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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Discovery of ML367, inhibitor of ATAD5 stabilization - 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="Discovery of ML367, inhibitor of ATAD5 stabilization">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/11/14">
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<meta name="citation_author" content="Jason M. Rohde">
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<meta name="citation_author" content="Ganesha Rai">
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<meta name="citation_author" content="Yong Jun Choi">
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<meta name="citation_author" content="Srilatha Sakamuru">
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<meta name="citation_author" content="Jennifer T. Fox">
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<meta name="citation_author" content="Ruili Huang">
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<meta name="citation_author" content="Menghang Xia">
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<meta name="citation_author" content="Kyungjae Myung">
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<meta name="citation_author" content="Matthew B. Boxer">
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<meta name="citation_author" content="David J. Maloney">
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<meta name="DC.Title" content="Discovery of ML367, inhibitor of ATAD5 stabilization">
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<meta name="DC.Contributor" content="Jason M. Rohde">
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<meta name="DC.Contributor" content="Ganesha Rai">
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<meta name="DC.Contributor" content="Yong Jun Choi">
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<meta name="DC.Contributor" content="Srilatha Sakamuru">
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<meta name="DC.Contributor" content="Jennifer T. Fox">
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<meta name="DC.Contributor" content="Ruili Huang">
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<meta name="DC.Contributor" content="Menghang Xia">
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<meta name="DC.Contributor" content="Kyungjae Myung">
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<meta name="DC.Contributor" content="Matthew B. Boxer">
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<meta name="DC.Contributor" content="David J. Maloney">
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<meta name="DC.Date" content="2013/11/14">
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<meta name="description" content="Encoding the genetic instructions essential to both our development and function as living organisms, our DNA must be maintained with exquisite precision and integrity, especially throughout replication [1, 2]. DNA can undergo damage in many different ways by both endogenous and exogenous agents. Thus, the numerous mechanisms by which DNA damage is both recognized and repaired are essential to cell survival. ATAD5 is involved in the DNA damage response, and its protein level increases in response to DNA damage without an increase in mRNA transcription [3, 4]. Identification of pathway(s) that stabilize ATAD5 protein levels in response to DNA damage and inhibitors of these pathway(s) would be beneficial to understanding a novel mechanism involved in the DNA damage response and introduce a new therapeutic approach for sensitizing cancer cells, respectively [5, 6]. However, no chemical matter is currently known that perturbs ATAD5 function. To understand the biology of ATAD5 and to evaluate its therapeutic potential, we conducted a quantitative high throughput screening campaign and subsequent medicinal chemistry optimization in pursuit of small molecules that destabilize ATAD5. Herein, we detail the discovery of ML367, a probe molecule that has low micromolar inhibitory activity in the ATAD5 destabilizer screen run with 10 μM 5-fluorouridine (5-FUrd) as the DNA damaging agent. Interestingly, ML367 was found to block general DNA damage responses including RPA32-phosphorylation and CHK1-phosphorylation in response to UV irradiation. In this regard, the probe molecule could block DNA repair pathways that function upstream of ATAD5. Additionally, the compound sensitized cells possessing a knock-out mutation of the PARP1 gene and as a result may serve as a sensitizer to kill cancer cells defective in the poly (ADP-ribose) polymerase 1 (PARP1)-dependent DNA repair pathway.">
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<meta name="og:description" content="Encoding the genetic instructions essential to both our development and function as living organisms, our DNA must be maintained with exquisite precision and integrity, especially throughout replication [1, 2]. DNA can undergo damage in many different ways by both endogenous and exogenous agents. Thus, the numerous mechanisms by which DNA damage is both recognized and repaired are essential to cell survival. ATAD5 is involved in the DNA damage response, and its protein level increases in response to DNA damage without an increase in mRNA transcription [3, 4]. Identification of pathway(s) that stabilize ATAD5 protein levels in response to DNA damage and inhibitors of these pathway(s) would be beneficial to understanding a novel mechanism involved in