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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Inhibitors of Protein Folding: DnaK - 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="Inhibitors of Protein Folding: DnaK">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2010/09/02">
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<meta name="citation_author" content="J Cellitti">
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<meta name="citation_author" content="Z Zhang">
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<meta name="citation_author" content="S Wang">
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<meta name="citation_author" content="B Wu">
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<meta name="citation_author" content="D Guiney">
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<meta name="citation_author" content="M Pellecchia">
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<meta name="citation_pmid" content="21433369">
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<meta name="DC.Title" content="Inhibitors of Protein Folding: DnaK">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="J Cellitti">
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<meta name="DC.Contributor" content="Z Zhang">
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<meta name="DC.Contributor" content="S Wang">
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<meta name="DC.Contributor" content="B Wu">
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<meta name="DC.Contributor" content="D Guiney">
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<meta name="DC.Contributor" content="M Pellecchia">
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<meta name="DC.Date" content="2010/09/02">
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<meta name="description" content="The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family. Specifically, these probes would alter DnaK through interactions with its substrate-binding domain (SBD). DnaK serves as a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression. The identified probe ML076 (CID-25105719) binds to DnaK at a novel allosteric site of the substrate binding domain, in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. ML076 and its closely related analogs described in this probe report might be useful as tools for dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. These molecular probes also might serve to validate DnaK as a possible target for novel antibacterial agents.">
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<meta name="og:title" content="Inhibitors of Protein Folding: DnaK">
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<meta name="og:description" content="The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family. Specifically, these probes would alter DnaK through interactions with its substrate-binding domain (SBD). DnaK serves as a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression. The identified probe ML076 (CID-25105719) binds to DnaK at a novel allosteric site of the substrate binding domain, in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. ML076 and its closely related analogs described in this probe report might be useful as tools for dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. These molecular probes also might serve to validate DnaK as a possible target for novel antibacterial agents.">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos 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="_NBK47353_"><span class="title" itemprop="name">Inhibitors of Protein Folding: DnaK</span></h1><p class="contribs">Cellitti J, Zhang Z, Wang S, et al.</p><p class="fm-aai"><a href="#_NBK47353_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>The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK, the E. coli bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family. Specifically, these probes would alter DnaK through interactions with its substrate-binding domain (SBD). DnaK serves as a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression. The identified probe <a href="/pcsubstance/?term=ML076[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML076</a> (CID-25105719) binds to DnaK at a novel allosteric site of the substrate binding domain, in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. <a href="/pcsubstance/?term=ML076[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML076</a> and its closely related analogs described in this probe report might be useful as tools for dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. These molecular probes also might serve to validate DnaK as a possible target for novel antibacterial agents.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> X01 MH078942-01</p><p><b>Screening Center Name & PI:</b>
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<i>Conrad Prebys</i> Center for Chemical Genomics (<i>formerly Burnham Center for Chemical Genomics</i>) & Dr. John C. Reed</p><p><b>Chemistry Center Name & PI:</b>
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<i>Conrad Prebys</i> Center for Chemical Genomics (<i>formerly Burnham Center for Chemical Genomics)</i> & Dr. John C. Reed</p><p><b>Assay Submitter & Institution: Dr.</b> Maurizio Pellecchia & Sanford-Burnham Medical Research Institute (<i>formerly Burnham Institute for Medical Research</i>)</p><p>
