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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="The Role of PHOSPHO1 in the Initiation of Skeletal Calcification" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/09/02" /><meta name="citation_author" content="Dereck Stonich" /><meta name="citation_author" content="Ying Su" /><meta name="citation_author" content="Shakeela Dad" /><meta name="citation_author" content="Santhi Reddy" /><meta name="citation_author" content="Yalda Mostofi" /><meta name="citation_author" content="Dahl Russell" /><meta name="citation_author" content="Thomas DY Chung" /><meta name="citation_author" content="Nicholas Michael Hedrick" /><meta name="citation_author" content="Justin Rascon" /><meta name="citation_author" content="Xochella Garcia" /><meta name="citation_author" content="Eduard Sergienko" /><meta name="citation_author" content="José Luis Millán" /><meta name="citation_author" content="Derek Stonich" /><meta name="citation_author" content="Nicholas Cosford" /><meta name="citation_pmid" content="21433371" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47355/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="The Role of PHOSPHO1 in the Initiation of Skeletal Calcification" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Dereck Stonich" /><meta name="DC.Contributor" content="Ying Su" /><meta name="DC.Contributor" content="Shakeela Dad" /><meta name="DC.Contributor" content="Santhi Reddy" /><meta name="DC.Contributor" content="Yalda Mostofi" /><meta name="DC.Contributor" content="Dahl Russell" /><meta name="DC.Contributor" content="Thomas DY Chung" /><meta name="DC.Contributor" content="Nicholas Michael Hedrick" /><meta name="DC.Contributor" content="Justin Rascon" /><meta name="DC.Contributor" content="Xochella Garcia" /><meta name="DC.Contributor" content="Eduard Sergienko" /><meta name="DC.Contributor" content="José Luis Millán" /><meta name="DC.Contributor" content="Derek Stonich" /><meta name="DC.Contributor" content="Nicholas Cosford" /><meta name="DC.Date" content="2010/09/02" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK47355/" /><meta name="description" content="Defective bone mineralization (osteomalacia) occurs in tissue non-specific alkaline phosphatase (TNAP) deficiency (hypophosphatasia). TNAP functions to maintain normal bone mineralization via hydrolysis of inorganic pyrophosphate (PPi), a bone mineralization inhibitor. This study sought to examine the role of PHOSPHO1, a soluble phosphatase that has specificity for phosphoethanolamine and phosphocholine, in increasing the inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) ratio inside matrix vesicles (MVs) and, thus, controlling the first step of initiation of hydroxyapatite crystal deposition inside MVs. The identified molecular probe ML086 (CID-1674999) is a biochemical inhibitor of PHOSPHO1. PHOSPHO1-specific inhibitors may serve as novel tools to examine the functional role of this enzyme in skeletal mineralization and soft tissue ossification abnormalities, as well as to help elucidate its mechanism of action in diseases such as osteoarthritis, osteoporosis, and arterial calcification." /><meta name="og:title" content="The Role of PHOSPHO1 in the Initiation of Skeletal Calcification" /><meta name="og:type" content="book" /><meta name="og:description" content="Defective bone mineralization (osteomalacia) occurs in tissue non-specific alkaline phosphatase (TNAP) deficiency (hypophosphatasia). TNAP functions to maintain normal bone mineralization via hydrolysis of inorganic pyrophosphate (PPi), a bone mineralization inhibitor. This study sought to examine the role of PHOSPHO1, a soluble phosphatase that has specificity for phosphoethanolamine and phosphocholine, in increasing the inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) ratio inside matrix vesicles (MVs) and, thus, controlling the first step of initiation of hydroxyapatite crystal deposition inside MVs. The identified molecular probe ML086 (CID-1674999) is a biochemical inhibitor of PHOSPHO1. PHOSPHO1-specific inhibitors may serve as novel tools to examine the functional role of this enzyme in skeletal mineralization and soft tissue ossification abnormalities, as well as to help elucidate its mechanism of action in diseases such as osteoarthritis, osteoporosis, and arterial calcification." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK47355/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml086/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK47355/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK47355_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK47355_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml088/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml085/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK47355_"><span class="title" itemprop="name">The Role of PHOSPHO1 in the Initiation of Skeletal Calcification</span></h1><p class="contrib-group"><span itemprop="author">Dereck Stonich</span>, <span itemprop="author">Ying Su</span>, <span itemprop="author">Shakeela Dad</span>, <span itemprop="author">Santhi Reddy</span>, <span itemprop="author">Yalda Mostofi</span>, <span itemprop="author">Dahl Russell</span>, <span itemprop="author">Thomas DY Chung</span>, <span itemprop="author">Nicholas Michael Hedrick</span>, <span itemprop="author">Justin Rascon</span>, <span itemprop="author">Xochella Garcia</span>, <span itemprop="author">Eduard Sergienko</span>, <span itemprop="author">José Luis Millán</span>, <span itemprop="author">Derek Stonich</span>, and <span itemprop="author">Nicholas Cosford</span>.</p><p class="small">Received: <span itemprop="datePublished">April 18, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Defective bone mineralization (osteomalacia) occurs in tissue non-specific alkaline phosphatase (TNAP) deficiency (hypophosphatasia). TNAP functions to maintain normal bone mineralization via hydrolysis of inorganic pyrophosphate (PPi), a bone mineralization inhibitor. This study sought to examine the role of PHOSPHO1, a soluble phosphatase that has specificity for phosphoethanolamine and phosphocholine, in increasing the inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) ratio inside matrix vesicles (MVs) and, thus, controlling the first step of initiation of hydroxyapatite crystal deposition inside MVs. The identified molecular probe ML086 (CID-1674999) is a biochemical inhibitor of PHOSPHO1. PHOSPHO1-specific inhibitors may serve as novel tools to examine the functional role of this enzyme in skeletal mineralization and soft tissue ossification abnormalities, as well as to help elucidate its mechanism of action in diseases such as osteoarthritis, osteoporosis, and arterial calcification.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH084086-01</p><p><b>Screening Center Name & PI:</b>
|
||
<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>
|
||
<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:</b> Dr José Luis Millán &
|
||
Sanford-Burnham Medical Research Institute (<i>formerly Burnham Institute for
|
||
Medical Research</i>)</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1574" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1574</a></p><div id="ml086.s2"><h2 id="_ml086_s2_">Probe Structure & Characteristics</h2><div id="ml086.fu1" class="figure"><div class="graphic"><img src="/books/NBK47355/bin/ml086fu1.jpg" alt="Image ml086fu1" /></div></div><div id="ml086.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47355/table/ml086.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml086.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml086.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml086.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml086.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_ml086.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_ml086.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM)