the DNA damage response and introduce a new therapeutic approach for sensitizing cancer cells, respectively [5, 6]. However, no chemical matter is currently known that perturbs ATAD5 function. To understand the biology of ATAD5 and to evaluate its therapeutic potential, we conducted a quantitative high throughput screening campaign and subsequent medicinal chemistry optimization in pursuit of small molecules that destabilize ATAD5. Herein, we detail the discovery of ML367, a probe molecule that has low micromolar inhibitory activity in the ATAD5 destabilizer screen run with 10 μM 5-fluorouridine (5-FUrd) as the DNA damaging agent. Interestingly, ML367 was found to block general DNA damage responses including RPA32-phosphorylation and CHK1-phosphorylation in response to UV irradiation. In this regard, the probe molecule could block DNA repair pathways that function upstream of ATAD5. Additionally, the compound sensitized cells possessing a knock-out mutation of the PARP1 gene and as a result may serve as a sensitizer to kill cancer cells defective in the poly (ADP-ribose) polymerase 1 (PARP1)-dependent DNA repair pathway.">
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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="_NBK179831_"><span class="title" itemprop="name">Discovery of ML367, inhibitor of ATAD5 stabilization</span></h1><p class="contribs">Rohde JM, Rai G, Choi YJ, et al.</p><p class="fm-aai"><a href="#_NBK179831_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>Encoding the genetic instructions essential to both our development and function as living organisms, our DNA must be maintained with exquisite precision and integrity, especially throughout replication [<a class="bibr" href="#ml367.r1" rid="ml367.r1">1</a>, <a class="bibr" href="#ml367.r2" rid="ml367.r2">2</a>]. DNA can undergo damage in many different ways by both endogenous and exogenous agents. Thus, the numerous mechanisms by which DNA damage is both recognized and repaired are essential to cell survival. ATAD5 is involved in the DNA damage response, and its protein level increases in response to DNA damage without an increase in mRNA transcription [<a class="bibr" href="#ml367.r3" rid="ml367.r3">3</a>, <a class="bibr" href="#ml367.r4" rid="ml367.r4">4</a>]. Identification of pathway(s) that stabilize ATAD5 protein levels in response to DNA damage and inhibitors of these pathway(s) would be beneficial to understanding a novel mechanism involved in the DNA damage response and introduce a new therapeutic approach for sensitizing cancer cells, respectively [<a class="bibr" href="#ml367.r5" rid="ml367.r5">5</a>, <a class="bibr" href="#ml367.r6" rid="ml367.r6">6</a>]. However, no chemical matter is currently known that perturbs ATAD5 function. To understand the biology of ATAD5 and to evaluate its therapeutic potential, we conducted a quantitative high throughput screening campaign and subsequent medicinal chemistry optimization in pursuit of small molecules that destabilize ATAD5. Herein, we detail the discovery of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>, a probe molecule that has low micromolar inhibitory activity in the ATAD5 destabilizer screen run with 10 μM 5-fluorouridine (5-FUrd) as the DNA damaging agent. Interestingly, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> was found to block general DNA damage responses including RPA32-phosphorylation and CHK1-phosphorylation in response to UV irradiation. In this regard, the probe molecule could block DNA repair pathways that function upstream of ATAD5. Additionally, the compound sensitized cells possessing a knock-out mutation of the <i>PARP1</i> gene and as a result may serve as a sensitizer to kill cancer cells defective in the poly (ADP-ribose) polymerase 1 (PARP1)-dependent DNA repair pathway.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> <a href="/nuccore/1523394213" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">MH092164</a></p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Kyungjae Myung, National Human Genome Research Institute, NIH</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493125" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493125</a></p><div id="ml367.s1"><h2 id="_ml367_s1_">Probe Structure & Characteristics</h2><div id="ml367.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK179831/bin/ml367fu1.jpg" alt="ML367." /></div><h3><span class="title">ML367</span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml367tu1"><a href="/books/NBK179831/table/ml367.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml367tu1" rid-ob="figobml367tu1"><img class="small-thumb" src="/books/NBK179831/table/ml367.tu1/?report=thumb" src-large="/books/NBK179831/table/ml367.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml367.tu1"><a href="/books/NBK179831/table/ml367.tu1/?report=objectonly" target="object" rid-ob="figobml367tu1">Table</a></h4></div></div></div><div id="ml367.s2"><h2 id="_ml367_s2_">1. Recommendations for Scientific Use of the Probe</h2><p><i>ATAD5</i> is a known suppressor of genomic instability and tumor formation in mice. ATAD5 protein levels increase in response to DNA damage and thus an inhibitor of ATAD5 stabilization, such as <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>, could sensitize cancer cells to DNA damaging agents. In this study, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> exhibited inhibition of ATAD5 stabilization in HEK293T cells as well as destabilization of the protein by western blot analysis. Moreover, our results demonstrate that treatment of cells deficient in DNA damage repair proteins (e.g. PARP1, Lig3, Lig4, FancM, FancG, and Rad54b) with <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> results in significant growth inhibition in colony formation assays. The <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> can therefore be used by investigators as a tool to further understand the role of ATAD5 in repair mechanism. Moreover, these data suggest a potential use of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> in combination with inhibitors of DNA repair proteins (e.g. PARP1) and/or cancer cells deficient in enzymes involved in the DNA repair response.