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This is a subset of the Currency Management Software, and tracks the vault contents of Numismatic (Collectors') Currency Items, including the categories of General Stock, Consignment Stock (available for sale), Redeemed Stock, Gratis Stock (items to be given as gifts), and Archive Stock (the Bank's Collection). It allows for the entry of requests for numismatic items for sale or as gifts, and tracks the process of their removal from the vault and delivery to the proper department, with full audit capabilities.
Centralize and manage all aspects of client communications including sales/marketing campaigns, and history of service across multiple MIMICS modules. Read More ^
Ensures that your data and system is protected and can be fully accessed or restored in the event of catastrophe. Read More ^
Allows customer forms such as confirmations and statements to be encrypted and emailed automatically. Read More ^
A customized web interface that allows your customers to view history and to place requests, with the look and aesthetics to match your own website. Read More ^
Allows secure, read-only access to aspects of the database of the related MIMICS module, for data mining or to allow you to create your own reports or exports. Read More ^
Includes functionality for general ledger, accounts receivable/payable, fixed assets, payment processing, purchasing and account reconciliation. Can interface with any other MIMICS module. Read More ^
You deserve better financial software. We have been thoughtfully crafting intuitive, easy-to-use software for decades to give you more of what you want, less of what you don't.
Software should not be one-size-fits-all. We take the time to get to know what makes you you, so that we can customize our software to offer a product that is exactly to your specifications and intuitively easy to use.
Our software does much of the "heavy lifting" of calculating, processing and automating to help greatly boost your productivity, streamline workflow operations and get you moving at optimal efficiency.
Beyond the labor you will save with the wide-ranging functionality our software provides, our customizations give you more of what you want for a lower price than our competition.
We respect the sensitive nature of your information. That's why we offer industry-exceeding security, including fully-encrypted databases and data tokenization.
We truly pride ourselves on our friendly, responsive customer support, day or night. We love our customers and strive for 100% client satisfaction. But don't take our word for it; read what our actual users say.
We are very happy with the MIMICS product that we are currently using. Our staff is very complimentary of the customer service there, as well as the response time.
We are happy with MIMICS. The Repo system we use has been very stable and customer support is readily available if needed.
Software is very customizable, allowing us address needs and changes as they arise.
The method used prior to MIMICS' installation in 2004 was a manual ledger. I'd say 100% increase in efficiency!
After working with MIMICS for over 15 years, I can honestly say their customer service is above and beyond expectations. Whether working on an upgrade to an existing product or requesting an additional service, the response time is exceptional. The staff is always pleasant and accessible!
[MIMICS Item Collections system is] a top-of-the-line domestic item collections software that we not only use for form printing, but also to balance to our general ledger account…we are very pleased with the quality of the product and the service we receive.
Since 2004, the Central Bank of The Bahamas has engaged MIMICS to develop several solutions to automate the processing of dormant accounts, to integrate cash management activities with straight-through processing capabilities, and to provide bondholder accounting for its registrar activities. Through the automation of these activities, the bank has achieved greater efficiency in its operations, with the desired audit trails.
The Bank is pleased with the quality of service support delivered by MIMICS and its demonstrated commitment to ongoing product enhancement in a cost-effective manner.
Saves on time both tracking collections and paying collections. It takes me about 5 minutes every time it is used, compared to 30 to 1 hour-plus by hand.
The system is very efficient for our daily use. It functions exactly the way we need it to.
The Repo software completely automates the pledging of securities to our sweep customers and communicates to these customers their daily positions.
Customer service is fantastic!
Born of the simple idea that your software should do exactly what you need it to do, MIMICS has been making customized financial software solutions since 1976. We craft systems that interface seamlessly with one another as well as with third-party programs to make your business operations run at optimum efficiency. Dedicated to customer satisfaction, we offer remote or on-site installations, trainings and outstanding people support. We are a company built on ingenuity and fueled by possibilities.