|
||
[SID, AID]</th><th id="hd_h_ml086.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml086.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
|
||
[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml086.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-16749996<br /><a href="/pcsubstance/?term=ML086[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML086</a></td><td headers="hd_h_ml086.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PHOSPHO1</td><td headers="hd_h_ml086.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">139 nM IC50 <br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57287582</a>
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1666" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1666</a></td><td headers="hd_h_ml086.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">TNAP</td><td headers="hd_h_ml086.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>100 μM IC50
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57287582</a>
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1056" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1056</a></td><td headers="hd_h_ml086.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>719</td><td headers="hd_h_ml086.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PMM2: <br />>100,000 nM IC50
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57287582</a>
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1655" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1655</a><br /><br />PMI:
|
||
<br />>50,000 nM IC50 <br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57287582</a>
|
||
<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1535" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1535</a></td></tr></tbody></table></div></div></div><div id="ml086.s3"><h2 id="_ml086_s3_">Recommendations for the scientific use of this probe</h2><p>A newly identified soluble phosphatase, PHOSPHO1, with specificity for
|
||
phosphoethanolamine (PEA) and phosphoserine (PS) present in matrix vesicles (MVs),
|
||
is responsible for increasing the local concentration of Pi inside MVs to change the
|
||
Pi/PPi ratio to favor precipitation of hydroxyapatite (HA) seed crystals. As tissue
|
||
non-specific alkaline phosphatase (TNAP) is also in the same biological compartment,
|
||
PHOSPHO1-specific inhibitors will enable us to probe and clarify the functional
|
||
involvement of this enzyme in skeletal mineralization and soft tissue ossification
|
||
abnormalities. This will help to elucidate the mechanism of action for diseases such
|
||
as osteoarthritis, osteoporosis, and arterial calcification.</p></div><div id="ml086.s4"><h2 id="_ml086_s4_">1. Scientific Rationale for Project</h2><div id="ml086.s5"><h3>Specific Aims</h3><p>The main aim of this project was to screen large comprehensive chemical libraries
|
||
to identify lead compounds for PHOSPHO1-specific inhibitors that will enable the
|
||
elucidation of the functional involvement of this enzyme in skeletal
|
||
mineralization and related biological phenomena.</p></div><div id="ml086.s6"><h3>Background and Significance</h3><p>During the process of endochondral bone formation, osteoblasts mineralize the
|
||
extracellular matrix (ECM) by promoting the initial formation of crystalline
|
||
hydroxyapatite in the sheltered interior of membrane-limited matrix vesicles
|
||
(MVs) and by modulating matrix composition to further promote propagation of
|
||
apatite outside of the MVs. All available data indicate that controlled bone
|
||
mineralization depends on a regulated balance of the following factors: the
|
||
concentrations of Ca<sup>2+</sup> and inorganic phosphate (Pi), the
|
||
presence of fibrilar collagens (e.g., type I in bone; Types II and X in
|
||
cartilage) and the presence of adequate concentrations of mineralization
|
||
inhibitors, <i>i.e</i>., inorganic pyrophosphate (PPi), and
|
||
osteopontin (OPN). Three molecules present in osteoblasts have so far been
|
||
identified as affecting the controlled deposition of bone mineral by regulating
|
||
the extracellular levels of PPi, and in turn of OPN, <i>i.e</i>.,
|
||
tissue-nonspecific alkaline phosphatase (TNAP); NPP1 (a nucleotide
|
||
pyrophosphatase/phosphodiesteraseisozyme) and the ANK gene product. A deficiency
|
||
in the TNAP isozyme causes the inborn-error-of-metabolism known as
|
||
hypophosphatasia and the study of this disease has provided the best evidence of
|
||
the importance of TNAP for bone mineralization. TNAP is the only
|
||
tissue-nonrestrictedisozyme of a family of four homologous human alkaline
|
||
phosphatase (AP) genes (EC. 3.1.3.1) (<a class="bk_pop" href="#ml086.r1">1</a>) that additionally includes the placental (PLAP), germ cell (GCAP)
|
||
and intestinal (IAP) AP genes. Expressed as an ecto-enzyme anchored
|
||
<i>via</i> a phosphatidylinositolglycan moiety, TNAP has been
|
||
demonstrated to play an essential physiological role during bone matrix
|
||
mineralization. Specifically, defective bone mineralization (osteomalacia)
|
||
occurs in TNAP deficiency (hypophosphatasia) (<a class="bk_pop" href="#ml086.r2">2</a>). The severity of hypophosphatasia is variable and modulated by the
|
||
nature of the TNAP mutation (<a class="bk_pop" href="#ml086.r3" data-bk-pop-others="ml086.r4 ml086.r5 ml086.r6">3–6</a>). Unlike most types of rickets or
|
||
osteomalacia neither calcium nor inorganic phosphate levels in serum are
|
||
subnormal in hypophosphatasia. In fact hypercalcemia and hyperphosphatemia may
|
||
exist and hypercalciuria is common in infantile hypophosphatasia (<a class="bk_pop" href="#ml086.r7">7</a>). The clinical severity in
|
||
hypophosphatasia patients varies widely. The different syndromes, listed from
|
||
the most severe to the mildest forms, are: perinatalhypophosphatasia, infantile
|
||
hypophosphatasia, childhood hypophosphatasia, adult hypophosphatasia,
|
||
odontohypophosphatasia and pseudohypophosphatasia (<a class="bk_pop" href="#ml086.r7">7</a>). These phenotypes range from complete absence of
|
||
bone mineralization and stillbirth to spontaneous fractures and loss of decidual
|
||
teeth in adult life. Inactivation of the mouse TNAP gene (<i>Akp2</i>)
|
||
phenocopies the infantile form of human hypophosphatasia (<a class="bk_pop" href="#ml086.r8">8</a>, <a class="bk_pop" href="#ml086.r9">9</a>). In
|
||
bone, TNAP is confined to the cell surface of osteoblasts and chondrocytes,
|
||
including the membranes of their shed MVs (<a class="bk_pop" href="#ml086.r10">10</a>, <a class="bk_pop" href="#ml086.r11">11</a>). In fact, by an
|
||
unknown mechanism, MVs are markedly enriched in TNAP compared to both whole
|
||
cells and the plasma membrane (<a class="bk_pop" href="#ml086.r12">12</a>). It
|
||
has been proposed that the role of TNAP in the bone matrix is to generate the
|
||
inorganic phosphate needed for hydroxyapatite crystallization (<a class="bk_pop" href="#ml086.r13" data-bk-pop-others="ml086.r14 ml086.r15">13–15</a>). However, TNAP has also been hypothesized to hydrolyze
|
||
the mineralization inhibitor PPi (<a class="bk_pop" href="#ml086.r16">16</a>)
|
||
to facilitate mineral precipitation and growth (<a class="bk_pop" href="#ml086.r17" data-bk-pop-others="ml086.r18 ml086.r19">17–19</a>). Electron microscopy revealed that TNAP-deficient MVs,
|
||
in both humans and mice, contain apatite crystals, but that extravesicular
|
||
crystal propagation is retarded (<a class="bk_pop" href="#ml086.r20">20</a>).