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<b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1501" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1501</a></p><div id="ml076.s2"><h2 id="_ml076_s2_">Probe Structure & Characteristics</h2><p><b>Chemical name:</b> N-(1-benzothiophen-7-ylmethyl) thiophene-2-carboxamide</p><p><b>Reference name:</b> BI-88D10</p><p><b>MLS:</b> MLS0315930</p><p><b>SID:</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56427267</a></p><p><b>CID:</b> CID-25105719</p><p><b>Supplier:</b> Internal BCCG Synthesis</p><div id="ml076.fu1" class="figure"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu1.jpg" alt="Image ml076fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml076tu1"><a href="/books/NBK47353/table/ml076.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml076tu1" rid-ob="figobml076tu1"><img class="small-thumb" src="/books/NBK47353/table/ml076.tu1/?report=thumb" src-large="/books/NBK47353/table/ml076.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml076.tu1"><a href="/books/NBK47353/table/ml076.tu1/?report=objectonly" target="object" rid-ob="figobml076tu1">Table</a></h4></div></div></div><div id="ml076.s3"><h2 id="_ml076_s3_">Recommendations for the scientific use of this probe</h2><p>The described probe CID-25105719, binds to DnaK, the <i>E. coli</i> bacterial homolog of the eukaryotic protein-folding chaperone of the Hsp70 family, at a novel allosteric site of the substrate binding domain in a region that has been suggested as being involved in the allosteric communication with the adjacent ATPase domain. The reported compound and its closely related analogs described in this probe report may be useful as tools in dissecting the underlying mechanisms of the ATP-dependant substrate binding and release. In principle, the compounds can find applications also in validating DnaK as possible target for novel antibacterial agents.</p></div><div id="ml076.s4"><h2 id="_ml076_s4_">1. Scientific Rationale for Project</h2><p>The specific aim of this project was to identify small molecule binders that would modulate/alter the function of DnaK by specifically interacting with its substrate-binding domain (SBD). DnaK is a molecular chaperone essential to processes such as protein folding, translocation, degradation, and even gene expression (<a class="bibr" href="#ml076.r8" rid="ml076.r8">Schiene-Fischer 2002</a>, <a class="bibr" href="#ml076.r1" rid="ml076.r1">Bukau 2006</a>, <a class="bibr" href="#ml076.r14" rid="ml076.r14">Young 2004</a>). The central hypothesis is based on recent observations with some antimicrobial peptides from insects that bind to the SBD of DnaK and that consequently have demonstrated activity against clinical bacterial strains, including fluoroquinoline (<a class="bibr" href="#ml076.r6" rid="ml076.r6">Otvos 2000</a>) resistant ones. At least one peptide did not bind to Hsp70 suggesting that it may be feasible to identify selective small molecules inhibitors of DnaK with antimicrobial activity targeting its SBD. DnaK is also very interesting from a protein structure function perspective. It comprises two structural domains (<a class="figpopup" href="/books/NBK47353/figure/ml076.f1/?report=objectonly" target="object" rid-figpopup="figml076f1" rid-ob="figobml076f1">Figure 1, A and B</a>), an N-terminal nucleotide binding ATPase domain (NBD) and a C-terminal substrate binding domain (SBD) which contains a β-strand (β-domain) region that binds the protein substrate and an α-helical lid region that closes over bound substrate (<a class="bibr" href="#ml076.r10" rid="ml076.r10">Swain 2007</a>, <a class="bibr" href="#ml076.r2" rid="ml076.r2">Chang 2008</a>). DnaK cycles between two main states: an ATP bound state with low affinity for substrate, that is characterized by a tight interaction of the two domains, and an ADP bound state that has high substrate affinity with less interaction between the domains (<a class="bibr" href="#ml076.r3" rid="ml076.r3">Hu 2006</a>, <a class="bibr" href="#ml076.r11" rid="ml076.r11">Szabo 1994</a>). Structural and biochemical data have shown that residues from both domains (including loop L2,3 of the β-domain), as well as from the linker between them are required for communicating the nucleotide or substrate occupancy to the other respective domain (<a class="bibr" href="#ml076.r10" rid="ml076.r10">Swain 2007</a>, <a class="bibr" href="#ml076.r12" rid="ml076.r12">Vogel 2006a</a>,<a class="bibr" href="#ml076.r13" rid="ml076.r13">b</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml076f1" co-legend-rid="figlgndml076f1"><a href="/books/NBK47353/figure/ml076.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml076f1" rid-ob="figobml076f1"><img class="small-thumb" src="/books/NBK47353/bin/ml076f1.gif" src-large="/books/NBK47353/bin/ml076f1.jpg" alt="Figure 1" /></a><div class="icnblk_cntnt" id="figlgndml076f1"><h4 id="ml076.f1"><a href="/books/NBK47353/figure/ml076.f1/?report=objectonly" target="object" rid-ob="figobml076f1">Figure 1</a></h4></div></div><p>With the experience of protein kinase inhibitors in mind (<a class="bibr" href="#ml076.r5" rid="ml076.r5">Orchard 2002</a>), screens for Hsp70 inhibitors have focused on compounds targeting the ATP binding site, however, the ATP binding site of Hsp70 is rather hydrophilic and most interaction energy between the nucleotide (ATP or ADP) and the protein is derived from the phosphate groups (<a class="bibr" href="#ml076.r4" rid="ml076.r4">Liu 2007</a>). Hence, traditional HTS approaches targeting the ATP binding pocket of Hsp70 or DnaK are unlikely to produce viable hits. Therefore, the proposed NMR-based screen takes the novel approach of directly targeting the substrate binding domain of DnaK. Binders are subsequently used to interrogate in biophysical, biochemical and cell-based assay, the effect of the small molecule on any functional and/or phenotypical parameters.