml341t2" rid-ob="figobml341t2"><img class="small-thumb" src="/books/NBK179828/table/ml341.t2/?report=thumb" src-large="/books/NBK179828/table/ml341.t2/?report=previmg" alt="Table 1. Summary of Known Probe Properties in PubChem OR Properties Computed from Structure." /></a><div class="icnblk_cntnt"><h4 id="ml341.t2"><a href="/books/NBK179828/table/ml341.t2/?report=objectonly" target="object" rid-ob="figobml341t2">Table 1</a></h4><p class="float-caption no_bottom_margin">Summary of Known Probe Properties in PubChem OR Properties Computed from Structure. </p></div></div><p>After preparation as described in <a href="#ml341.s18">Section 2.3</a>, the probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was analyzed by UPLC, <sup>1</sup>H and <sup>13</sup>C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicated an isolated purity of >95%.</p><p>The solubility of the probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was experimentally determined to be 1 μM in phosphate buffered saline (PBS, pH 7.4, 23° C) solution. Plasma protein binding (PPB) was determined to be 98% bound in human plasma. The probe is stable in human plasma, with approximately 97% remaining after a 5-hour incubation period.</p><p>The stability of the probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) in PBS (0.1% DMSO) was measured over 48 hours. Due to the low solubility of the probe, the ion count of the PBS solution was insufficient for analysis. Thus, we decided to determine the total amount of the probe present in the well after the probe was treated with PBS alone for a given length of time. We added acetonitrile at various time points to wells containing the probe in PBS and measured the total amount of the probe. This result is shown in <a class="figpopup" href="/books/NBK179828/figure/ml341.f2/?report=objectonly" target="object" rid-figpopup="figml341f2" rid-ob="figobml341f2">Figure 1</a>. From these results, the probe seems to be stable in PBS since more than 97% is still present after 48 hours of incubation.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml341f2" co-legend-rid="figlgndml341f2"><a href="/books/NBK179828/figure/ml341.f2/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml341f2" rid-ob="figobml341f2"><img class="small-thumb" src="/books/NBK179828/bin/ml341f2.gif" src-large="/books/NBK179828/bin/ml341f2.jpg" alt="Figure 1. Stability Data for the Probe (ML341) in PBS Buffer (pH 7.4, 23°C) followed by addition of acetonitrile after 48 Hours." /></a><div class="icnblk_cntnt" id="figlgndml341f2"><h4 id="ml341.f2"><a href="/books/NBK179828/figure/ml341.f2/?report=objectonly" target="object" rid-ob="figobml341f2">Figure 1</a></h4><p class="float-caption no_bottom_margin">Stability Data for the Probe (ML341) in PBS Buffer (pH 7.4, 23°C) followed by addition of acetonitrile after 48 Hours. </p></div></div></div><div id="ml341.s18"><h3>2.3. Probe Preparation</h3><p>The probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was synthesized from 4-(tert-butoxycarbonyl(methyl)amino)-3-(tert-butyldimethylsilyloxy)-2-methylbutanoic acid in nine steps (see <a class="figpopup" href="/books/NBK179828/figure/ml341.f3/?report=objectonly" target="object" rid-figpopup="figml341f3" rid-ob="figobml341f3">Scheme 1</a>). Synthesis of core scaffold <b>5</b> [<a class="bibr" href="#ml341.r25" rid="ml341.r25">25</a>] and further elaboration to library synthesis including the synthesis of compound <b>9</b> [<a class="bibr" href="#ml341.r26" rid="ml341.r26">26</a>] have been previously reported from Broad Institute. The full synthetic scheme is presented below.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml341f3" co-legend-rid="figlgndml341f3"><a href="/books/NBK179828/figure/ml341.f3/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml341f3" rid-ob="figobml341f3"><img class="small-thumb" src="/books/NBK179828/bin/ml341f3.gif" src-large="/books/NBK179828/bin/ml341f3.jpg" alt="Scheme 1. Synthesis of the Probe (ML341)." /></a><div class="icnblk_cntnt" id="figlgndml341f3"><h4 id="ml341.f3"><a href="/books/NBK179828/figure/ml341.f3/?report=objectonly" target="object" rid-ob="figobml341f3">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of the Probe (ML341). </p></div></div></div><div id="ml341.s19"><h3>Additional Analytical Analysis</h3><p>The probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was found to be 98% and 99% bound to human and mouse plasma respectively and stable in human and mouse plasma with 97% and 89% remaining after 5 hours. The probe was also found to be stable to mouse microsomes and hepatocytes with 77% and 95% remaining after 1 hour respectively. The probe inhibits hERG in a radioligand assay 26% at 10 μM. The probe was shown to be permeable in a Caco-2 assay with A-B:B-A of 14:7 (10<sup>-6</sup> cm/s) and non-toxic to <i>HepG2</i> cells with an IC<sub>50</sub> >30 μM. The hit compound, its seven stereoisomers of the hit (including <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) and several analogs were subjected to mouse microsomes for stability analysis (<a class="figpopup" href="/books/NBK179828/table/ml341.t3/?report=objectonly" target="object" rid-figpopup="figml341t3" rid-ob="figobml341t3">Table 2</a> and <a class="figpopup" href="/books/NBK179828/table/ml341.t4/?report=objectonly" target="object" rid-figpopup="figml341t4" rid-ob="figobml341t4">3</a>). The hit and its seven stereoisomers were also tested for inhibition of CYP3A4.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml341t3"><a href="/books/NBK179828/table/ml341.t3/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml341t3" rid-ob="figobml341t3"><img class="small-thumb" src="/books/NBK179828/table/ml341.t3/?report=thumb" src-large="/books/NBK179828/table/ml341.t3/?report=previmg" alt="Table 2. Microsomal Stability and CYP3A4 of Hit and Stereoisomers." /></a><div class="icnblk_cntnt"><h4 id="ml341.t3"><a href="/books/NBK179828/table/ml341.t3/?report=objectonly" target="object" rid-ob="figobml341t3">Table 2</a></h4><p class="float-caption no_bottom_margin">Microsomal Stability and CYP3A4 of Hit and Stereoisomers. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml341t4"><a href="/books/NBK179828/table/ml341.t4/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml341t4" rid-ob="figobml341t4"><img class="small-thumb" src="/books/NBK179828/table/ml341.t4/?report=thumb" src-large="/books/NBK179828/table/ml341.t4/?report=previmg" alt="Table 3. Microsomal Stability of Select Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml341.t4"><a href="/books/NBK179828/table/ml341.t4/?report=objectonly" target="object" rid-ob="figobml341t4">Table 3</a></h4><p class="float-caption no_bottom_margin">Microsomal Stability of Select Analogs. </p></div></div></div></div><div id="ml341.s20"><h2 id="_ml341_s20_">3. Results</h2><div id="ml341.s21"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml341.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Dose-dependent%20Activity%20of%20the%20Probe%20(CID%2044493436%2F%20ML341)%20and%20CID%2044492448.&p=BOOKS&id=179828_ml341f11.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK179828/bin/ml341f11.jpg" alt="Figure 2. Dose-dependent Activity of the Probe (CID 44493436/ ML341) and CID 44492448." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Dose-dependent Activity of the Probe (CID 44493436/ ML341) and CID 44492448</span></h3></div><p>Dose-dependent Activities of Probe <a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> and primary screen hit, CID44492448 in primary assay (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> IC<sub>50</sub> =0.9 nM (A), and CID44492448 IC<sub>50</sub> =1.2 nM (C)) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651845" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651845</a>) and NIH3T3 toxicity assays (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> IC50= 8200 nM (B) and CID44492448 IC<sub>50</sub>= 6600 nM (D)) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651844" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651844</a>).</p></div><div id="ml341.s22"><h3>3.2. Cellular Activity</h3><p>Both the primary and secondary assays in this project are cell-based; therefore the probe clearly displays cellular activity and permeability. Additionally, the probe has been tested in a Caco-2 assay and shown to be permeable with an A-B:B-A of 14:7 (10<sup>-6</sup> cm/s).</p></div><div id="ml341.s23"><h3>3.3. Profiling Assays</h3><p>The probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was screened against a panel of 68 targets in radioligand binding assays that are commonly used in drug discovery for lead profiling. The assays were done by EuroFins PanLabs Taiwan, Limited and include targets from various areas such as, GPCRs, ion channels, and transporters. The probe (<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a>) was active in 6 of the 68 assays displaying inhibition at 10 μM of the cannabinoid CB<sub>1</sub> (60%), Glucocorticoid (69%), Opiate K (OP2. KOP) (77%), sigma (sigma<sub>1</sub>) (76%) receptors along with inhibition of sodium channel, site 2 (66%), and the Norepinephrine transporter (NET) (61%). These assays only assess binding to the above mentioned targets and the functional consequence of that binding will need to be assessed.</p></div></div><div id="ml341.s24"><h2 id="_ml341_s24_">4. Discussion</h2><div id="ml341.s25"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p><a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a> provides a much more potent cidal compound than what has been previously tested. Also, there is no mammalian cell toxicity observed in any cell line tested (NIH3T3, L6, BESM, or HepG2 cells). Benznidazole is cidal with an IC<sub>50</sub> of 6600 nM after a 20 day treatment and IC<sub>50</sub> of 3300nM after a 29 day compound treatment. This is significant because benznidazole must be administered for long periods of time to human patients to achieve ‘cure’ and causes multitudes of side effects. By developing a compound and potentially a drug that could be given at lower doses for shorter periods of time would greatly increase the quality of a patient.</p></div></div><div id="ml341.s26"><h2 id="_ml341_s26_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml341.r1"><span class="ref-journal">Research Priorities for Chagas Disease, Human African Trypanosomiasis and Leishmaniasis: Technical Report of the TDR Disease Reference Group on Chagas Disease, Human African Trypanosmiasis and Leishmaniasis.</span> World Health Organization; 2012. </div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml341.r2">De Souza W. Basic cell biology of Trypanosoma cruzi. <span><span class="ref-journal">Curr Pharm Des. </span>2002;<span class="ref-vol">8</span>:269–285.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11860366" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11860366</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml341.r3">Tarleton RL, Reithinger R, Urbina JA, Kitron U, Gurtler RE. The challenges of Chagas Disease-- grim outlook or glimmer of hope. <span><span class="ref-journal">PLoS Med. </span>2007;<span class="ref-vol">4</span>:e332.</span> [<a href="/pmc/articles/PMC2222930/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2222930</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18162039" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18162039</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml341.r4">Bettiol E, Samanovic M, Murkin AS, Raper J, Buckner F, et al. Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening. <span><span class="ref-journal">PLoS Negl Trop Dis. </span>2009;<span class="ref-vol">3</span>:e384.</span> [<a href="/pmc/articles/PMC2639639/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2639639</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19238193" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19238193</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml341.r5">Carmody LC, Germain A, Barker D, Galan-Rodriguez C, Bettiol E, et al. <span class="ref-journal">Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 3.</span> 2010. </div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml341.r6">Carmody LC, Germain A, Barker D, Galan-Rodriguez C, Bettiol E, et al. <span class="ref-journal">Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1.</span> 2010. </div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml341.r7">Carmody LC, Germain A, Barker D, Galan-Rodriguez C, Bettiol E, et al. <span class="ref-journal">Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection.</span> 2010. </div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml341.r8">Konkle ME, Hargrove TY, Kleshchenko YY, von Kries JP, Ridenour W, et al. Indomethacin amides as a novel molecular scaffold for targeting Trypanosoma cruzi sterol 14alpha-demethylase. <span><span class="ref-journal">J Med Chem. </span>2009;<span class="ref-vol">52</span>:2846–2853.</span> [<a href="/pmc/articles/PMC2744100/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2744100</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19354253" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19354253</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml341.r9">Brak K, Kerr ID, Barrett KT, Fuchi N, Debnath M, et al. Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:1763–1773.</span> [<a href="/pmc/articles/PMC2838180/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2838180</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20088534" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20088534</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml341.r10">Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KK, et al. Identification and optimization of inhibitors of Trypanosomal cysteine proteases: cruzain, rhodesain, and TbCatB. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:52–60.</span> [<a href="/pmc/articles/PMC2804034/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2804034</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19908842" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19908842</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml341.r11">Germain AR, Carmody LC, Dockendorff C, Galan-Rodriguez C, Rodriguez A, et al. Identification of small-molecule inhibitors of Trypansoma cruzi replication. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2011;<span class="ref-vol">21</span>:7197–7200.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22018462" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22018462</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml341.r12">Engel JC, Doyle PS, Dvorak JA. Trypanosoma cruzi: biological characterization of clones derived from chronic chagasic patients. II. Quantitative analysis of the intracellular cycle. <span><span class="ref-journal">J Protozool. </span>1985;<span class="ref-vol">32</span>:80–83.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3921699" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3921699</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml341.r13">Buckner FS, Verlinde CL, La Flamme AC, Van Voorhis WC. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1996;<span class="ref-vol">40</span>:2592–2597.</span> [<a href="/pmc/articles/PMC163582/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC163582</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8913471" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8913471</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml341.r14">Huber W, Koella JC. A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. <span><span class="ref-journal">Acta Trop. </span>1993;<span class="ref-vol">55</span>:257–261.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8147282" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8147282</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml341.r15">Mikus J, Steverding D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. <span><span class="ref-journal">Parasitol Int. </span>2000;<span class="ref-vol">48</span>:265–269.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11227767" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11227767</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml341.r16">Baltz T, Baltz D, Giroud C, Crockett J. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. <span><span class="ref-journal">EMBO J. </span>1985;<span class="ref-vol">4</span>:1273–1277.</span> [<a href="/pmc/articles/PMC554336/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC554336</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/4006919" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 4006919</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml341.r17">Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. <span><span class="ref-journal">Acta Trop. </span>1997;<span class="ref-vol">68</span>:139–147.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9386789" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9386789</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml341.r18">Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1979;<span class="ref-vol">16</span>:710–718.</span> [<a href="/pmc/articles/PMC352941/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC352941</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/394674" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 394674</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml341.r19">Ponnudurai T, Leeuwenberg AD, Meuwissen JH. Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. <span><span class="ref-journal">Trop Geogr Med. </span>1981;<span class="ref-vol">33</span>:50–54.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7018038" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7018038</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>20.</dt><dd><div class="bk_ref" id="ml341.r20">Ahmed SA, Gogal RM Jr., Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. <span><span class="ref-journal">J Immunol Methods. </span>1994;<span class="ref-vol">170</span>:211–224.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8157999" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8157999</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>21.</dt><dd><div class="bk_ref" id="ml341.r21">Page B, Page M, Noel C. A new fluorometric assay for cytotoxicity measurements in-vitro. <span><span class="ref-journal">Int J Oncol. </span>1993;<span class="ref-vol">3</span>:473–476.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21573387" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21573387</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>22.</dt><dd><div class="bk_ref" id="ml341.r22">Engel JC, Ang KK, Chen S, Arkin MR, McKerrow JH, et al. Image-based high-throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas' disease. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>2010;<span class="ref-vol">54</span>:3326–3334.</span> [<a href="/pmc/articles/PMC2916317/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2916317</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20547819" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20547819</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>23.</dt><dd><div class="bk_ref" id="ml341.r23">Doyle PS, Zhou YM, Hsieh I, Greenbaum DC, McKerrow JH, et al. The Trypanosoma cruzi protease cruzain mediates immune evasion. <span><span class="ref-journal">PLoS Pathog. </span>2011;<span class="ref-vol">7</span>:e1002139.</span> [<a href="/pmc/articles/PMC3164631/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3164631</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21909255" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21909255</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>24.</dt><dd><div class="bk_ref" id="ml341.r24">Ang KK, Ratnam J, Gut J, Legac J, Hansell E, et al. Mining a cathepsin inhibitor library for new antiparasitic drug leads. <span><span class="ref-journal">PLoS Negl Trop Dis. </span>2011;<span class="ref-vol">5</span>:e1023.</span> [<a href="/pmc/articles/PMC3086806/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3086806</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21572521" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21572521</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>25.</dt><dd><div class="bk_ref" id="ml341.r25">Marcaurelle LA, Comer E, Dandapani S, Duvall JR, Gerard B, et al. An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors. <span><span class="ref-journal">J Am Chem Soc. </span>2010;<span class="ref-vol">132</span>:16962–16976.</span> [<a href="/pmc/articles/PMC3004530/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3004530</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21067169" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21067169</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>26.</dt><dd><div class="bk_ref" id="ml341.r26">Chou DH, Duvall JR, Gerard B, Liu H, Pandya BA, et al. Synthesis of a novel suppressor of beta-cell apoptosis via diversity-oriented synthesis. <span><span class="ref-journal">ACS Med Chem Lett. </span>2011;<span class="ref-vol">2</span>:698–702.</span> [<a href="/pmc/articles/PMC3171963/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3171963</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21927648" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21927648</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>27.</dt><dd><div class="bk_ref" id="ml341.r27">Engel JC, Torres C, Hsieh I, Doyle PS, McKerrow JH. Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi. <span><span class="ref-journal">J Cell Sci. </span>2000;<span class="ref-vol">113</span>(Pt 8):1345–1354.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10725218" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10725218</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK179828_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Leigh C. Carmody</span>,<sup>1</sup><sup>,<img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup> <span itemprop="author">Andrew R. Germain</span>,<sup>1</sup> <span itemprop="author">Juan C. Engel</span>,<sup>2</sup> <span itemprop="author">Jiri Gut</span>,<sup>2</sup> <span itemprop="author">Marcel Kaiser</span>,<sup>2</sup> <span itemprop="author">Ivan Jewett</span>,<sup>1</sup> <span itemprop="author">Sebastian LeQuemen</span>,<sup>1</sup> <span itemprop="author">Jean-Charles Marie</span>,<sup>1</sup> <span itemprop="author">Sivaraman Dandapani</span>,<sup>1</sup> <span itemprop="author">Ana Rodriguez</span>,<sup>3</sup> <span itemprop="author">Jose R. Perez</span>,<sup>1</sup> <span itemprop="author">James H. McKerrow</span>,<sup>2</sup> <span itemprop="author">Michelle A. J. Palmer</span>,<sup>1</sup> <span itemprop="author">Benito Munoz</span>,<sup>1</sup> and <span itemprop="author">Stuart L. Schrieber</span><sup>1,4</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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The Broad Institute Probe Development Center, Cambridge, MA</div><div class="affiliation"><sup>2</sup>
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University of California, San Francisco, CA</div><div class="affiliation"><sup>3</sup>