|
||
This growth retardation could be due to either the lack of
|
||
TNAP'spyrophosphatase function or the lack of inorganic
|
||
phosphate-generation. Our recent studies have provided compelling proof that the
|
||
function of TNAP in bone tissue consists of hydrolyzing PPi to maintain a proper
|
||
concentration of this mineralization inhibitor to ensure normal bone
|
||
mineralization. PPi is primarily generated by the members of the nucleotide
|
||
pyrophosphatase/phosphodiesterase (NPP) family of isozymes. Plasma cell membrane
|
||
glycoprotein-1 (PC-1; more correctly termed NPP1) is plasma membrane-bound,
|
||
whereas autotaxin (NPP2) is secreted and B10 (NPP3) is abundant in intracellular
|
||
spaces (<a class="bk_pop" href="#ml086.r21">21</a>). All three isozymes
|
||
are expressed in a wide variety of tissues, including bone and cartilage (<a class="bk_pop" href="#ml086.r22">22</a>), and they all have the common
|
||
ability to hydrolyze diesters of phosphoric acid into phosphomonoesters. NPPs
|
||
have been implicated in various processes, including bone mineralization,
|
||
signaling by insulin and by nucleotides, and the differentiation and motility of
|
||
cells (<a class="bk_pop" href="#ml086.r23">23</a>). However, NPPs are
|
||
known primarily as suppliers of intra- and extracellular PPi (<a class="bk_pop" href="#ml086.r24">24</a>). Similar to skeletal TNAP
|
||
expression, NPP1 is highly abundant on the surfaces of osteoblasts and
|
||
chondrocytes as well as on the membrane of their MVs (<a class="bk_pop" href="#ml086.r25">25</a>, <a class="bk_pop" href="#ml086.r26">26</a>).
|
||
NPP1 has a role in inhibiting hydroxyapatite precipitation by its PPi-generating
|
||
property. This proposed function has been supported by <i>in vitro</i>
|
||
studies where cells transfected with the NPP1 cDNA resulted in elevated levels
|
||
of PPi in osteoblast-derived MVs, accompanied by decreased matrix
|
||
mineralization (<a class="bk_pop" href="#ml086.r27">27</a>). It is also
|
||
thought that glycoprotein-1 participates in osteblastic mineralization (<a class="bk_pop" href="#ml086.r28">28</a>). Moreover,
|
||
<i>Enpp1</i>−/− mice develop
|
||
hypermineralization abnormalities such as ossification of the posterior
|
||
longitudinal ligament of the spine, diffuse idiopathic skeletal hyperostosis,
|
||
ankylosing spinal hyperostosis and pathological soft-tissue ossification,
|
||
including arterial calcification (<a class="bk_pop" href="#ml086.r29">29</a>, <a class="bk_pop" href="#ml086.r30">30</a>). Conversely,
|
||
while NPP1 deficiency results in hypermineralization, elevated expression of
|
||
NPP1 is associated with MV-mediated calcium pyrophosphate dihydrate (CPPD)
|
||
matrix calcification in the knee meniscal cartilage during aging (<a class="bk_pop" href="#ml086.r31">31</a>, <a class="bk_pop" href="#ml086.r32">32</a>). Interestingly, as in NPP1 overexpressing tissues,
|
||
abnormal CPPD precipitation has also been observed in association with TNAP
|
||
deficiency. While the excess of PPi in hypophosphatasia patients is primarily
|
||
linked with rickets and osteomalacia, subjects with the disease may also develop
|
||
pathological CPPD mineralization of the articular cartilage (<a class="bk_pop" href="#ml086.r33">33</a>).</p><p>Similar to NPP1, the ankylosis protein (ANK) has a role in mineralization by
|
||
contributing to the extracellular supply of PPi. However, unlike NPP1, ANK
|
||
appears to function as a transmembrane PPi-channeling protein, allowing PPi
|
||
molecules to passage through the plasma membrane from the cytoplasm to the
|
||
outside of the cell (<a class="bk_pop" href="#ml086.r34">34</a>). ANK protein
|
||
is detectable in many tissues, yet its expression is particularly strong in the
|
||
cartilage of joints (<a class="bk_pop" href="#ml086.r34">34</a>). Cell
|
||
surfaces of osteoblasts and chondrocytes appear to be abundant in ANK
|
||
protein (<a class="bk_pop" href="#ml086.r36">36</a>), but in contrast to
|
||
NPP1 and TNAP, it is not present in the membranes of MVs. ANK was identified in
|
||
a naturally occurring mutant mouse strain that had characteristics of
|
||
progressive ankylosis, thus the designation <i>ank/ank</i>mice (<a class="bk_pop" href="#ml086.r35">35</a>). These animals develop
|
||
hydroxyapatite crystals in articular surfaces and synovial fluids. As
|
||
<i>Enpp1</i>−/− mice,
|
||
<i>ank/ank</i>mice display pathological abnormalities that mimic
|
||
several arthritic diseases, including ectopic calcification, cartilage erosion
|
||
and osteophyte formation seen in osteoarthritis, and vertebral fusion observed
|
||
in ankylosisspondylitis patients (<a class="bk_pop" href="#ml086.r37" data-bk-pop-others="ml086.r38 ml086.r39 ml086.r40">37–40</a>).</p><p>It has also been shown that normalizing calcium and Pi concentrations corrects
|
||
the hyperosteoidosis of rickets patients (<a class="bk_pop" href="#ml086.r41">41</a>). Since extracellular calcium is more tightly regulated than
|
||
extracellular Pi concentrations, Pi may be the critical element in the induction
|
||
of mineral crystals in a given ECM. The production by osteoblasts of an
|
||
inhibitor of ECM mineralization like PPi is counter-intuitive. It suggests a
|
||
model whereby the removal of an inhibitor rather than the synthesis of an
|
||
inducer of mineralization would explain why ECM mineralization occurs in bone.