</p></div><div id="ml076.s5"><h2 id="_ml076_s5_">2. Project Description</h2><div id="ml076.s6"><h3>a. The original goal for probe characteristics</h3></div><div id="ml076.s7"><h3>b. Assay implementation and screening</h3><div id="ml076.s8"><h4>i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)</h4><p>The primary assay was an NMR-based chemical shift perturbation assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1033" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1033</a>) Spectra were acquired on a 600 MHz Bruker Avance equipped with TCI cryoprobe. Ligand binding was monitored by comparing the aliphatic region of 1D <sup>13</sup>C-filtered <sup>1</sup>H NMR spectra of 20 μM DnaK β-domain only protein solution (20 mM sodium phosphate buffer at pH 7.5 containing 90%/10% H<sub>2</sub>O/D<sub>2</sub>O or 99.5% D<sub>2</sub>O; T= 300 K) in the presence or absence of 80 μM mixtures of 10 compounds, and then individual compound mixtures that caused significant perturbations in the spectrum were characterized further. Simple 1D <sup>1</sup>H NMR experiments of the protein measured in presence and absence of mixtures of potential ligands, observing the aliphatic region of the spectra δ<1 ppm). Compound or mixtures causing spectral perturbation above a threshold of 0.08 ppm were deconvoluted.</p></div><div id="ml076.s9"><h4>ii. Assay Rationale & Description</h4><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml076tu2"><a href="/books/NBK47353/table/ml076.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml076tu2" rid-ob="figobml076tu2"><img class="small-thumb" src="/books/NBK47353/table/ml076.tu2/?report=thumb" src-large="/books/NBK47353/table/ml076.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml076.tu2"><a href="/books/NBK47353/table/ml076.tu2/?report=objectonly" target="object" rid-ob="figobml076tu2">Table</a></h4></div></div><p>The first secondary assay was to confirm binding by NMR by obtaining K<sub>d</sub>’s using isothermal calorimetry (ITC; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1495" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1495</a>). Titrations were done using a VP-ITC from MicroCal (Northampton, MA). Full-length DnaK or SBD were used at 100 μM in 20 mM sodium phosphate buffer (pH 7.4) and 0.5–5% DMSO. Compounds were used at 10–15x in the same buffer.</p><p>The ATPase activity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1494" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1494</a>) of confirmed binders were determined using the ADAPTA™ kit from Invitrogen at 200 µM of compounds. Positive inhibition would suggest allosteric cross-talk between the domains of DnaK even though compounds bind to only the SBD. This assay cannot be considered a bona fide secondary assay because the other co-chaperone proteins are involved in the process and substrate and cofactor binding and release.</p></div><div id="ml076.s10"><h4>iii. Summary of Results</h4><p>Using a construct of DnaK corresponding to the β-domain only (residues 393–507) whose solution structures with and without bound peptide are known (<a class="bibr" href="#ml076.r7" rid="ml076.r7">Pellecchia 2000</a>, <a class="bibr" href="#ml076.r9" rid="ml076.r9">Stevens 2003</a>) a simple 1D <sup>1</sup>H NMR based screen of an assembled a scaffold library composed of ~4,000 member was completed by monitoring the aliphatic region of the spectra δ<1 ppm) in the presence and absence pools of 10 compounds at 80 µM per compound binding to 10–20 µM of protein. Mixtures causing spectral perturbation above the threshold of 0.08 ppm were deconvoluted, and additionally reconfirmed for binding by ITC. The compound BI-88B12 emerged as a hit from the screen (Table I, in appendices).</p><p>After identifying initial binders, chemical shift mapping studies with <sup>13</sup>C and <sup>15</sup>N labeled protein suggested that these compounds do not bind to the substrate binding pocket but rather to a pocket located around loop L2,3 on the opposite side of the SBD (<a class="figpopup" href="/books/NBK47353/figure/ml076.f1/?report=objectonly" target="object" rid-figpopup="figml076f1" rid-ob="figobml076f1">Figure 1, A and B</a>). Further molecular docking studies predict binding of compounds BI-88B12 and BI-88D7 in a deep pocket near residues Leu484 and Pro419 reported to be essential for the allosteric communication between the substrate binding and the ATPase domains (<a class="bibr" href="#ml076.r10" rid="ml076.r10">Swain 2007</a>, <a class="bibr" href="#ml076.r12" rid="ml076.r12">Vogel 2006a</a>,<a class="bibr" href="#ml076.r13" rid="ml076.r13">b</a>). These studies also suggested these two compounds bind in different orientations. BI-88B12 places its thiophene ring outside the pocket, this moiety is inserted into the pocket in BI-88D7. For BI-88B12, the fluorobenzene group is inserted deep into the hydrophobic pocket as evidenced by the strongest perturbation of residues there. With its thiophene group in the pocket, the indole ring of BI-88D7 is also able to interact with Asp481, located further out near the edge of the pocket. BI-88D7 causes equal perturbation of both Leu484 and Asp481, possibly due to its increased size that allows it to bridge both residues. This result possibly explains the much lower <i>K</i><sub>d</sub> of BI-88D7 as determined by ITC (see <a class="figpopup" href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-figpopup="figml076t1" rid-ob="figobml076t1">Table 1</a>).</p><p>Interestingly, BI-88B12 was indeed found to be an inhibitor of the DnaK ATPase activity with an IC50 of 2 mM, whereas BI-88D7 did not give significant ATPase activity even at 20 mM (Table, below). Interestingly, further studies suggest that the binding of probe BI88D10 alters allosterically the affinity for ATP by ITC (please refer to table 2 of the attached pre-publication manuscript from the assay provider.</p></div></div><div id="ml076.s11"><h3>c. Probe Optimization</h3><div id="ml076.s12"><h4>i. SAR & chemistry strategy that led to the probe</h4><p>An additional 5 commercially available BI-88B12 analogs were qualitatively assessed for their