We appreciate that without our customers, we would never have made it this far, and we will never take them for granted now or in the future. Ensuring that our customers are always well taken care of is a founding principle of this company, and we will continue always with a customer-first state of mind.
We work hard to create a workplace that encourages inclusion, collaboration, respect and open communication. We have fun together. We listen to one another. We work hard, but maintain a good work-life balance. We always treat ourselves and customers with respect, and have no tolerance for intolerance or discrimination in any form. We support, we help, we drive one another to succeed.
We strive to be leaders of the financial software industry. To us, this means thinking ahead, challenging and pushing ourselves to be better, to do more. Asking questions like 'How can we make this better for our customers?' Trying new things and taking chances. We don't want to keep up with technology, we want technology to follow our lead.
We want our actions to match up with our core beliefs on a daily basis. We also understand the many facets of integrity, from product design, to delivering customer satisfaction as promised, to acting financially responsible. Our reputation has been built on integrity, and we will continue to bolster it with thoughtful intentions and actions.
All of our software is fully customizable, and we work with you personally to tailor our programs to meet the individual needs of your business. Our software will save you time and labor and increase your company's overall efficiency.
We have been creating software solutions for our clients for forty years now, continuously evolving to meet the demands of an ever-changing technoscape. We offer more functionality and customizations than our competitors, at a competitive price. And our product support and customer service are second-to-none.
We appreciate that our clients often work daily with sensitive information, and the privacy and security of your data is our top priority. We offer SSL (Secure Sockets Layer) and a highly-encrypted database that exceeds industry standards. Also, vulnerable data such as Tax IDs and SSNs are only hosted locally and NEVER reach our database.
We design our products with our customers in mind, making sure they are user-friendly and intuitive. We also full give full tutorials at installation, and our customer service team is always available to help.
Yes! We have clients all over the world, and we can customize any of our programs to be in full compliance of local statutes.
Due to the customizable features, it is virtually impossible to say until we have a better idea of what your needs are. Of course we are more than happy to provide free detailed quotes once we have more information. Annual maintenance fees, as a rule of thumb, are approximately 15-20% of the license fees. Please contact firstname.lastname@example.com or fill out our contact card on the site.
This will vary depending on the programs commissioned, the data conversion, specified customizations, interfacing and accounting specifications unique to you. After we have a good idea of what you want, we can give you a more specific quote, but the average is around 60 days.
Absolutely! We offer live online demonstrations via Webex, and can schedule one at your convenience. We also offer on-site demonstrations at any of our offices, and remote demonstrations if you prefer. For any one of these options, please fill out our online contact card, or email us directly at firstname.lastname@example.com.
Of course! Due to the variety of specific arenas we operate in, we would just want to make sure we have enough information so that we can put you in touch with an appropriate client. Please contact our sales team at email@example.com to facilitate.
Great! You can contact us in whichever manner is most comfortable for you. We would just like some initial information as to your specific needs and goals, and we can go from there. You can contact us via phone, email, or simply fill out the contact card here on our site and we will be in touch shortly.


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Oct. 7, 2007
There are victories and then there are signature victories.
Every member of the Lady Bisons volleyball time signed the 3-2 Atlantic Sun match win over Florida Gulf Coast Sunday afternoon at Allen Arena. The Lady Bisons lost the first game 30-26, rallied to win game two 30-23 and game three 30-24 before falling 31-29 in game four. That set up a game five that required the Lady Bisons to come from behind for a 15-13 game five win.
"We talked about being steady and being aggressive," said Lady Bisons coach Brandon Rosenthal. "If we could be aggressive and stay steady with a point here and a point there we would be in good shape. It was a great win for us."
It was the first Atlantic Sun loss for Florida Gulf Coast and the third straight A-Sun match win for the Lady Bisons.
"I felt good the whole match," said Rosenthal. "I turned to Jenna Panatier (assistant coach) and said before a point was played that this program needs one big win. We had been close, but we had never been able to get that big win."