|
||
Consistent with this model we have shown that removal of PPi via TNAP action and
|
||
the presence of a fibrilar collagen-rich scaffold are two conditions necessary
|
||
to induce mineralization of bone or any ECM. Our data also indicate that the
|
||
Pi/PPi ratio is of fundamental significance for bone ECM mineralization. This is
|
||
in agreement with the observation that
|
||
<i>Akp2</i><sup>−</sup><i>
|
||
<sup>/</sup>
|
||
</i><sup>−</sup>mice that have abnormally high extracellular
|
||
PPi levels have hyperosteoidosis, while
|
||
[<i>Akp2</i><sup>−</sup><i>
|
||
<sup>/</sup>
|
||
</i><sup>−</sup><i>;
|
||
Enpp1</i><sup>−</sup><i>
|
||
<sup>/</sup>
|
||
</i><sup>−</sup>] and
|
||
[<i>Akp2</i><sup>−</sup><i>
|
||
<sup>/</sup>
|
||
</i><sup>−</sup><i>; ank/ank</i>] mice
|
||
have normal mineralization of the skull and normal extracellular PPi
|
||
concentration (<a class="bk_pop" href="#ml086.r42">42</a>,<a class="bk_pop" href="#ml086.r43">43</a>). Thus, in the bone ECM, while the
|
||
extracellular Pi concentration is fairly constant, TNAP’s enzymatic
|
||
degradation of PPi controls the Pi/PPi ratio to favor crystallization of HA
|
||
outside the MVs along collagen fibrils. But, why are
|
||
<i>Akp2</i>−/− mice born with a mineralized
|
||
skeleton and still contain HA crystals inside their MVs? As TNAP sits on the
|
||
outer surface of the MV membrane there is no TNAP-mediated hydrolysis of PPi
|
||
inside the MVs. Therefore, it is likely that another enzyme is responsible for
|
||
either cleaving PPi or elevating the intravesicular concentration of Pi so as to
|
||
achieve a Pi/PPi ratio conducive for crystallization. We proposed and tested our
|
||
hypothesis in this research that PHOSPHO1, a soluble phosphatase (<a class="bk_pop" href="#ml086.r44">44</a>, <a class="bk_pop" href="#ml086.r45">45</a>) that has specificity for phosphoethanolamine and
|
||
phosphocholine (<a class="bk_pop" href="#ml086.r46">46</a>), plays the
|
||
important role of increasing the Pi/PPi ratio inside MVs and thus controls the
|
||
first step of initiation of HA crystal deposition inside MVs.</p></div></div><div id="ml086.s7"><h2 id="_ml086_s7_">2. Project Description</h2><div id="ml086.s8"><h3>a. The original goal for probe characteristics</h3><p>The original goal was to find compounds that had minimally less that 1 µM
|
||
potency, but were selective against tissue specific alkaline phosphatase. None
|
||
of the existing three inhibitors (SCH-20676, lansoprazole, ebselen) from the
|
||
original CPDP met the 1 µM potency barrier.</p></div><div id="ml086.s9"><h3>b. Assay implementation and screening</h3><div id="ml086.s10"><h4>i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen,
|
||
Secondary)</h4><div id="ml086.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47355/table/ml086.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml086.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChemBioAssay Name</th><th id="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">AIDs</th><th id="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe Type</th><th id="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Type</th><th id="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Format</th><th id="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Detection &
|
||
wellformat</th></tr></thead><tbody><tr><td headers="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">uHTS absorbance assay for the
|
||
identification of compounds that inhibit PHOSPHO1.</td><td headers="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1565" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1565</a></td><td headers="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibitor</td><td headers="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">biochemical</td><td headers="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Absorbance (@620 nm,
|
||
BIOMOL)1536</td></tr><tr><td headers="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SAR assay for compounds that
|
||
inhibit PHOSPHO1</td><td headers="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1666" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1666</a></td><td headers="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibitor</td><td headers="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SAR</td><td headers="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">biochemical</td><td headers="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Absorbance at 630 nm, 384</td></tr><tr><td headers="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target in Vitro TNAP Dose
|
||
Response Luminescent Assay for SAR Study</td><td headers="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1056" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1056</a></td><td headers="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibitor</td><td headers="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Counterscn</td><td headers="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">biochemical</td><td headers="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence, 384</td></tr><tr><td headers="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Counter screen SAR assay for
|
||
PMM2 inhibitors via a fluorescence intensity assay</td><td headers="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1655" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1655</a></td><td headers="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibitor</td><td headers="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay for
|
||
specificity</td><td headers="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">biochemical</td><td headers="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence 384</td></tr><tr><td headers="hd_h_ml086.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Confirmation of compounds
|
||
inhibiting phosphomannoseisomerase (PMI) via a fluorescence
|
||
intensity assay</td><td headers="hd_h_ml086.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1535" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1535</a></td><td headers="hd_h_ml086.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibitor</td><td headers="hd_h_ml086.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay for
|
||
specificity</td><td headers="hd_h_ml086.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">biochemical</td><td headers="hd_h_ml086.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence 384</td></tr></tbody></table></div></div></div><div id="ml086.s11"><h4>ii. Assay Rationale & Description</h4><p>This biochemical assay employs a colorimetric readout based on the
|
||
enzyme's ability to liberate phosphate from phosphoethanolamine and
|
||
its reaction with the Biomol Green reagent. The primary screening protocol
|
||
is described below.</p><div id="ml086.s12"><h5>Assay materials</h5><ol><li class="half_rhythm"><div>PHOSPHO1 was obtained from the assay provider's
|
||
laboratory. The construct was designed to express PHOSPHO1 fused
|
||
to a V5 epitope and 6 His-tag at the C-terminus.</div></li><li class="half_rhythm"><div>Assay Buffer: 20mM MES-NaOH pH 6.7, 2mM MgCl<sub>2</sub>,
|
||
0.0125% Tween-20, 0.01% BSA.</div></li></ol><div id="ml086.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Reagents used for the uHTS experiments</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47355/table/ml086.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml086.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml086.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Reagent</th><th id="hd_h_ml086.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Vendor</th></tr></thead><tbody><tr><td headers="hd_h_ml086.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Recombinant human PHOSPHO1
|
||
produced in <i>E. coli</i> from an arabinose
|
||
inducible obtained expression clone</td><td headers="hd_h_ml086.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen-- pBAD TOPO TA
|
||
vector from a cDNA corresponding to Met19-Cys267 of
|
||
human PHOSPHO1</td></tr><tr><td headers="hd_h_ml086.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">BIOMOL Green (AK-111)</td><td headers="hd_h_ml086.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">BIOMOL</td></tr><tr><td headers="hd_h_ml086.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SCH-202676: previously
|
||
identified as an inhibitor of PHOSPHO1 as positive
|
||
control</td><td headers="hd_h_ml086.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Calbiochem, Cat No.