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ability to bind the β-domain of DnaK by the magnitude of the chemical shifts induced upon
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complexation on the <sup>13</sup>C<sup>δ</sup>,<sup>1</sup>H<sup>δ</sup> resonances
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of Leu484 (1:10 protein to ligand ratio, at 70 μM protein concentration) by 2D
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[<sup>13</sup>C, <sup>1</sup>H] correlation spectra. Hit compounds with estimated
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<i>K</i><sub>d</sub> values of 200 μM or less were selected for further SAR
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studies including <i>K</i><sub>d</sub> determination by ITC. These data were further
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analyzed to design 10 additional analogues that were for chemical syntheses. The various ID numbers,
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structures, molecular weights, activity data and source (commercial or synthesized) for the selected
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compounds are summarized in <a class="figpopup" href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-figpopup="figml076t1" rid-ob="figobml076t1">Table 1</a> below:</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml076t1"><a href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml076t1" rid-ob="figobml076t1"><img class="small-thumb" src="/books/NBK47353/table/ml076.t1/?report=thumb" src-large="/books/NBK47353/table/ml076.t1/?report=previmg" alt="Table 1. Selected purchased & synthesized compounds for SAR Development of confirmed hit BI-88B12." /></a><div class="icnblk_cntnt"><h4 id="ml076.t1"><a href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-ob="figobml076t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Selected purchased & synthesized compounds for SAR Development of confirmed hit BI-88B12. </p></div></div><p>Of the commercial analogs obtained, BI-88E3 (containing a furan moiety) improved the potency of binding to the SBD of DnaK, but with a loss of ATPase activity. However, BI-88C5 uncovered a compound with improved binding affinity to the DnaK SBD and showed binding to the full-length DnaK, while also recovering a significant amount of ATPase activity, and suggested that substitution of the furan by a thiophene may be a good strategy to improve potency and ATPase activity, though the previous docking studies indicated that despite a shared thiophene ring compounds could bind in different orientations with respect to this thiophene.</p><p>Of the ten additional synthesized compounds, significant improvement in affinity to DnaK of about 10-fold was achieved with compounds BI-88D5 and BI-88D9 over the original hit BI-88B12. The most dramatic improvement (>200-fold) was achieved with BI-88D10, though with loss of significant ATPase activity.</p></div></div></div><div id="ml076.s13"><h2 id="_ml076_s13_">3. Probe</h2><div id="ml076.s14"><h3>a. Chemical name</h3><p>N-(1-benzothiophen-7-ylmethyl)thiophene-2-carboxamide <b>[<a href="/pcsubstance/?term=ML076[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML076</a>]</b></p></div><div id="ml076.s15"><h3>b. Probe chemical structure</h3><div id="ml076.fu2" class="figure"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu1.jpg" alt="Image ml076fu1" /></div></div></div><div id="ml076.s16"><h3>c. Structural Verification Information of probe SID</h3><p><sup>1</sup>H NMR (CDCl<sub>3</sub>, 600MHz): δ 8.35 (s, 1H), 7.82 (m, 5H), 7.47 (s, 1H), 6.97 (s, 1H), 5.30 (s, 2H); HRESI-TOF-MS: calcd for C<sub>14</sub>H<sub>11</sub>NOS<sub>2</sub> 274.0355 [M + H]<sup>+</sup>, found 274.0361.</p></div><div id="ml076.s17"><h3>d. PubChem CID (corresponding to the SID)</h3><p>CID-25105719</p></div><div id="ml076.s18"><h3>e. Availability from a vendor</h3><p>Probe was synthesized internally. A 15 mg sample of the probe compound and 5 analogs have been submitted to the MLSMR.</p></div><div id="ml076.s19"><h3>f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml076t2"><a href="/books/NBK47353/table/ml076.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml076t2" rid-ob="figobml076t2"><img class="small-thumb" src="/books/NBK47353/table/ml076.t2/?report=thumb" src-large="/books/NBK47353/table/ml076.t2/?report=previmg" alt="Table 2. Probe and analog submission." /></a><div class="icnblk_cntnt"><h4 id="ml076.t2"><a href="/books/NBK47353/table/ml076.t2/?report=objectonly" target="object" rid-ob="figobml076t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Probe and analog submission. </p></div></div></div><div id="ml076.s20"><h3>g. Mode of action for biological activity of probe</h3><p>By attempting to target the substrate binding β-domain of DnaK we have discovered compounds that bind not in the substrate-binding pocket but rather in a cavity involved in allosteric communication between protein domains. We also have discovered compounds with sub-micromolar affinity for DnaK, the most potent to date is the probe molecule CID25105719, as demonstrated by NMR and ITC against the β-domain. Several related analogs also bind the β-domain and the full length protein with single digit micromolar potency. The assay provider intends to use the probe molecule and analogs to “probe” the allosteric communication between domains <i>in vitro</i> and eventually the role of DnaK on bacterial growth in cell and <i>in vivo</i>.</p></div><div id="ml076.s21"><h3>h. Detailed synthetic pathway for making probe</h3><p><i>Chemical Synthesis</i>- The actual probe molecule <i>BI-88D10 CID-25105719 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56427267</a>]</i> was synthesized.</p><p>The details for synthesized analogs in <a class="figpopup" href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-figpopup="figml076t1" rid-ob="figobml076t1">Table 1</a> are