It was the fifth five-game match of the season, and the third in the A-Sun, for the Lady Bisons. They have beaten South Alabama and lost to Evansville, Mercer and ETSU. They drew on those experiences to overcome setbacks against Florida Gulf Coast.
"Our players had a confidence about them," Rosenthal said. "They said, `hey, we can play with whoever we want and we are the team'. It was matter of how we put those other five-game matches aside. Everything was moving forward in this match.
"We had a great week of practice. And we had won against Stetson on Friday. I stepped back and let them play."
In game four the Lady Bisons watched a 29-24 lead disappear as Florida Gulf Coast made seven straight points.
"I thought we were ready to go," Rosenthal said. "In the first game we just made a couple of mental errors. From there on out I thought we had a great game plan and our players executed it well. In games two, three and four I thought we were in great control.
"In game four, being up 29-24 and losing is something you never want to go through. But to the credit of Florida Gulf Coast they made a lot of great plays at the end."
As has been the case all season junior outside hitter Sarah Snider and senior libero Ann Mullins set the pace for the Lady Bisons. Snider had a match-high 26 kills and also made 20 digs. Mullins made a team-leading 25 digs.
"Sarah got out to a really quick start," Rosenthal said. "I thought she did a phenomenal job and not just on offense. She was just solid on both offense and defense.
"She was being asked to do things in tougher situations than most of our players. She had 74 total attacks. She was a calming force for the team." Also in double figures in kills were senior outside hitter Jessica Enderle with a season high 21, freshman middle blocker Alex Kelly with 15 and senior middle blocker Alex Scruggs with 14.
Finishing in double figures in digs were freshman setter Stefine Pease with 15 and defensive specialists Jessica Turner and Grace Gilmore with 13 each.
Scruggs finished with a .433 hitting percentage. She also made a big play to end the match.
"It was a gutsy set by Jake Pease to go to Alex for the match point," Rosenthal said. "She had our best hitter on the outside. Alex Scruggs ran the play exactly how it should have been played.
"We talked at the end of game four after we lost/ We told our players they had to want the ball and when they got the ball they had to do something with it. I was proud of Alex."
The Lady Bisons are 5-4 at home this season. They close out a six-game homestand Tuesday night at 7 against Murray State from the Ohio Valley Conference.
The Lady Bisons are 9-9, 3-2 in the A-Sun. Florida Gulf Coast is 14-2, 4-1 in the conference.
"There is a still a lot of season left," Rosenthal said. "We are going to have some tough road matches ahead."

subset: commoncrawl


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Looks like you are using an old version of Internet Explorer - Please update your browser
The praying mantis is one of the most recognizable and striking members of the insect kingdom.
No results found in .
Answers in Genesis is an apologetics ministry, dedicated to helping Christians defend their faith and proclaim the gospel of Jesus Christ.


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Adena Anesthesiology
Anesthetics are used to reduce or prevent pain. Adena Anesthesiology, located at the Adena Regional Medical Center in Chillicothe, Ohio, is committed to providing excellent and compassionate anesthesia care. Our highly skilled and experienced clinical team provides state-of-the-art anesthesia and post-operative pain management.
The physicians at Adena Anesthesiology are experts in all aspects of anesthesiology, including general anesthesia, spinal anesthesia, regional anesthesia and nerve blocks, IV sedation, monitored anesthesia care (MAC), epidural anesthesia for labor and delivery, and postoperative pain management.
There are four main types of anesthesia. The type of procedure and health of the patient are some of the factors considered to determine the appropriate anesthesia. The general categories of anesthesia include:
- Local anesthesia: Numbs a small area of the body. Patient is awake and alert.
- Conscious or intravenous sedation (IV): A mild sedative is used to relax and relieve pain. Patient is awake but may not remember the procedure afterward.
- Regional anesthesia: Blocks pain in an area of the body, such as an arm or leg. Epidural anesthesia, which numbs sensation from the waist down, is a type of regional anesthesia.