|
||
565645</td></tr></tbody></table></div></div></div><div id="ml086.s13"><h5>The following uHTS protocol was developed</h5><ol><li class="half_rhythm"><div>1.5ul of assay buffer (20mM MES-NaOH, 2mM MgCl<sub>2</sub>,
|
||
0.0125% Tween 20, 0.01% BSA) are added
|
||
to each well in columns 1 and 2 of a black/clear bottom Corning
|
||
assay plate (cat # 3891).</div></li><li class="half_rhythm"><div>1.5ul of assay buffer containing 2.5ng/ul PHOSPHO1 are added to
|
||
the wells in columns 3–48.</div></li><li class="half_rhythm"><div>Using a 1536 pintool, 20nl of 2mM compound are added to each well
|
||
in columns 5–48.</div></li><li class="half_rhythm"><div>20nl of DMSO are added to the wells in columns
|
||
1–4.</div></li><li class="half_rhythm"><div>1.5ul of assay buffer containing 450µM PEA is added to all
|
||
wells.</div></li><li class="half_rhythm"><div>The plate is centrifuged and incubated at room temperature for 1
|
||
hour.</div></li><li class="half_rhythm"><div>3ul of Biomol Green is added to each well.</div></li><li class="half_rhythm"><div>The plate is centrifuged and incubated for 30 min at room
|
||
temperature.</div></li><li class="half_rhythm"><div>The plate is read on a Viewlux plate reader in absorbance mode
|
||
@630nm.</div></li></ol><p>The average Z’ for the screen was 0.56, the
|
||
signal-to-background was 11.5, signal-to-noise was 21.8 and
|
||
signal-to-window was 6.6.</p><div id="ml086.s14"><h5>Rationale for confirmatory, counter and selectivity
|
||
assays</h5><p>Compounds that looked promising based on their selectivity profile
|
||
were then tested in dose-response secondary assays using 10-point
|
||
serial dilution in duplicate. The assay was based on detection of
|
||
phosphate release from phosphoethanolamine using the Biomol reagent.
|
||
We also developed another secondary assay for PHOSPHO1 which was
|
||
based on the detection of choline released in the dephosphorylation
|
||
of phosphocholine, an endogenous substrate for PHOSPHO1. Phosphate
|
||
was also detected with the Biomol reagent (see <a class="figpopup" href="/books/NBK47355/table/ml086.t2/?report=objectonly" target="object" rid-figpopup="figml086t2" rid-ob="figobml086t2">Table 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml086t2"><a href="/books/NBK47355/table/ml086.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml086t2" rid-ob="figobml086t2"><img class="small-thumb" src="/books/NBK47355/table/ml086.t2/?report=thumb" src-large="/books/NBK47355/table/ml086.t2/?report=previmg" alt="Table 2. Assays used in PHOSPHO1 studies." /></a><div class="icnblk_cntnt"><h4 id="ml086.t2"><a href="/books/NBK47355/table/ml086.t2/?report=objectonly" target="object" rid-ob="figobml086t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Assays used in PHOSPHO1 studies. </p></div></div><p>Knowledge of specificity is important for the delineation of PHOSPHO1
|
||
biological functions, specifically the selectivity of inhibitors for
|
||
PHOSPHO1 versus TNAP present in the same tissue and participating in
|
||
similar biological processes.</p></div></div></div><div id="ml086.s15"><h4>iii. Summary of Results</h4><p>The uHTS assay was developed and implemented in 1536-well plates. After
|
||
successful implementation the assay underwent uHTS with a library of ~55K
|
||
compounds. Approximately 5000 compounds showed >50%
|
||
activity in the assay at that point. This gave a hit rate of ~
|
||
3%, which was unexpectedly high and difficult to follow up.
|
||
Interestingly, more than 50% of the hits on two representative
|
||
plates that were cherry-picked using our acoustic Echo dispenser confirmed
|
||
in both single-concentration and dose-response modes.</p><p>During the preparation for this probe project we realized that PHOSPHO1, an
|
||
orphan phosphatase with little information available, belongs to a family of
|
||
halo-acid dehydrogenases. Thus, it was expected to share some common
|
||
features with other members of the same family, including
|
||
phosphomannomutase-2 (PMM2). We had already developed an assay for PMM2 and
|
||
utilized it as a counter screen for phosphomannoseisomerase (PMI), an MLSCN
|
||
probe project. We also noticed that in the PHOSPHO1R03 grant application
|
||
three compounds had previously been identified as potential hits. We had
|
||
already demonstrated that one of them (ebselen) inhibits both PMI and PMM2.
|
||
Since we had previously generated analogues by purchase and synthesis we had
|
||
a large number of dry powder compounds based on this scaffold available for
|
||
testing. We therefore decided to test these compounds against PHOSPHO1 and
|
||
found that several of them were very potent and selective for PHOSPHO1, not
|
||
PMM or PMI active, but also satisfyingly not active against TNAP, the
|
||
original anti-target of most concern for PHOPHOSPHI activity.</p></div></div><div id="ml086.s16"><h3>c. Probe Optimization</h3><div id="ml086.s17"><h4>i. SAR & chemistry strategy that led to the probe</h4><p>The goals for optimization of the PHOSPHO1 hit compounds from the primary HTS
|
||
screen were two-fold: (1) find analogues with increased potency in the
|
||
PHOSPHO1 enzyme inhibition assay and (2) optimize the potent PHOSPHO1
|
||
inhibitors for selectivity against TNAP. Additionally, as it was realized
|
||
that PHOSPHO1 was a relative of the haloacid dehydrogenases, PMM2 and PMI,
|
||
therefore, we also included 2 specifity assays for the class of ebselen-like
|
||
inhibitors we had previously tested for: PMM and PMI enzyme inhibition, and
|
||
indeed we used these to actually select and define the final desired
|
||
compound, since TNAP activity was minimal for these series (see below and
|
||
<a class="figpopup" href="/books/NBK47355/table/ml086.t3/?report=objectonly" target="object" rid-figpopup="figml086t3" rid-ob="figobml086t3">Table 3</a>). HTS revealed
|
||
multiple potent hits in the benzoisothiazolone compound class, with a
|
||
majority of the inhibitors containing a sulfonamide group as in CID-4089709
|
||
(MLS-0315803), CID-2325813 (MLS-0315794), CID-5040456 (MLS-0263839), and
|
||
CID-2327953 (MLS-0022297) (<a class="figpopup" href="/books/NBK47355/table/ml086.t3/?report=objectonly" target="object" rid-figpopup="figml086t3" rid-ob="figobml086t3">Table
|
||
3</a>). Select representatives of these potent hits were
|
||
re-synthesized in house and activity was confirmed with synthesized
|
||
analogues. All of the data for these analogs and the probe were obtained
|
||
from powder samples from the same batch for each replicate experiments.