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described in <a href="#ml076.s25">Appendix 4a</a> reported below and the attached pre-print manuscript (see <a href="#ml076.s29">appendix 4c.</a> and Table I below). The QC data for the probe molecule are also listed therein.</p></div><div id="ml076.s22"><h3>i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)</h3></div><div id="ml076.s23"><h3>j. Properties Computed from Structure</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml076tu3"><a href="/books/NBK47353/table/ml076.tu3/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml076tu3" rid-ob="figobml076tu3"><img class="small-thumb" src="/books/NBK47353/table/ml076.tu3/?report=thumb" src-large="/books/NBK47353/table/ml076.tu3/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml076.tu3"><a href="/books/NBK47353/table/ml076.tu3/?report=objectonly" target="object" rid-ob="figobml076tu3">Table</a></h4></div></div></div></div><div id="ml076.s24"><h2 id="_ml076_s24_">4. Appendices</h2><div id="ml076.s25"><h3>a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes</h3><p>See <a class="figpopup" href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-figpopup="figml076t1" rid-ob="figobml076t1">Table 1</a>.</p><p>The general Scheme for synthesis (<a class="figpopup" href="/books/NBK47353/figure/ml076.f2/?report=objectonly" target="object" rid-figpopup="figml076f2" rid-ob="figobml076f2">Scheme 1</a>) and the detailed synthetic pathway for making selected analogs of <a class="figpopup" href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object" rid-figpopup="figml076t1" rid-ob="figobml076t1">Table 1</a> and <a class="figpopup" href="/books/NBK47353/table/ml076.t2/?report=objectonly" target="object" rid-figpopup="figml076t2" rid-ob="figobml076t2">2</a> are described below.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml076f2" co-legend-rid="figlgndml076f2"><a href="/books/NBK47353/figure/ml076.f2/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml076f2" rid-ob="figobml076f2"><img class="small-thumb" src="/books/NBK47353/bin/ml076f2.gif" src-large="/books/NBK47353/bin/ml076f2.jpg" alt="Scheme 1. General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives." /></a><div class="icnblk_cntnt" id="figlgndml076f2"><h4 id="ml076.f2"><a href="/books/NBK47353/figure/ml076.f2/?report=objectonly" target="object" rid-ob="figobml076f2">Scheme 1</a></h4><p class="float-caption no_bottom_margin">General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives. </p></div></div><div id="ml076.s26"><h4>Chemical Synthesis</h4><p>To a stirred solution of free amine (1.0 equiv.) and Et<sub>3</sub>N (2.0 equiv.) in DCM was added a solution of thiophene-2-carbonyl chloride (1.1 equiv.) in DCM at −30 °C. The resulting solution was stirred for 1h and then allowed to warm up to room temperature. After removal of the solvent, the residue was purified by flash column chromatography to provide the correspondent product (yield 75–95%).</p></div><div id="ml076.s27"><h4>Probe molecule (CID-25105719)</h4><p><i>BI-88D10</i>-N-(benzo[b]thiophen-7-ylmethyl)thiophene-2-carboxamide <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600MHz): δ 8.35 (s, 1H), 7.82 (m, 5H), 7.47 (s, 1H), 6.97 (s, 1H), 5.30 (s, 2H); HRESI-TOF-MS: calcd for C<sub>14</sub>H<sub>11</sub>NOS<sub>2</sub> 274.0355 [M + H]<sup>+</sup>, found 274.0361.</p><p><i>BI-88D5</i>- N-(naphthalen-1-ylmethyl)thiophene-2-carboxamide <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600MHz): δ 8.17 (s, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.46 (m, 6H), 6.99 (s, 1 H), 6.39 (s, 1H), 5.01 (s, 2H); HRESI-TOF-MS: calcd for C<sub>16</sub>H<sub>13</sub>NOS 268.0791 [M + H]+, found 268.0796.</p><p><i>BI-88D7</i>- N-(1H-indol-5-yl)thiophene-2-carboxamide <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600MHz): δ 8.19 (s, 1H), 7.92 (s, 1H), 7.71 (s, 1H), 7.62 (s, 1H), 7.52 (s, 1H), 7.33 (m, 2H), 7.23 (s, 1H), 7.12 (s, 1H), 6.54 (s, 1H); HRESI-TOF-MS: calcd for C<sub>13</sub>H<sub>10</sub>N<sub>2</sub>OS 243.0587 [M + H]+, found 243.0592. <i>BI-88D6</i>- Is an isomer and side product of BI-88D7 synthesis.</p><p><i>BI-88D9</i>- N-(quinazolin-2-yl)thiophene-2-carboxamide <sup>1</sup>H NMR (CDCl<sub>3</sub>, 600MHz): δ 9.35 (s, 1H), 7.90 (m, 3H), 7.61 (m, 4H), 6.89 (s, 1H); HRESI-TOF-MS: calcd for C<sub>13</sub>H<sub>9</sub>N<sub>3</sub>OS 256.0539 [M + H]<sup>+</sup>, found 256.0545.</p></div></div><div id="ml076.s28"><h3>b. Comparative data showing probe specificity for target</h3><p>See <a class="figpopup" href="/books/NBK47353/table/ml076.t2/?report=objectonly" target="object" rid-figpopup="figml076t2" rid-ob="figobml076t2">Table 2</a>.