</p></div><div id="ml367.s3"><h2 id="_ml367_s3_">2. Materials and Methods</h2><p><b>General Methods for Chemistry:</b> All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, <i>N,N</i>-dimethylforamide (DMF), acetonitrile, methanol, and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. <sup>1</sup>H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d<sub>6</sub> at 2.49 ppm) as internal standard for DMSO-<i>d</i><sub>6</sub> solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).</p><p><b>High-throughput Screen Materials:</b> Dimethyl sulfoxide (DMSO) ACS grade was obtained from Fisher, while ferrous ammonium sulfate, Xylenol Orange (XO), sulfuric acid, and Triton X-100 were obtained from Sigma-Aldrich.</p><div id="ml367.s4"><h3>2.1. Assays</h3><p><b>qHTS ATAD5 Assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504467" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504467</a>).</b> ATAD5-luc cells were dispensed at 2,000/4 μL/well into a tissue culture treated 1,536-well white/solid bottom assay plates (Greiner Bio-One) using a Multidrop Combi dispenser (Thermo Scientific). After the assay plates were incubated for 3–4 hr at 37 °C for the cell adherence, 23 nL of each compound was transferred via Pin Tool (Kalypsys) to columns 5–48 of the assay plates, resulting in the final concentrations ranging from 1.0 μM to 46 μM. DMSO was only included in columns 1 to 4. For antagonist screening, the compound transfer was followed by the addition of either 1 μL of culture medium (columns 1 and 3) or 5-FUrd (10 μM final concentration in rest of the columns), a known stabilizer of ATAD5. The assay plates were incubated for 16 hr at 37 °C, followed by the addition of Amplite Luciferase reagent (AAT Bioquest, Inc.) at 5 μL/well using a Flying Reagent Dispenser (FRD) (Aurora Discovery). After 30 min incubation at room temperature, the luminescence intensity was quantified using a ViewLux CCD-based plate reader (Perkin Elmer). Raw plate reads for each titration point were first normalized relative to FUrd control (10 μM, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plates.</p><p><b>Biochemical Luciferase Counter Screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686933</a>).</b> A biochemical was used to validate the inhibitor identified from the primary screen. Three μL of 10 μM substrate (50 mM Tris acetate, 13.3 mM Magnesium acetate, 0.01 mM D-Luc, 0.01 mM ATP, 0.01% Tween, 0.05% BSA and dH<sub>2</sub>O) was dispensed into 1,536-well white/solid bottom assay plates (Greiner Bio-One) using a Flying Reagent Dispenser (FRD) (Aurora Discovery). Twenty three nanoliter of each compound was transferred via Pin Tool (Kalypsys) to rows 1 – 30 of the assay plates resulting in the final concentrations ranging from 0.2 nM to 46 μM and DMSO only was transferred to rows 31 – 32. Then compound addition was followed by adding 1 μL of buffer (row 32 only) or enzyme; “Pyralis Luciferase” (0.04 μM, final concentration) for rest of the plate. After 5 min of incubation at room temperature, the luminescence intensity was quantified using a ViewLux CCD-based plate reader (Perkin Elmer). Raw plate reads for each titration point were first normalized relative to Pyralis luciferase control (0.04 μM, 100%) and DMSO only wells (basal, 0%) and then corrected by applying a pattern correction algorithm using compound free control plates (DMSO) plate.</p><p><b>Cell Viability Assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686921" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686921</a>).</b> To evaluate the cytotoxic effect of the inhibitors, a cell viability assay using HCT116 cell line was developed. 1 × 10<sup>4</sup> HCT116 or PARP-1 deficient cells were seeded into each well of a 96-well. After allowing the cells to attach to the bottom of the plate for 24 hours, compounds were added at serial dilutions from starting concentration of 40 μM. 48 hours following treatment, cell viability was determined using Cell Titer-Glo (Promega) according to the manufacturer’s protocol and quantified on a Fluoroskan Ascent Luminometer (Thermo Scientific).</p><p><b>FLAG-ATAD5 Transfections and Western Blotting.</b> To evaluate the cellular activity of the probe, a secondary assay using a different cell line was developed. HEK293T cells were transfected with FLAG-tagged ATAD5 using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s protocol. 48 hours post-transfection, the cells were treated with the indicated compounds for 16 hours. To obtain total lysate, the cells were resuspended in lysis buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, protease inhibitors (Roche)] and lysed on ice for 30 min. Proteins were separated by SDS-PAGE using a 4–15% Tris-glycine gel (Bio-Rad) and transferred to a Polyvinylidene difluoride membrane. FLAG-ATAD5 protein levels were detected by the ECL Western Blotting Detection System (GE Healthcare) using an HRP-conjugated antibody against FLAG (Sigma). Equal protein loading was confirmed using an antibody against tubulin (Abcam). The ratio of FLAG/Tubulin was quantified using ImageJ.