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New York University, New York, NY</div><div class="affiliation"><sup>4</sup>
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Howard Hughes Medical Institute, Chemistry and Chemical Biology, Harvard University, Cambridge, MA</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author email:
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<a href="mailto:dev@null" data-email="gro.etutitsnidaorb@ydomracl" class="oemail">gro.etutitsnidaorb@ydomracl</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">December 17, 2012</span>; Last Update: <span itemprop="dateModified">November 7, 2013</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Carmody LC, Germain AR, Engel JC, et al. Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi. 2012 Dec 17 [Updated 2013 Nov 7]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml342/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml340/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml341f1"><div id="ml341.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml341f1&p=BOOKS&id=179828_ml341f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK179828/bin/ml341f1.jpg" alt="Image ml341f1" class="tileshop" title="Click on image to zoom" /></a></div></div></article><article data-type="table-wrap" id="figobml341t1"><div id="ml341.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179828/table/ml341.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml341.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml341.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml341.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml341.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml341.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Antitarget Name(s)</th><th id="hd_h_ml341.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml341.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective</th><th id="hd_h_ml341.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml341.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 44493436/<a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a></td><td headers="hd_h_ml341.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>Trypanosoma cruzi</i> replication</td><td headers="hd_h_ml341.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.9 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/13421649" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 13421649</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651845" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651845</a>]</td><td headers="hd_h_ml341.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>NIH3T3 host cell toxicity</i></td><td headers="hd_h_ml341.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134216491</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651844" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651844</a>]</td><td headers="hd_h_ml341.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.9 nM vs. inactive</td><td headers="hd_h_ml341.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Tulahuen Amastigote, CA-I/72 Amastigote, Cidal, <i>P. falc., L. don., T.brucei rhod.</i>, Cruzain, L6 Toxicity [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134216491</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651890" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651890</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651892" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651892</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651894" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651894</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/68195" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 68195</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651885</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651877</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651869" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651869</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651897" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651897</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651889" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651889</a>]</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml341t2"><div id="ml341.t2" class="table"><h3><span class="label">Table 1</span><span class="title">Summary of Known Probe Properties in PubChem OR Properties Computed from Structure</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179828/table/ml341.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml341.t2_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml341.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">IUPAC Chemical Name</th><td headers="hd_b_ml341.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>N</i>-((2<i>S</i>,3<i>S</i>)-5-((<i>S</i>)-1-hydroxypropan-2-yl)-3-methyl-2-((methyl(4-phenoxybenzyl)amino)methyl)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][<a class="bibr" href="#ml341.r1" rid="ml341.r1">1</a>,<a class="bibr" href="#ml341.r5" rid="ml341.r5">5</a>]oxazocin-10-yl)isonicotinamide</td></tr><tr><th id="hd_b_ml341.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PubChem CID</th><td headers="hd_b_ml341.