</p></div><div id="ml076.s29"><h3>c. Additional information</h3><p>The assay provider has completed more detailed studies that have been summarized in this probe report. They have also completed additional hypothesis generating experiments that use the probe and some of it’s related analogs as tools to dissect the relative contributions of small molecule binding, ATP nucleotide occupancy, and peptide substrate binding on the binding and function of the full length DnaK protein, that are beyond the scope of the initial MLSCN probe uncover. These do provide a good illustration of the use of small molecule probes on exploring complex interactions related to the specific mechanism of protein allostery for DnaK, and of protein allostery in general.</p></div></div><div id="ml076.s30"><h2 id="_ml076_s30_">5. Bibliography</h2><ol><li><div class="bk_ref" id="ml076.r1">Bukau B, Weissman J, Horwich A. <span><span class="ref-journal">Cell . </span>2006;<span class="ref-vol">125</span>:443–451.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16678092" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16678092</span></a>]</div></li><li><div class="bk_ref" id="ml076.r2">Chang YW, Sun YJ, Wang C, Hsiao CD. <span><span class="ref-journal">J Biol Chem . </span>2008;<span class="ref-vol">283</span>:15502–15511.</span> [<a href="/pmc/articles/PMC3258884/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3258884</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18400763" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18400763</span></a>]</div></li><li><div class="bk_ref" id="ml076.r3">Hu B, Mayer MP, Tomita M. <span><span class="ref-journal">Biophys J . </span>2006;<span class="ref-vol">91</span>:496–507.</span> [<a href="/pmc/articles/PMC1483108/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1483108</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16648174" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16648174</span></a>]</div></li><li><div class="bk_ref" id="ml076.r4">Liu Q, Hendrickson WA. <span><span class="ref-journal">Cell. </span>2007;<span class="ref-vol">131</span>:106–120.</span> [<a href="/pmc/articles/PMC2041797/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2041797</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17923091" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17923091</span></a>]</div></li><li><div class="bk_ref" id="ml076.r5">Orchard S. <span><span class="ref-journal">Curr Opin Drug Discov Devel. </span>2002;<span class="ref-vol">5</span>:713–717.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12630291" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12630291</span></a>]</div></li><li><div class="bk_ref" id="ml076.r6">Otvos LOI Jr, Rogers ME, Consolvo PJ, Condie BA, Lovas S, Bulet P, Blaszczyk-Thurin M. <span><span class="ref-journal">Biochemistry . </span>2000;<span class="ref-vol">39</span>:14150–14159.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11087363" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11087363</span></a>]</div></li><li><div class="bk_ref" id="ml076.r7">Pellecchia M, Montgomery DL, Stevens SY, Vander
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Inhibitors of Protein Folding: DnaK. 2009 Jan 15 [Updated 2010 Sep 2]. 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/ml077/?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/ml075/?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="figobml076fu1"><div id="ml076.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK47353/bin/ml076fu1.jpg" alt="Image ml076fu1" /></div></div></article><article data-type="table-wrap" id="figobml076tu1"><div id="ml076.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47353/table/ml076.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml076.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml076.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml076.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml076.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml076.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s )</th><th id="hd_h_ml076.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC5 0 (μM) [SID, AID]</th><th id="hd_h_ml076.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml076.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s)<br />Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml076.tu1_1_1_1_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:top;">CID-25105719<br /><br /><a href="/pcsubstance/?term=ML076[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML076</a></td><td headers="hd_h_ml076.tu1_1_1_1_2" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">DnaK<br />NMR</td><td headers="hd_h_ml076.tu1_1_1_1_3" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;"><200,000 nM) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56427267</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1033" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1033</a>]<br /><br /><i>Significantly perturbs chemical shift > 0.08 ppm</i></td><td headers="hd_h_ml076.tu1_1_1_1_4" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml076.tu1_1_1_1_5" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml076.tu1_1_1_1_6" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml076.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ITC Binding, 200 nM (Kd) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56427267</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1495" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1495</a>]</td></tr><tr><td headers="hd_h_ml076.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATPase, >20,000,000 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56427267</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1494" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1494</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml076f1"><div id="ml076.