|
||
While the sulfonamide compounds were indeed potent PHOSPHO1 inhibitors,
|
||
counterscreening against TNAP, PMM and PMI enzymes showed most to be
|
||
non-selective. For example, CID-4089709 (MLS-0315803) showed an
|
||
IC<sub>50</sub> of 0.81 µM against PHOSPHO1. However, this compound
|
||
inhibited PMM with an IC<sub>50</sub> of 5.17 µM and was even more potent
|
||
against PMI with an IC<sub>50</sub> of 0.71 µM. Thus, the SAR strategy for
|
||
medicinal chemistry included variations of the sulfonamide moiety on the
|
||
aryl ring as well as replacing the sulfonamide with suitable, drug-like
|
||
isosteres, specifically amide and ester analogues. Regarding the SAR around
|
||
the sulfonamide analogues, it was observed that large sulfonamide
|
||
substituents, as in CID-4089709 (MLS-0315803), whilst proving to be potent
|
||
at inhibiting PHOSPHO1, also showed activity against PMM and PMI. Thus, the
|
||
focus shifted to smaller substituents. Selectivity began to emerge as these
|
||
smaller, less polar substituents were incorporated, as in CID-5040456
|
||
(MLS-0263839), CID-2327953 (MLS-0022297), and CID-2325813 (MLS-0315794), all
|
||
of which had PHOSPHO1 selectivities from 4- to 100-fold over both PMM and
|
||
PMI. However, additional selectivity for PHOSPHO1 was sought. Gratifyingly,
|
||
CID-2381612 (MLS-0315805), which had an IC<sub>50</sub> of 0.56 µM against
|
||
PHOSPHO1, showed no inhibition against TNAP and PMM up to 100 µM, and a PMI
|
||
IC<sub>50</sub> of 61.9 µM. While this compound demonstrated desirable
|
||
potency and selectivity, we envisaged that potency could be further
|
||
increased since we had previously observed non-selective compounds that were
|
||
2- to 3-fold more potent against PHOSPHO1. Encouraging potency and
|
||
selectivity results were seen when the sulfonamide group was replaced with
|
||
either an amide or ester isostere. As was previously observed with the
|
||
sulfonamide series, larger substituents, as in CID-16749997 (MLS-0390837),
|
||
were potent (PHOSPHO1 IC<sub>50</sub> = 2.33 µM), yet lacked the
|
||
required selectivity against PMM and PMI. Subsequent synthesis of esters and
|
||
amides containing smaller substituents facilitated a means to achieve the
|
||
required selectivity while maintaining potency, as shown in CID-2381612
|
||
(MLS-0315805). Thus, a final round of synthesis of amides afforded the probe
|
||
compound. CID-16749996 MLS-0390838) proved to be the most potent and
|
||
selective PHOSPHO1 compound synthesized, having an IC<sub>50</sub> of 0.14
|
||
µM and negligible inhibition of PMM (IC<sub>50</sub> = 76.4 µM)
|
||
or TNAP and PMI (IC<sub>50</sub>> 100 µM).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml086t3"><a href="/books/NBK47355/table/ml086.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml086t3" rid-ob="figobml086t3"><img class="small-thumb" src="/books/NBK47355/table/ml086.t3/?report=thumb" src-large="/books/NBK47355/table/ml086.t3/?report=previmg" alt="Table 3. Representative PHOSPHO1 inhibitors: SAR and selectivity." /></a><div class="icnblk_cntnt"><h4 id="ml086.t3"><a href="/books/NBK47355/table/ml086.t3/?report=objectonly" target="object" rid-ob="figobml086t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Representative PHOSPHO1 inhibitors: SAR and selectivity. </p></div></div></div></div></div><div id="ml086.s18"><h2 id="_ml086_s18_">3. Probe</h2><div id="ml086.s19"><h3>a. Chemical name</h3><p>N,N-dimethyl-3-(3-oxo-1,2-benzothiazol-2-yl)benzamide
|
||
<b>[<a href="/pcsubstance/?term=ML086[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML086</a>]</b></p></div><div id="ml086.s20"><h3>b. Probe chemical structure</h3><div id="ml086.fu2" class="figure"><div class="graphic"><img src="/books/NBK47355/bin/ml086fu12.jpg" alt="Image ml086fu12" /></div></div></div><div id="ml086.s21"><h3>c. Structural Verification Information of probe SID</h3><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57287582</a></p><div id="ml086.fu3" class="figure"><div class="graphic"><img src="/books/NBK47355/bin/ml086fu13.jpg" alt="Image ml086fu13" /></div></div><div id="ml086.fu4" class="figure"><div class="graphic"><img src="/books/NBK47355/bin/ml086fu14.jpg" alt="Image ml086fu14" /></div></div></div><div id="ml086.s22"><h3>d. PubChem CID (corresponding to the SID)</h3><p>CID-16749996</p></div><div id="ml086.s23"><h3>e. Availability from a vendor</h3><p>This probe is not commercially available from vendors.</p></div><div id="ml086.s24"><h3>f. MLS#'s of probe molecule and five
|
||
related samples that were submitted to the SMR collection</h3><div id="ml086.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47355/table/ml086.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml086.tu3_lrgtbl__"><table><thead><tr><th id="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe/Analog</th><th id="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS-#
|
||
(BCCG#)</th><th id="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml086.tu3_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_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Amt (mg)</th><th id="hd_h_ml086.tu3_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_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0390838</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16749996</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287582" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">57287582</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4/15/09</td></tr><tr><td headers="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 1</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0390837</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16749997</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/57287581" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">57287581</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4/15/09</td></tr><tr><td headers="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 2</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315861</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">25067463</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56405542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56405542</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BCCG syn</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4/15/09</td></tr><tr><td headers="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 3</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0263839</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5040456</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56373499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56373499</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Enamine</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4/15/09</td></tr><tr><td headers="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 4</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0022297</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2327953</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56373866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56373866</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Enamine</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Backorder</td></tr><tr><td headers="hd_h_ml086.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog 5</td><td headers="hd_h_ml086.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0315803</td><td headers="hd_h_ml086.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4089709</td><td headers="hd_h_ml086.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56373872" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56373872</a></td><td headers="hd_h_ml086.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Enamine</td><td headers="hd_h_ml086.tu3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td><td headers="hd_h_ml086.tu3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Backorder</td></tr></tbody></table></div></div></div><div id="ml086.s25"><h3>g. Mode of action for biological activity of probe</h3><p>CID-16749996 is a biochemical inhibitor of PHOSPHO1. The biological mode of