- General anesthesia: Affects the whole body. Patient is asleep for the procedure and doesn't feel any pain. There is no memory of the procedure afterward.
Anesthesiology services are also provided at Adena Greenfield Medical Center and Adena Pike Medical Center.
Phone Number: 740-779-7540


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Texas Russell Rescue is the Texas group of
Russell Rescue, Inc. We are a network of volunteers located throughout Texas
who are dedicated to rescuing and rehoming homeless Jack Russell Terriers. We foster
them in our homes until we find them their perfect forever home.
You will find our available dogs on the RRI website and on Adopt-A-Pet.
You can also see our dogs on this website by clicking on the "Available Dogs†tab
at the top of the page.
If you are interested in adopting one of our dogs, please submit an adoption application.
To learn more about Jack Russell's, please read Bad Dog Talk. A
Jack Russell is not the right dog for everyone. Please read Bad Dog Talk carefully
to see if a JR is right for you.
The adoption fee is $200.00. All of our JR's are spayed/neutered, current on vaccinations,
and on heart worm preventative. Every adopter must sign a contract agreeing that
they will provide good care for the dog.
Donations Needed!


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The Editor's Back Fence
Which Berkeley Is It in the San Francisco Chronicle?
Which Berkeley article do you read in the Comical?
A couple of weeks ago, they published a Sunday front page piece by Carolyn Jones that took a dim view of the situation on Telegraph Avenue, one of Berkeley's traditionally most profitable shopping streets. It noted that things on Teley have gone badly downhill in the last few years (oddly enough during the decade when Tom Bates was mayor):"… sales tax revenues on Telegraph have plummeted, and empty storefronts abound. The intersection of Haste and Telegraph is marred by two vacant lots, and the former Cody's bookstore - once a hub of intellectual life - has sat empty since 2006."
But this week, only two weeks later, it seems everything's actually coming up roses in Berkeley. This Sunday's front page proclaims that "Berkeley mayor pushes city to prosperity."
Jones glows approvingly in a long gushy puff piece: "Call it Pax Berkeleyana. Crime has plummeted to its lowest level in 50 years. Schools have improved. Home values have remained high (the median home price is $720,000, according to the most recent census figures). Major hubs for environmental and disability rights groups have opened. Hotels, theaters and galleries are all thriving, and more are on the way."
I guess it's possible to have it both ways if you hire the right PR firm to hold hands with the press. Does anyone out there know who's flacking for Tom Bates these days? Both his chief of staff and the city's public information officer quit recently, and if you believe the city web site they haven't been replaced.
I'm not a betting woman, but if I were I'd bet that Bates is finally poised to make a play for the appointment to UC's Board of Regents that he's rumored to have been coveting for a while now. That would create an opening for him to anoint a successor to the Mayor's job, in the time-honored tradition of the Bates-Hancock dynasty.


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Animation is nothing but to bestow essence to any character. It is the art of bringing motion to such a thing that cannot be moved. Animation emulates rapid movements through a series of pictures that have objects in various positions. Time and space play a crucial role in animation. The object of animation could be a 2D painting, a clay statue, a picture of a person/ animal/ thing – just about anything at all.
Web designing is a process about conceptualizing, arranging, molding, and implementing electronic media content delivery by means of Internet appropriate for elucidation and display by a web browser. The purpose of web designing is to create a web site in order to portray content to the end user in the form of web. Elements such as text, forms, and bit-mapped images (GIFs, JPEGs, PNGs) can be placed on the page using HTML, XHTML, or XML tags.


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15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 184.108.40.206) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies.
Principal Findings
To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action.
Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways.
Enhanced version
This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2.