</p></div><div id="ml367.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml367.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK179831/bin/ml367fu2.jpg" alt="Probe ML367 (CID 921541)." /></div><h3><span class="title">Probe ML367 (CID 921541)</span></h3><div class="caption"><p>*Purity > 98% as determined by LC/MS and <sup>1</sup>H NMR analyses.</p></div></div><p><b><i>N</i>-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine (<a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a></b>): LC-MS Retention Time: t<sub>1</sub> (Method 1) = 4.368 min and t<sub>2</sub> (Method 2) = 2.845 min; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 10.20 (s, 1H), 8.90 – 8.83 (m, 2H), 8.59 (d, <i>J</i> = 8.3 Hz, 1H), 8.46 – 8.40 (m, 2H), 8.09 (ddd, <i>J</i> = 13.2 Hz, 7.5 Hz & 2.6 Hz, 1H), 8.01 – 7.87 (m, 2H) and 7.80 – 7.44 (m, 3H); <sup>13</sup>C (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 158.18, 158.03, 155.98, 150.10, 149.97, 149.81, 148.74, 147.68, 147.55, 147.16, 147.10, 146.99, 144.74, 144.61, 135.88, 135.85, 135.78, 133.95, 128.42, 127.59, 123.17, 123.09, 122.98, 122.92, 122.83, 119.00, 117.53, 117.20, 114.36, 111.86, 111.81, 111.66 and 111.60; HRMS: <i>m/z</i> (M+H)<sup>+</sup> = 335.1086 (Calculated for C<sub>19</sub>H<sub>13</sub>F<sub>2</sub> N<sub>4</sub> = 335.1103).</p><div id="ml367.f1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK179831/bin/ml367f1.jpg" alt="Figure 2. Structures of ML367 analogs with their corresponding Compound IDs listed in Table 1." /></div><h3><span class="label">Figure 2</span><span class="title">Structures of ML367 analogs with their corresponding Compound IDs listed in <a class="figpopup" href="/books/NBK179831/table/ml367.t1/?report=objectonly" target="object" rid-figpopup="figml367t1" rid-ob="figobml367t1">Table 1</a></span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml367t1"><a href="/books/NBK179831/table/ml367.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml367t1" rid-ob="figobml367t1"><img class="small-thumb" src="/books/NBK179831/table/ml367.t1/?report=thumb" src-large="/books/NBK179831/table/ml367.t1/?report=previmg" alt="Table 1. List of the ML367 probe and related analogs." /></a><div class="icnblk_cntnt"><h4 id="ml367.t1"><a href="/books/NBK179831/table/ml367.t1/?report=objectonly" target="object" rid-ob="figobml367t1">Table 1</a></h4><p class="float-caption no_bottom_margin">List of the ML367 probe and related analogs. </p></div></div></div><div id="ml367.s6"><h3>2.3. Probe Preparation</h3><p>Preparation of <i>N</i>-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine (<a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>) is a two-step process described below and illustrated in <a class="figpopup" href="/books/NBK179831/figure/ml367.f5/?report=objectonly" target="object" rid-figpopup="figml367f5" rid-ob="figobml367f5">Scheme 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml367f5" co-legend-rid="figlgndml367f5"><a href="/books/NBK179831/figure/ml367.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml367f5" rid-ob="figobml367f5"><img class="small-thumb" src="/books/NBK179831/bin/ml367f5.gif" src-large="/books/NBK179831/bin/ml367f5.jpg" alt="Scheme 1. Synthetic route to ML367 is a 2 step process." /></a><div class="icnblk_cntnt" id="figlgndml367f5"><h4 id="ml367.f5"><a href="/books/NBK179831/figure/ml367.f5/?report=objectonly" target="object" rid-ob="figobml367f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to ML367 is a 2 step process. </p></div></div><ol><li class="half_rhythm"><div>A mixture of 2,4-dichloroquinazoline, 3,4-difluoroaniline and diisopropylethylamine ((<i>i</i>Pr)<sub>2</sub>NEt) in 2-propanol was heated at reflux with stirring for 16 hr. The solvent was removed and the crude product was triturated with water and sonicated, which caused the brown oil to become a tan solid. The solid was removed by filtration and was washed with water. 2-chloro-<i>N</i>-(3,4-difluorophenyl)quinazolin-4-amine was isolated as a tan solid and used in the next step without further purification.</div></li><li class="half_rhythm"><div>A mixture of 2-chloro-<i>N</i>-(3,4-difluorophenyl)quinazolin-4-amine, pyridin-4-ylboronic acid, and 2 molar solution of sodium carbonate and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh<sub>3</sub>)<sub>4</sub>) in dimethoxyethane was degassed with argon for 5 min then heated in a microwave for 30 min at 150 °C. The solvent was removed by forced air and the crude product was dissolved in dimethyl sulfoxide (DMSO) then stirred with palladium scavenger for 30 min. The solution was passed through a thiol cartridge and finally purified in preparative high-performance liquid chromatography (HPLC) to provide <i>N</i>-(3,4-difluorophenyl)-2-(pyridin-4-yl)quinazolin-4-amine as a trifluoroacetate (TFA) salt.</div></li></ol></div></div><div id="ml367.s7"><h2 id="_ml367_s7_">3. Results</h2><div id="ml367.s8"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml367.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK179831/bin/ml367f2.jpg" alt="Figure 3. Dose response curves for the probe ML367 against the ATAD5-Luc primary screen (green) and the cell viability assay (red)." /></div><h3><span class="label">Figure 3</span><span class="title">Dose response curves for the probe ML367 against the ATAD5-Luc primary screen (green) and the cell viability assay (red)</span></h3><div class="caption"><p>Results showed <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>’s dose response inhibition of ATAD5 activity without any significant cytotoxic effect.