|
||
action of this probe has not yet been elucidated.</p></div><div id="ml086.s26"><h3>h. Detailed synthetic pathway for making probe</h3><div id="ml086.fu5" class="figure"><div class="graphic"><img src="/books/NBK47355/bin/ml086fu15.jpg" alt="Image ml086fu15" /></div></div><p><b>Synthesis of</b>
|
||
CID-16749996.<i>N</i>,<i>N</i>-dimethyl-3-(3-oxobenzo[<i>d</i>]isothiazol-2(3H)-yl)benzamide.</p></div><div id="ml086.s27"><h3>i. Summary of probe properties (solubility, absorbance/fluorescence,
|
||
reactivity, toxicity, etc.)</h3><p>No obvious reactivities or toxicities.</p></div><div id="ml086.s28"><h3>j. Properties Computed from Structure</h3><div id="ml086.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47355/table/ml086.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml086.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Property</th><th id="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:bottom;">Value</th></tr></thead><tbody><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">298.35956
|
||
[g/mol]</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">C<sub>16</sub>H<sub>14</sub>N<sub>2</sub>O<sub>2</sub>S</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2.5</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">298.077598</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">298.077598</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Topological Polar Surface Area</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">65.9</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">21</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">427</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml086.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</td><td headers="hd_h_ml086.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1</td></tr></tbody></table></div></div></div></div><div id="ml086.s29"><h2 id="_ml086_s29_">4. Appendices</h2><div id="ml086.s30"><h3>a. Comparative data on (1) probe, (2) similar compound structures (establishing
|
||
SAR) and (3) prior probes</h3><p>The three compounds (<a class="figpopup" href="/books/NBK47355/figure/ml086.fu6/?report=objectonly" target="object" rid-figpopup="figml086fu6" rid-ob="figobml086fu6">Fig. 1A</a>) on the left were obtained during pilot screening of
|
||
two well-known chemical libraries, LOPAC and Spectrum. Their calculated IC50
|
||
values against PHOSPHO1-mediated hydrolysis of PEA were 1.97 ± 0.01
|
||
μM (SCH 202676), 4.71 ± 0.1 μM
|
||
(Lansoprazole) and 2.81 ± 0.04 μM (Ebselen) (<a class="figpopup" href="/books/NBK47355/figure/ml086.fu6/?report=objectonly" target="object" rid-figpopup="figml086fu6" rid-ob="figobml086fu6">Fig. 1B</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml086fu6" co-legend-rid="figlgndml086fu6"><a href="/books/NBK47355/figure/ml086.fu6/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml086fu6" rid-ob="figobml086fu6"><img class="small-thumb" src="/books/NBK47355/bin/ml086fu16.gif" src-large="/books/NBK47355/bin/ml086fu16.jpg" alt="Figure 1" /></a><div class="icnblk_cntnt" id="figlgndml086fu6"><h4 id="ml086.fu6"><a href="/books/NBK47355/figure/ml086.fu6/?report=objectonly" target="object" rid-ob="figobml086fu6">Figure 1</a></h4></div></div><p>None of these meet the current minimal definition of less than 1 µM potency for a
|
||
biochemical assay. The nominated probe meets the potency requirement and also
|
||
meets the original need for TNAP selectivity. It also exceeds the specification
|
||
with the added selectivity against two related in-family enzyme members.
|
||
However, the biological relevance of these homologous enzyme to calcification is
|
||
not known. The probe provided has however does have this added selectivity,
|
||
which presumably minimize potential off-target effects.</p></div><div id="ml086.s31"><h3>b. Comparative data showing probe specificity for target</h3><p>See <a class="figpopup" href="/books/NBK47355/table/ml086.t3/?report=objectonly" target="object" rid-figpopup="figml086t3" rid-ob="figobml086t3">Table 3</a> above for
|
||
selectivity.</p></div></div><div id="ml086.s32"><h2 id="_ml086_s32_">5. Bibliography</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml086.r1">Millán JL. <span class="ref-journal">Mammalian alkaline phosphatases From biology to applications in
|
||
medicine and biotechnology.</span> Wiley-VCH Verlag GmbH & Co; Weinheim, Germany: 2006. pp. 1–322.</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml086.r2">Whyte MP. Hypophosphatasia and the role of alkaline phosphatase in
|
||
skeletal mineralization. <span><span class="ref-journal">Endocr Rev. </span>1994;<span class="ref-vol">15</span>:439–461.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7988481" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7988481</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml086.r3">Henthorn PS, Raducha M, Fedde KN, Lafferty MA, Whyte MP. Different missense mutations at the tissue-nonspecific
|
||
alkaline phosphatase gene locus in autosomal recessively inherited forms
|
||
of mild and severe hypophosphatasia. <span><span class="ref-journal">Proc Natl Acad Sci USA. </span>1992;<span class="ref-vol">89</span>:9924–8.</span> [<a href="/pmc/articles/PMC50246/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC50246</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/1409720" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1409720</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml086.r4">Fukushi M, Amizuka N, Hoshi K, Ozawa H, Kumagai H, Omura S, Misumi Y, Ikehara Y, Oda K. Intracellular retention and degradation of tissue-nonspecific
|
||
alkaline phosphatase with a Gly317-->Asp substitution associated
|
||
with lethal hypophosphatasia. <span><span class="ref-journal">Biochem. Biophys. Res. Comm. </span>1998;<span class="ref-vol">246</span>:613–618.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9618260" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9618260</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml086.r5">Shibata H, Fukushi M, Igarashi A, Misumi Y, Ikehara Y, Ohashi Y, Oda K. Defective intracellular transport of tissue-nonspecific
|
||
alkaline phosphatase with an Ala162-->Thr mutation associated
|
||
with lethal hypophosphatasia. <span><span class="ref-journal">J Biochem. </span>1988;<span class="ref-vol">123</span>:968–977.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9562633" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9562633</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml086.r6">Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon-Bouy B, Serre JL, Mornet E. Correlations of genotype and phenotype in
|
||
hypophosphatasia. <span><span class="ref-journal">Hum Mol Genet. </span>1999;<span class="ref-vol">8</span>:1039–1046.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10332035" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10332035</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml086.r7">Whyte MP. Hypophosphatasia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. <span class="ref-journal">The metabolic and molecular bases of inherited disease.</span> 7th ed. McGraw-Hill; New York: 1995. pp. 4095–4112.</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml086.r8">Narisawa S, Fröhlander N, Millán JL. Inactivation of two mouse alkaline phosphatase genes and