Citation: Niesen FH, Schultz L, Jadhav A, Bhatia C, Guo K, Maloney DJ, et al. (2010) High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships. PLoS ONE 5(11): e13719. https://doi.org/10.1371/journal.pone.0013719
Editor: Nick Gay, University of Cambridge, United Kingdom
Received: July 22, 2010; Accepted: October 4, 2010; Published: November 2, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This research was supported in part by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research, the Intramural Research Program of the NHGRI, NIH, and the Oxford Biomedical Research Unit. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Eicosanoids are arachidonic acid derivatives that comprise distinct functional classes including prostaglandins (PGs), lipoxins and leukotrienes [1]–[3]. These bioactive fatty acids control a multitude of physiological functions including inflammation and differentiation. Dysregulation of the enzymes responsible for the generation and metabolism of active prostaglandins and lipoxins is associated with malignant transformation and progression in a variety of cancer types, such as breast, colon, lung and bladder cancers [4]–[10]. The intracellular levels of prostaglandins are controlled mainly by the interplay between the cyclooxygenase enzymes (COX-1 and COX-2) on the one hand, and 15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 220.127.116.11) and other inactivating enzymes, on the other. COX-1 and COX-2 are bifunctional enzymes that, through their fatty acid cyclooxygenase- and prostaglandin hydroxyperoxidase activities, ultimately catalyze the generation of prostaglandin H2 (PGH2) from arachidonic acid [11]. The other prostaglandins are then generated and transformed into one another by several isomerases and synthases, producing, e.g., prostacyclin (PGI2) or thromboxane A2 (TX) [11], [12]. The wide variety of effects of prostaglandins in many different cell types derives from binding to a variety of mainly G-protein coupled receptors, of which eleven are currently known [12]. In addition, prostaglandins also interact with nuclear hormone receptors, thereby eliciting direct transcriptional effects [13], [14].
15-PGDH represents the key enzyme in the inactivation of a number of active prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) (e.g., by catalyzing oxidation of PGE2 to 15-keto-prostaglandin E2, (15k-PGE)) [15]. Thus far, two forms of 15-PGDH have been identified: NAD+-dependent type-I 15-PGDH, and the NADP+-dependent type-II 15-PGDH [15], also known as carbonyl reductase 1 (CBR1, SDR21C1) [16], [17]. The preference for NADP+ and the high Km values of CBR1 for most PGs suggest that the majority of the in vivo activity can be attributed to type-I 15-PGDH [15]. Human type-I 15-PGDH is encoded by the HPGD gene and belongs to the evolutionarily conserved superfamily of short-chain dehydrogenase/reductase enzymes (SDRs) [18], within which it is classified as SDR36C1 [17]. The enzyme has been purified from human placenta and its primary structure determined by Edman degradation [19]; it was subsequently cloned [20] and characterized as a homodimer with subunits of a size of 29 kDa [20], [21].
The critical importance of 15-PGDH for the inactivation of prostaglandins makes the enzyme an attractive target for studying the details of interactions and signaling events in inflammation and cancer. However, all inhibitors that have been identified so far lack potency and specificity. Several thiazolidinedione peroxisome proliferator-activated receptor γ (PPARγ) agonists, including pioglitazone and ciglitazone, have been shown to inhibit recombinant human placental 15-PGDH. Ciglitazone showed an IC50 of 2.7 µM [22], while an optimized derivative, CT-8, had a Ki of ∼90 nM [23]. Other clinically approved drugs that also act as inhibitors of 15-PGDH with micromolar potencies include non-steroidal anti-inflammatory drugs (NSAIDs) and COX inhibitors, e.g., indomethacin, sulindac, and niflumic acid [22]. Finally, a group of compounds called sulphasalazines have also been shown to inhibit human 15-PGDH, with the most effective compound, CAY10397, having a Ki of 110 nM [24].