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">44493436</td></tr><tr><th id="hd_b_ml341.t2_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Molecular Weight</th><td headers="hd_b_ml341.t2_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">594</td></tr><tr><th id="hd_b_ml341.t2_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Molecular Formula</th><td headers="hd_b_ml341.t2_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">C<sub>35</sub>H<sub>38</sub>N<sub>4</sub>O<sub>5</sub></td></tr><tr><th id="hd_b_ml341.t2_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">XLogP3-AA</th><td headers="hd_b_ml341.t2_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">4.6</td></tr><tr><th id="hd_b_ml341.t2_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">H-Bond Donor</th><td headers="hd_b_ml341.t2_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">2</td></tr><tr><th id="hd_b_ml341.t2_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">H-Bond Acceptor</th><td headers="hd_b_ml341.t2_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">6</td></tr><tr><th id="hd_b_ml341.t2_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Rotatable Bond Count</th><td headers="hd_b_ml341.t2_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10</td></tr><tr><th id="hd_b_ml341.t2_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Exact Mass</th><td headers="hd_b_ml341.t2_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">594.28</td></tr><tr><th id="hd_b_ml341.t2_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Topological Polar Surface Area</th><td headers="hd_b_ml341.t2_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">104</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml341f2"><div id="ml341.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Stability%20Data%20for%20the%20Probe%20(ML341)%20in%20PBS%20Buffer%20(pH%207.4%2C%2023%B0C)%20followed%20by%20addition%20of%20acetonitrile%20after%2048%20Hours.&p=BOOKS&id=179828_ml341f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK179828/bin/ml341f2.jpg" alt="Figure 1. Stability Data for the Probe (ML341) in PBS Buffer (pH 7.4, 23°C) followed by addition of acetonitrile after 48 Hours." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Stability Data for the Probe (ML341) in PBS Buffer (pH 7.4, 23°C) followed by addition of acetonitrile after 48 Hours</span></h3></div></article><article data-type="fig" id="figobml341f3"><div id="ml341.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Scheme%201.%20Synthesis%20of%20the%20Probe%20(ML341).&p=BOOKS&id=179828_ml341f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK179828/bin/ml341f3.jpg" alt="Scheme 1. Synthesis of the Probe (ML341)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of the Probe (ML341)</span></h3></div></article><article data-type="table-wrap" id="figobml341t3"><div id="ml341.t3" class="table"><h3><span class="label">Table 2</span><span class="title">Microsomal Stability and CYP3A4 of Hit and Stereoisomers</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179828/table/ml341.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml341.t3_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml341.t3_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml341.t3_1_1_1_1" style="text-align:center;vertical-align:middle;">No.</th><th id="hd_h_ml341.t3_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml341.t3_1_1_1_2" style="text-align:center;vertical-align:middle;">CID<br />SID<br />Broad ID</th><th id="hd_h_ml341.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure<br />
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f4.jpg" alt="Image ml341f4.jpg" /></div></th><th id="hd_h_ml341.t3_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml341.t3_1_1_1_4" style="text-align:center;vertical-align:middle;"><i>T. cruzi</i> growth inhibition<br /><br />(IC<sub>50</sub>, nM)</th><th id="hd_h_ml341.t3_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml341.t3_1_1_1_5" style="text-align:center;vertical-align:middle;">Mouse microsomal stability<br /><br />(% remaining after 1h)</th><th id="hd_h_ml341.t3_1_1_1_6" rowspan="2" colspan="1" headers="hd_h_ml341.t3_1_1_1_6" style="text-align:center;vertical-align:middle;">CYP3A4 Inhibition<br /><br />(10 μM)</th></tr><tr><th headers="hd_h_ml341.t3_1_1_1_3" id="hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Configuration (C<sub>2</sub>C<sub>5</sub>C<sub>6</sub>)</th></tr></thead><tbody><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">56589425<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216480</a><br />K64366758</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRR</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.3%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">99%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44493933<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360167" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360167</a><br />K87113270-</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">RRR</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.9%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">99%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44493436<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216491</a><br />K82092559-</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>SSS (</b><a href="/pcsubstance/?term=ML341[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML341</a><b>)</b></td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">77%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">99%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44202487<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216488" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216488</a><br />K02920722</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">RSS</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">70%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">99%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44495351<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216474" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216474</a><br />K46532897</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRS</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">450</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.4%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">98%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44494873<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216489" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216489</a><br />K28800414</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SSR</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.1%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">98%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44495386<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134216490" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134216490</a><br />K24861700</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">RRS</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">260</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">92%</td></tr><tr><td headers="hd_h_ml341.