</p></div></div></div><div id="ml367.s9"><h3>3.2. Cellular Activity</h3><div id="ml367.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK179831/bin/ml367f3.jpg" alt="Figure 4. Inhibition of FLAG-ATAD5 stabilization by ML367." /></div><h3><span class="label">Figure 4</span><span class="title">Inhibition of FLAG-ATAD5 stabilization by ML367</span></h3><div class="caption"><p>HEK293T cells were transiently transfected with FLAG-tagged ATAD5 and treated with the indicated amount of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> for 16 hours in the presence or absence of 20 μM 5-FUrd. ATAD5 protein levels were visualized by western blotting using an antibody against FLAG (top panel), and quantified using ImageJ (bottom panel). Results showed inhibition of ATAD5 expression upon treatment of the probe.</p></div></div></div><div id="ml367.s10"><h3>3.3. Profiling Assays</h3><p>The preliminary absorption, distribution, metabolism, excretion (ADME) profile of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> supports its use as a valuable probe for ATAD5 destabilization. While both its microsomal stability (in rat and human) and solubility (in PBS buffer) are moderate, the latter was above the IC<sub>50</sub> determined in the cell based assay. <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> has good PAMPA permeability, and its overall ADME profile is consistent with its measured Log D of 1.58 (<a class="figpopup" href="/books/NBK179831/table/ml367.t2/?report=objectonly" target="object" rid-figpopup="figml367t2" rid-ob="figobml367t2">Table 2</a>). Additionally, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> showed good stability in mouse plasma as well as a series of aqueous stability assessments including pH 2 and pH 10 buffers and aqueous 5 mM glutathione (<a class="figpopup" href="/books/NBK179831/figure/ml367.f4/?report=objectonly" target="object" rid-figpopup="figml367f4" rid-ob="figobml367f4">Figure 1</a>). While the overall ADME profile may limit the utility of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>
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<i>in vivo</i>, results of solubility, permeability and biological activity showed good results.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml367t2"><a href="/books/NBK179831/table/ml367.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml367t2" rid-ob="figobml367t2"><img class="small-thumb" src="/books/NBK179831/table/ml367.t2/?report=thumb" src-large="/books/NBK179831/table/ml367.t2/?report=previmg" alt="Table 2. ADME profile of ML367." /></a><div class="icnblk_cntnt"><h4 id="ml367.t2"><a href="/books/NBK179831/table/ml367.t2/?report=objectonly" target="object" rid-ob="figobml367t2">Table 2</a></h4><p class="float-caption no_bottom_margin">ADME profile of ML367. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml367f4" co-legend-rid="figlgndml367f4"><a href="/books/NBK179831/figure/ml367.f4/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml367f4" rid-ob="figobml367f4"><img class="small-thumb" src="/books/NBK179831/bin/ml367f4.gif" src-large="/books/NBK179831/bin/ml367f4.jpg" alt="Figure 1. Stability of ML367 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS buffer pH 7.4, (B) assay buffer, (C) pH 2 and (D) pH 10 buffers; and (E) 5 mM solution of Glutathione over 24 hr." /></a><div class="icnblk_cntnt" id="figlgndml367f4"><h4 id="ml367.f4"><a href="/books/NBK179831/figure/ml367.f4/?report=objectonly" target="object" rid-ob="figobml367f4">Figure 1</a></h4><p class="float-caption no_bottom_margin">Stability of ML367 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS buffer pH 7.4, (B) assay buffer, (C) pH 2 and (D) pH 10 buffers; and (E) 5 mM solution of <a href="/books/NBK179831/figure/ml367.f4/?report=objectonly" target="object" rid-ob="figobml367f4">(more...)</a></p></div></div></div></div><div id="ml367.s11"><h2 id="_ml367_s11_">4. Discussion</h2><div id="ml367.s12"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>As no prior art for inhibitors of ATAD5 stabilization exist, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> represents an important tool for the scientific community to begin to understand the protein’s role in DNA repair as well as other biological modalities. First, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> can be used to dissect initial events in the DNA damage response. The molecular mechanism of action in which <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> destabilizes TEL2 will unveil how DNA damage can activate TEL2 and its downstream targets. Many of these targets have been suggested to play important roles in tumorigenesis. Additionally, a number of genetic disorders are the result of mutations in these genes. For instance, ataxia telansiectasia (AT) is caused by a mutation in ATM, Seckel syndrome is caused by a hypomorphic mutation in ATR, and severe combined immunodeficiency is caused by a mutation in DNA-PKcs. As no prior art exists for inhibitors of ATAD5 or TEL2 stabilization, <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> is positioned to be a novel tool to study the molecular mechanism of DNA damage response and the resultant signal cascade with a potentially wide application to cancer and other genetic diseases.