Here we describe the identification of chemotypes that potently inhibit human 15-PGDH, with either competitive or noncompetitive kinetics, and strongly stabilize the enzyme in a cofactor-dependent manner. The lead compounds show remarkable selectivity based on accumulated data from a large number of high-throughput screens against a wide range of targets. The determination of the crystal structure of 15-PGDH, also reported in this work, enabled us to propose a binding mode for the competitive inhibitor in the active site pocket that confirms its mechanism of inhibition. For the other inhibitors, the observations from the 15-PGDH crystal structure characterized the mechanism of inhibition as being noncompetitive.
High-throughput identification of inhibitors of 15-PGDH
To screen for inhibitors in a high-throughput format, we adopted the standard assay used for this enzyme which involved monitoring the increase in sample fluorescence corresponding to the conversion of the non-fluorescent NAD+ cofactor into the fluorescent NADH upon oxidation of the 15-PGDH substrate 15-hydroxyprostaglandin type-E2 (PGE2) (Figure 1). The assay was miniaturized to a 4-µL volume in 1536-well format: enzyme (3 µL) was dispensed first, followed by a pintool transfer of library compounds dissolved in DMSO. After equilibration, a substrate dispense (1 µL) initiated the enzymatic reaction (see Materials and Methods, for detailed protocol description). A robotic validation run consisting of a concentration-response screen of the LOPAC1280 library, performed in triplicate, yielded excellent assay statistics and hit reproducibility (supplementary information Figures S1A and S1B). The concentration-response screen of the entire collection comprising 895 1536-well plates was completed in 5 days. The Z' screening factor [25] associated with each plate remained high and stable throughout the screen (average Z' = 0.86, Figure 1A). A concentration response of the control inhibitor GW5074, identified in an earlier pilot screen (PubChem AID 894, supplementary information Figure S1C), added as a 16-point dilution series in duplicate between 57.5 µM and 3.5 nM into the second column of every assay plate displayed consistent inhibition throughout the screen (supplementary information Figure S1D): the average IC50 was 10.4 µM and the associated minimum significant ratio was 1.5, indicating a highly stable run according to the definition given by Eastwood and co-workers [26]. The hits identified ranged in inhibitory potency from submicromolar to double-digit micromolar (Figure 1B). In order to progress only the highest-confidence primary screening hits to the subsequent investigations, samples exhibiting weak/noisy responses, as well as those representing potential false positives, were eliminated during the initial analysis. The testing of each library compound in dose-response mode (see example in Figures 1C and 1D) permitted a detailed examination of the type and quality of the inhibition response (IC50, concentration-response curve shape, efficacy, presence of asymptotes). Hits associated with low-efficacy (<40% maximum inhibition), incomplete curves, as well as those displaying only a single-point inhibition at the top concentration tested were eliminated from further consideration. Similarly, the collection of real-time kinetic data allowed the autofluorescence of each compound to be evaluated by comparing the initial fluorescence read at top concentration with the mean value of uninhibited assay controls: compounds whose top-concentration initial well fluorescence exceeded six standard deviations over the mean were flagged as potential autofluorescent artifacts [27], [28] (see example in supplementary information Figure S1E). The complete screening and followup results are available in PubChem (PubChem Assay ID 894).
A. Plot of the Z' factor associated with each plate, showing high stability over the entire duration of the screen (completed in five days). The average Z' was 0.86. B. Hit frequency for the library of tested compounds, measured as the distribution of compounds according to binned potencies. C. Typical effect of a non-fluorescent screening hit (inhibitor 13, titrated between 3.5 nM and 57.5 µM) on the time course of NAD+-reduction upon addition of PGE2. D. Dose-dependent reduction in enzyme activity caused by compound 13, as detected during the screen.
Re-testing of HTS hits
Similarity clustering of the high-confidence compounds resulting from the above triaging process performed using LEADSCOPE (Columbus, OH, USA) yielded 23 clusters and 15 singletons. A total of 87 representative members were chosen for re-sourcing and re-testing in a miniaturized screening assay as 24-point dilution series [29] where the majority of the compounds confirmed. Visual inspection of these retested hits (supplementary information Table S1, a subset shown in Figure 2) allowed further merging of clusters. Based on these clusters, 50 representative compounds that fulfilled stringent criteria for confidence (i.e., complete concentration-response curves comprising two clear asymptotes, ≥80% max. inhibition, and R2>0.9) were selected for further evaluation in the protein stabilization experiments described below.