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47353/bin/ml076f1.jpg" alt="Figure 1" /></div><h3><span class="label">Figure 1</span></h3></div></article><article data-type="table-wrap" id="figobml076tu2"><div id="ml076.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47353/table/ml076.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml076.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml076.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Assay</th><th id="hd_h_ml076.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary or Secondary</th><th id="hd_h_ml076.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</th><th id="hd_h_ml076.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml076.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1D NMR screen</td><td headers="hd_h_ml076.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary</td><td headers="hd_h_ml076.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DnaK β-domain (residues 393–507) w/(MGSSHHHHHHGLVPRGS) at the N-terminus</td><td headers="hd_h_ml076.tu2_1_1_1_4" rowspan="2" colspan="1" style="text-align:left;vertical-align:top;">Assay provider Expressed in <i>E. coli</i> BL21(DE3) pLysS Purified by Ni<sup>2+</sup> affinity column</td></tr><tr><td headers="hd_h_ml076.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2D NMR</td><td headers="hd_h_ml076.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td><td headers="hd_h_ml076.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DnaK β-domain 15N, 13C labeled</td></tr><tr><td headers="hd_h_ml076.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ITC</td><td headers="hd_h_ml076.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td><td headers="hd_h_ml076.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DnaK full - length</td><td headers="hd_h_ml076.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Stressgen</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml076t1"><div id="ml076.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Selected purchased & synthesized compounds for SAR Development of confirmed hit BI-88B12</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47353/table/ml076.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml076.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">BI88</th><th id="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID [SID]</th><th id="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">MLS – #</th><th id="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Structure</th><th id="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">MW</th><th id="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">ATPase IC<sub>50</sub> (mM)</th><th id="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">ITC <i>K</i><sub>d</sub> (μM)</th><th id="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">B12 <b>Initial Hit</b></td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1244199 [56427257]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0014807</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu2.jpg" alt="Image ml076fu2.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>235</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">2</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">64.1 ± 8.7 (SBD)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">ChemBridge</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">E3</td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">743472 [56427258]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0100672</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu3.jpg" alt="Image ml076fu3.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>260</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>20</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25 (SBD) 4.9</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">ChemBridge</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">D7</td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">2814135 [56427263]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0311872</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu4.jpg" alt="Image ml076fu4.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>242</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>20</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">(SBD) 12.7 (FL)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Internally Synthesized</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">D10 <b>Probe</b></td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25105719 [56427267]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0315930</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu5.jpg" alt="Image ml076fu5.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>273</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>20</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.2 (FL)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Internally Synthesized</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">D5</td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25105722 [56427270]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0315933</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu6.jpg" alt="Image ml076fu6.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>267</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>20</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1.0 (SBD)<br />1.7 (FL)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Internally Synthesized</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">D9</td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">25105724 [56427272]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0315935</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu7.jpg" alt="Image ml076fu7.