</p></div></div><div id="ml367.s13"><h2 id="_ml367_s13_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml367.r1">Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. <span><span class="ref-journal">Molecular cell. </span>2010;<span class="ref-vol">40</span>(2):179–204.</span> [<a href="/pmc/articles/PMC2988877/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2988877</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20965415" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20965415</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml367.r2">Zhou T, et al. Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts. <span><span class="ref-journal">Molecular cancer research: MCR. </span>2007;<span class="ref-vol">5</span>(8):813–22.</span> [<a href="/pmc/articles/PMC3607384/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3607384</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17699107" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17699107</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml367.r3">Michod D, Widmann C. DNA-damage sensitizers: potential new therapeutical tools to improve chemotherapy. <span><span class="ref-journal">Crit Rev Oncol Hematol. </span>2007;<span class="ref-vol">63</span>(2):160–71.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17544289" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17544289</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml367.r4">Lee KY, et al. ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin. <span><span class="ref-journal">The Journal of cell biology. </span>2013;<span class="ref-vol">200</span>(1):31–44.</span> [<a href="/pmc/articles/PMC3542800/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3542800</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23277426" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23277426</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml367.r5">Bell DW, et al. Predisposition to cancer caused by genetic and functional defects of mammalian Atad5. <span><span class="ref-journal">PLoS genetics. </span>2011;<span class="ref-vol">7</span>(8):e1002245.</span> [<a href="/pmc/articles/PMC3161924/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3161924</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21901109" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21901109</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml367.r6">Michod D, Widmann C. DNA-damage sensitizers: potential new therapeutical tools to improve chemotherapy. <span><span class="ref-journal">Crit Rev Oncol Hematol. </span>2007;<span class="ref-vol">63</span>(2):160–71.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17544289" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17544289</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK179831_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Jason M. Rohde</span>,<sup>a</sup> <span itemprop="author">Ganesha Rai</span>,<sup>a</sup> <span itemprop="author">Yong Jun Choi</span>,<sup>b</sup> <span itemprop="author">Srilatha Sakamuru</span>,<sup>a</sup> <span itemprop="author">Jennifer T. Fox</span>,<sup>a</sup> <span itemprop="author">Ruili Huang</span>,<sup>a</sup> <span itemprop="author">Menghang Xia</span>,<sup>a</sup> <span itemprop="author">Kyungjae Myung</span>,<sup>b</sup> <span itemprop="author">Matthew B. Boxer</span>,<sup>a</sup><sup>,*</sup> and <span itemprop="author">David J. Maloney</span><sup>a</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD.</div><div class="affiliation"><sup>b</sup>
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Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD.</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@dyenolam" class="oemail">vog.hin.liam@dyenolam</a>,
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@mrexob" class="oemail">vog.hin.liam@mrexob</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">April 15, 2013</span>; Last Update: <span itemprop="dateModified">November 14, 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>Rohde JM, Rai G, Choi YJ, et al. Discovery of ML367, inhibitor of ATAD5 stabilization. 2013 Apr 15 [Updated 2013 Nov 14]. 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/ml368/?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/ml366/?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="figobml367fu1"><div id="ml367.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367fu1.jpg" alt="ML367." /></div><h3><span class="title">ML367</span></h3></div></article><article data-type="table-wrap" id="figobml367tu1"><div id="ml367.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179831/table/ml367.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml367.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml367.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml367.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml367.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml367.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml367.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml367.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml367.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml367.tu1_1_1_1_1" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">CID 921541/<a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a></td><td headers="hd_h_ml367.tu1_1_1_1_2" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">ATAD5</td><td headers="hd_h_ml367.tu1_1_1_1_3" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">1.2 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 161004434</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686921" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686921</a>]</td><td headers="hd_h_ml367.