Shown are the IC50 values for select compounds arranged by cluster number, along with their PubChem Chemical Identifiers (CID).
Compound stabilization of 15-PGDH
To investigate the ability of the inhibitors to thermally stabilize 15-PGDH, differential scanning fluorimetry (DSF [30]) was performed on the above 50 prioritized hits in the absence and in the presence of the cofactor in its oxidized or reduced state (NAD+ or NADH, respectively) (Figures 3 and 4). The melting point, Tm, of ligand-free 15-PGDH at pH 8.0 was 41.2±0.3°C (Figure 4A). The stability was increased by more than 4°C in the presence of NAD+ (Tm = 45.9±0.1°C) and by more than 10°C in the presence of NADH (Tm = 52.5±0.4°C). The substrate PGE2 failed to stabilize without cofactor, but stabilized by 2.4°C in the presence of NAD+ (Figure 4A). Interestingly, no stabilization was observed in the presence of NADH. Similarly to PGE2, none of the inhibitors stabilized the protein in the absence of cofactor (Figure 4A and supplementary information Table S1), suggesting that the cofactor may be necessary for the enzyme to assume a ligand-competent conformation. This observation is also in general concordance with the ordered bi-bi mechanism of the enzymatic reaction of short-chain dehydrogenases including 15-PGDH [31]. In the presence of cofactor, the 50 selected hits elicited a range of thermal stability enhancements up to 12.2°C in the presence of NAD+ and up to 13.5°C in the presence of NADH (expressed as a shift in the transition midpoint temperature, ΔTm) (Figure 3). A correlation between the ΔTm and the inhibitory potency has been shown in a number of protein-ligand systems, particularly for kinases [32]–[34]. In the case of 15-PGDH, the correlation between inhibitory pIC50s and ΔTm in the presence of cofactor appears to depend on chemotype: across the multiple clusters the correlation is generally weak (Figures 3A and 3B), while for individual chemotype clusters a meaningful correlation is observed. For the largest cluster 1, a moderate correlation is apparent in the presence of NAD+, while the correlation in the presence of NADH is more robust (R2 = 0.886): in general, compounds in this cluster show greater Tm shifts with NADH than with NAD+, as exemplified by the most potent analogue, compound 13, [1-(3-methylphenyl)-1H-benzimidazol-5-yl)(piperidin-1-yl)methanone (a.k.a. ML148), (IC50 of 56 nM, ΔTm = 7.3±0.2°C or 13.5±0.9°C with NAD+ or NADH, respectively). Compound 72 (a.k.a. ML149), belonging to a different cluster, showed a similar profile with a significantly larger ΔTm in the presence of NADH. Conversely, members of the smaller cluster 5 showed the reverse profile: the most potent analogue 61 (a.k.a. ML147) gave an IC50 of 141 nM and Tm shifts of 12.2±0.1°C or 2.9±0.5°C with NAD+ or NADH, respectively.
Thermal stabilization is expressed as a shift in the midpoint of the unfolding transition of the protein (ΔTm). ΔTm and pIC50 (-log(IC50) values are means of at least three independent measurements. The inhibitors are represented by color-coded symbols according to their chemical clusters, respectively (supplementary information Table S1): blue, cluster 1; dark green, cluster 2; orange, cluster 3; purple, cluster 4; light blue, cluster 5; light green, cluster 8; grey, cluster 9; dark yellow, cluster 11; red, singletons (a Tm could not be determined for the tested compounds in clusters 6, 7 & 10). The dotted line in each graph A & B denotes correlations for the compounds in cluster 1, respectively. Numbers within the graphs assign the positions for the inhibitors of special interest (see text).