|
||
establishment of a model of infantile hypophosphatasia. <span><span class="ref-journal">Dev Dyn. </span>1997;<span class="ref-vol">208</span>:432–446.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9056646" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9056646</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml086.r9">Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR, Whyte MP. Alkaline phosphatase knock-out mice recapitulate the metabolic
|
||
and skeletal defects of infantile hypophosphatasia. <span><span class="ref-journal">J Bone Miner Res. </span>1999;<span class="ref-vol">14</span>:2015–26.</span> [<a href="/pmc/articles/PMC3049802/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3049802</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10620060" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10620060</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml086.r10">Ali SY, Sajdera SW, Anderson HC. Isolation and characterization of calcifying matrix vesicles
|
||
from epiphyseal cartilage. <span><span class="ref-journal">Proc Natl Acad Sci USA. </span>1970;<span class="ref-vol">67</span>:1513–1520.</span> [<a href="/pmc/articles/PMC283384/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC283384</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/5274475" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 5274475</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml086.r11">Bernard GW. Ultrastructural localization of alkaline phosphatase in
|
||
initial membranous osteogenesis. <span><span class="ref-journal">Clin Orthop. </span>1978;<span class="ref-vol">135</span>:218–225.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/709934" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 709934</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml086.r12">Morris DC, Masuhara K, Takaoka K, Ono K, Anderson HC. Immunolocalization of alkaline phosphatase in osteoblasts and
|
||
matrix vesicles of human fetal bone. <span><span class="ref-journal">Bone Miner. </span>1992;<span class="ref-vol">19</span>:287–98.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1472898" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1472898</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml086.r13">Robison R. The possible significance of hexosephosphoric esters in
|
||
ossification. <span><span class="ref-journal">Biochem J. </span>1923;<span class="ref-vol">17</span>:286–293.</span> [<a href="/pmc/articles/PMC1259346/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1259346</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16743183" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16743183</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml086.r14">Majeska RJ, Wuthier RE. Studies on matrix vesicles isolated from chick
|
||
epiphysealcartilage. Association of pyrophosphatase and ATPase
|
||
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of phosphatases within the HAD superfamily. <span><span class="ref-journal">Protein Engineering. </span>2003;<span class="ref-vol">16</span>:889–895.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14983068" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14983068</span></a>]</div></dd><dt>45.</dt><dd><div class="bk_ref" id="ml086.r45">Houston B, Stewart AJ, Farquharson C. PHOSPHO1 - A novel phosphatase specifically expressed at
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK47355/?report=reader">PubReader</a></li><li><a href="/books/NBK47355/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK47355" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK47355" style="display:none" title="Cite this Page"><div class="bk_tt">Stonich D, Su Y, Dad S, et al. The Role of PHOSPHO1 in the Initiation of Skeletal Calcification. 2009 Apr 18 [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></div></div></li><li><a href="/books/NBK47355/pdf/Bookshelf_NBK47355.pdf">PDF version of this page</a> (332K)</li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml086.s2" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml086.s3" ref="log$=inpage&link_id=inpage">Recommendations for the scientific use of this probe</a></li><li><a href="#ml086.s4" ref="log$=inpage&link_id=inpage">Scientific Rationale for Project</a></li><li><a href="#ml086.s7" ref="log$=inpage&link_id=inpage">Project Description</a></li><li><a href="#ml086.s18" ref="log$=inpage&link_id=inpage">Probe</a></li><li><a href="#ml086.s29" ref="log$=inpage&link_id=inpage">Appendices</a></li><li><a href="#ml086.s32" ref="log$=inpage&link_id=inpage">Bibliography</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2359738" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=2359738" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2359738" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=2359738" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/20684022" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification.</a><span class="source">[J Bone Miner Res. 2011]</span><div class="brieflinkpop offscreen_noflow">Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Yadav MC, Simão AM, Narisawa S, Huesa C, McKee MD, Farquharson C, Millán JL. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Bone Miner Res. 2011 Feb; 26(2):286-97. Epub 2010 Aug 3.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23183786" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> The role of phosphatases in the initiation of skeletal mineralization.</a><span class="source">[Calcif Tissue Int. 2013]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> The role of phosphatases in the initiation of skeletal mineralization.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Millán JL. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Calcif Tissue Int. 2013 Oct; 93(4):299-306. Epub 2012 Nov 27.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/36185572" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Perspective on Dentoalveolar Manifestations Resulting From PHOSPHO1 Loss-of-Function: A Form of Pseudohypophosphatasia?</a><span class="source">[Front Dent Med. 2022]</span><div class="brieflinkpop offscreen_noflow">Perspective on Dentoalveolar Manifestations Resulting From PHOSPHO1 Loss-of-Function: A Form of Pseudohypophosphatasia?<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Mohamed FF, Chavez MB, de Oliveira FA, Narisawa S, Farquharson C, Millán JL, Foster BL. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Front Dent Med. 2022 Feb; 3. Epub 2022 Feb 3.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/17227223" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization.</a><span class="source">[J Bone Miner Res. 2007]</span><div class="brieflinkpop offscreen_noflow">Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Roberts S, Narisawa S, Harmey D, Millán JL, Farquharson C. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Bone Miner Res. 2007 Apr; 22(4):617-27. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24533943" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Role of bone-type tissue-nonspecific alkaline phosphatase and PHOSPO1 in vascular calcification.</a><span class="source">[Curr Pharm Des. 2014]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Role of bone-type tissue-nonspecific alkaline phosphatase and PHOSPO1 in vascular calcification.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Bobryshev YV, Orekhov AN, Sobenin I, Chistiakov DA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Curr Pharm Des. 2014; 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