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CMV-Luc Counter (cell-based)</td><td headers="hd_h_ml367.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 46 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 161004434</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686934" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686934</a>]</td><td headers="hd_h_ml367.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 40-fold</td><td headers="hd_h_ml367.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CMV Luc Counter (cell-based) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 161004434</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686934" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686934</a>]</td></tr><tr><td headers="hd_h_ml367.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Luc Counter (Biochemical)</td><td headers="hd_h_ml367.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 57 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 161004434</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686933</a>]</td><td headers="hd_h_ml367.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 50-fold</td><td headers="hd_h_ml367.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Luc Counter (Biochemical) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 161004434</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/686933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 686933</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml367fu2"><div id="ml367.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367fu2.jpg" alt="Probe ML367 (CID 921541)." /></div><h3><span class="title">Probe ML367 (CID 921541)</span></h3><div class="caption"><p>*Purity > 98% as determined by LC/MS and <sup>1</sup>H NMR analyses.</p></div></div></article><article data-type="fig" id="figobml367f5"><div id="ml367.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367f5.jpg" alt="Scheme 1. Synthetic route to ML367 is a 2 step process." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthetic route to ML367 is a 2 step process</span></h3></div></article><article data-type="fig" id="figobml367f4"><div id="ml367.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Stability%20of%20ML367%20measured%20as%20percent%20composition%20of%20probe%20molecule%20in%20aqueous%20solution%20(contains%2020%20%25%20acetonitrile)%20at%20room%20temperature%20over%2048%20hr%20in%20(A)%20DPBS%20buffer%20pH%207.4%2C%20(B)%20assay%20buffer%2C%20(C)%20pH%202%20and%20(D)%20pH%2010%20buffers%3B%20and%20(E)%205%20mM%20solution%20of%20Glutathione%20over%2024%20hr.&p=BOOKS&id=179831_ml367f4.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/NBK179831/bin/ml367f4.jpg" alt="Figure 1. Stability of ML367 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS buffer pH 7.4, (B) assay buffer, (C) pH 2 and (D) pH 10 buffers; and (E) 5 mM solution of Glutathione over 24 hr." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Stability of ML367 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at room temperature over 48 hr in (A) DPBS buffer pH 7.4, (B) assay buffer, (C) pH 2 and (D) pH 10 buffers; and (E) 5 mM solution of Glutathione over 24 hr.</span></h3><div class="caption"><p><a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> was observed to be relatively stable in these test buffers. The slight decrease in area over time in the assay and pH 10 buffers were observed to be a result of compound precipitation. No other peaks observed via LC/MS were formed over 48 hr.</p></div></div></article><article data-type="fig" id="figobml367f1"><div id="ml367.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367f1.jpg" alt="Figure 2. Structures of ML367 analogs with their corresponding Compound IDs listed in Table 1." /></div><h3><span class="label">Figure 2</span><span class="title">Structures of ML367 analogs with their corresponding Compound IDs listed in <a class="figpopup" href="/books/NBK179831/table/ml367.t1/?report=objectonly" target="object" rid-figpopup="figml367t1" rid-ob="figobml367t1">Table 1</a></span></h3></div></article><article data-type="table-wrap" id="figobml367t1"><div id="ml367.t1" class="table"><h3><span class="label">Table 1</span><span class="title">List of the ML367 probe and related analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179831/table/ml367.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml367.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Internal ID</th><th id="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ML #</th><th id="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Type</th><th