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8</td><td headers="hd_h_ml341.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44495835<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134958999" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134958999</a><br />K67798612-</td><td headers="hd_h_ml341.t3_1_1_1_3 hd_h_ml341.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">RSR</td><td headers="hd_h_ml341.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16</td><td headers="hd_h_ml341.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.2%</td><td headers="hd_h_ml341.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">99%</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div id="ml341.tfn1"><p class="no_margin">All values are the average of at least two replicates</p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobml341t4"><div id="ml341.t4" class="table"><h3><span class="label">Table 3</span><span class="title">Microsomal Stability of Select Analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179828/table/ml341.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml341.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No.</th><th id="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID<br />SID<br />Broad ID</th><th id="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>T. cruzi</i> growth inhibition (IC<sub>50</sub>, nM)</th><th id="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mouse microsomal stability (% remaining after 1h)</th></tr></thead><tbody><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">56962232<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/135676182" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">135676182</a><br />K56444260</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f5.jpg" alt="Image ml341f5.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6%</td></tr><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57345719<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136367238" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136367238</a><br />K54535512</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f6.jpg" alt="Image ml341f6.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><1</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0%</td></tr><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">56973436<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/135724423" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">135724423</a><br />K06077816</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f7.jpg" alt="Image ml341f7.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">45</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0%</td></tr><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57345701<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136367204" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">136367204</a><br />K97312028</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f8.jpg" alt="Image ml341f8.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">29</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0%</td></tr><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44495822<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/135724434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">135724434</a><br />K29134794</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f9.jpg" alt="Image ml341f9.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><1</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0%</td></tr><tr><td headers="hd_h_ml341.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml341.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44497028<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/135724433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">135724433</a><br />K60359902</td><td headers="hd_h_ml341.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179828/bin/ml341f10.jpg" alt="Image ml341f10.jpg" /></div></td><td headers="hd_h_ml341.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td><td headers="hd_h_ml341.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9%</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div id="ml341.tfn2"><p class="no_margin">All values are the average of at least two replicates</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml341f4"><div id="ml341.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Dose-dependent%20Activity%20of%20the%20Probe%20(CID%2044493436%2F%20ML341)%20and%20CID%2044492448.&p=BOOKS&id=179828_ml341f11.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK179828/bin/ml341f11.jpg" alt="Figure 2. Dose-dependent Activity of the Probe (CID 44493436/ ML341) and CID 44492448." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Dose-dependent Activity of the Probe (CID 44493436/ ML341) and CID 44492448</span></h3></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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