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>255</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>20</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1.5 (SBD)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Internally Synthesized</td></tr><tr><td headers="hd_h_ml076.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">C5</td><td headers="hd_h_ml076.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1246750 [56427262]</td><td headers="hd_h_ml076.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0315926</td><td headers="hd_h_ml076.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK47353/bin/ml076fu8.jpg" alt="Image ml076fu8.jpg" /></div></td><td headers="hd_h_ml076.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><i>296</i></td><td headers="hd_h_ml076.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.2–2</td><td headers="hd_h_ml076.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">14.3 (SBD)<br />39.3 (FL)</td><td headers="hd_h_ml076.t1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">ChemBridge</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml076fu2"><div id="ml076.fu2" class="figure"><div class="graphic"><img data-src="/books/NBK47353/bin/ml076fu1.jpg" alt="Image ml076fu1" /></div></div></article><article data-type="table-wrap" id="figobml076t2"><div id="ml076.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Probe and analog submission</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47353/table/ml076.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml076.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe/Analog</th><th id="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS-# (BCCG#)</th><th id="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Source (vendor or BCCG syn)</th><th id="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Amt (mg)</th><th id="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Date ordered/submitted</th></tr></thead><tbody><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe (D10)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315930</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">25105719</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427267" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427267</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 1 (B12)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0014807</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1244199</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427257" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427257</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ChemBridge</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 2 (D7)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0311872</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2814135</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427263" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427263</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 3 (D5)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315933</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">25105722</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427270" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427270</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 4 (D9)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315935</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">25105724</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427272" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427272</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr><tr><td headers="hd_h_ml076.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 5 (C5)</td><td headers="hd_h_ml076.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315926</td><td headers="hd_h_ml076.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1246750</td><td headers="hd_h_ml076.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56427262" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56427262</a></td><td headers="hd_h_ml076.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ChemBridge</td><td headers="hd_h_ml076.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15</td><td headers="hd_h_ml076.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7/9/09</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml076tu3"><div id="ml076.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47353/table/ml076.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml076.tu3_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">273.37324 [g/mol]</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">C<sub>14</sub>H<sub>11</sub>NOS<sub>2</sub></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">3.7</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tautomer Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">273.028205</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">273.028205</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">85.6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">18</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">310</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</td><td rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml076f2"><div id="ml076.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47353/bin/ml076f2.jpg" alt="Scheme 1. General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives." /></div><h3><span class="label">Scheme 1</span><span class="title">General synthesis path for the preparation of the thiophene-2-carbonyl amide derivatives</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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