id="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00262816</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820361</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">161004434</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">921541</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Probe</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00345418</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820362</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162010064" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162010064</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71295991</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00273992</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820363</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004441" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">161004441</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">9921994</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00345058</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820364</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004458" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">161004458</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">70789683</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00273492</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820365</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004437" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">161004437</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">70789685</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml367.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00345075</td><td headers="hd_h_ml367.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004820366</td><td headers="hd_h_ml367.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/161004464" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">161004464</a></td><td headers="hd_h_ml367.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">70789693</td><td headers="hd_h_ml367.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml367.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml367.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml367f2"><div id="ml367.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367f2.jpg" alt="Figure 3. Dose response curves for the probe ML367 against the ATAD5-Luc primary screen (green) and the cell viability assay (red)." /></div><h3><span class="label">Figure 3</span><span class="title">Dose response curves for the probe ML367 against the ATAD5-Luc primary screen (green) and the cell viability assay (red)</span></h3><div class="caption"><p>Results showed <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a>’s dose response inhibition of ATAD5 activity without any significant cytotoxic effect.</p></div></div></article><article data-type="fig" id="figobml367f3"><div id="ml367.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK179831/bin/ml367f3.jpg" alt="Figure 4. Inhibition of FLAG-ATAD5 stabilization by ML367." /></div><h3><span class="label">Figure 4</span><span class="title">Inhibition of FLAG-ATAD5 stabilization by ML367</span></h3><div class="caption"><p>HEK293T cells were transiently transfected with FLAG-tagged ATAD5 and treated with the indicated amount of <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> for 16 hours in the presence or absence of 20 μM 5-FUrd. ATAD5 protein levels were visualized by western blotting using an antibody against FLAG (top panel), and quantified using ImageJ (bottom panel). Results showed inhibition of ATAD5 expression upon treatment of the probe.</p></div></div></article><article data-type="table-wrap" id="figobml367t2"><div id="ml367.t2" class="table"><h3><span class="label">Table 2</span><span class="title">ADME profile of ML367</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179831/table/ml367.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml367.t2_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml367.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml367.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PBS buffer (pH 7.4) Solubility (μM)</th><th id="hd_h_ml367.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Microsomal Stability (T<sub>1/2</sub>)<sup>a</sup> or % remaining<sup>b</sup></th><th id="hd_h_ml367.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Permeability (PAMPA)</th><th id="hd_h_ml367.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Log D</th><th id="hd_h_ml367.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mouse Plasma Stability (T<sub>1/2</sub>)</th><th id="hd_h_ml367.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PBS buffer (pH 7.4) Stability at 48 h</th></tr></thead><tbody><tr><td headers="hd_h_ml367.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a></td><td headers="hd_h_ml367.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.9 μM</td><td headers="hd_h_ml367.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11 min<sup>a</sup> (rat)</td><td headers="hd_h_ml367.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57.9%<sup>b</sup> (human)</td><td headers="hd_h_ml367.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">376 × 10<sup>−6</sup> cm/s</td><td headers="hd_h_ml367.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.58</td><td headers="hd_h_ml367.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 90 min</td><td headers="hd_h_ml367.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100%</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml367.tfn1"><p class="no_margin">Represents the stability to rat liver microsomes (T<sub>1/2</sub>) in the presence of NADPH.</p></div></dd></dl><dl class="bkr_refwrap"><dt>b</dt><dd><div id="ml367.tfn2"><p class="no_margin">Represents the stability to human liver microsomes (% remaining after 30 minutes) in the presence of NADPH. <a href="/pcsubstance/?term=ML367[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML367</a> showed no degradation without NADPH present over a 1 hr period.</p></div></dd></dl></dl></div></div></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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