A. Thermal stability of the protein in dependence of substrate and inhibitors, without cofactor (grey bars), and in presence of NAD+ (light blue bars) as well as in presence of NADH (purple bars). Shown are sets of these data, respectively, for each of the tested compounds as denoted on the Y axis. Bars represent averages of three independent experiments, with standard deviations displayed as error bars. B. Inhibitor dose-dependent reduction of the enzyme activity caused by compounds 13 (black triangles), 72 (blue squares) and 61 (red diamonds), respectively. Plotted values are means of three independent experiments, with standard deviations displayed as vertical error bars. Dashed or dotted lines, in colors corresponding to the data points, result from least-squares non-linear fitting of the data to the Hill equation (see Materials & Methods), respectively. C & D. Michaelis-Menten plots of the enzyme activity at varying concentrations of substrate, PGE2, in absence of inhibitor (black symbols) or in presence of compounds 61 (C) or 13 (D) at concentrations close to (blue symbols) or above (red symbols) their IC50 values, specified in table 3. The inset graph in D shows the data from the main graph as Lineweaver-Burk plot, demonstrating competitive inhibition from independence of the Y-axis intercept (1/Vmax) of the inhibitor concentration. All values are means of three independent measurements; standard deviations are displayed as vertical error bars.
Verification of inhibitor activity after re-synthesis
The contrasting stabilization profiles alerted us to the possibility of differing mechanisms of inhibition, and we selected compounds 13, 61 and 72 for further study. In order to definitively confirm identity and activity and to provide high quality material for in-depth kinetic studies, these three compounds were re-synthesized (see Materials and Methods). The effects of the re-synthesized compounds on the protein stability (Figure 4A) were found to be similar to the results obtained previously. To achieve an optimal signal window in the detailed kinetic measurements with the new compounds, a higher enzyme concentration was employed and the reactions were run at a lower percent substrate conversion than used in both the qHTS and the re-tests. As a result, higher apparent potencies were observed for all three compounds (Figure 4B): The IC50 for compound 13 was 7.9±0.8 nM, compared to 56 nM calculated according to the re-test results, and for compounds 61 and 72, respectively, values of IC50 of 26.4±2.4 and 15.0±1.2 nM were determined (compared to 141 nM and 82 nM, respectively). All three compounds caused a maximum of 100% inhibition (Figure 4B).
Catalytic constants in presence of inhibitors
In order to explore the mechanisms of action of 13, 61 and 72, we determined the dependence of the enzyme activity on the PGE2 substrate and NAD+ cofactor concentrations, first in the absence of inhibitor, and then at inhibitor concentrations close to their IC50, respectively (Table 1). The Michaelis constant, Km, for PGE2 was calculated from the data as 5.5±0.6 µM, comparing favorably with previously published values [21], [35], [36]. The maximal rate, Vmax, for the oxidation of PGE2 was 28.1 µmol/(min·mg), and the catalytic constant, kcat, was about 14 per second; the catalytic efficiency, kcat/Km, was 2.5·106. Compound 61 had almost no effect on the Vmax (Figure 4C), while it caused an increase in the Km, by 100% at 10 nM and 3-fold at 50 nM, suggesting a competitive mechanism of inhibition with respect to PGE2 [37]. Global fitting of the results to the four possible modes of inhibition (competitive, uncompetitive, noncompetitive and mixed-type inhibition) returned the highest probability for the mixed-type mode (Table 2). However, the fitting results are also consistent with a competitive mode for inhibition by compound 61, particularly when the residual Chi2 value for this mode is compared to those of the other two compounds. Together with the observation from the Lineweaver-Burk analysis of the data (inset in Figure 4C), the most consistent mode of inhibition for compound 61 is competitive inhibition with respect to the substrate PGE2. On this basis, considering the concentration of the substrate in the experiment, the observed IC50 corresponds to an inhibition constant, Ki, of approximately 5 nM [38].
In contrast, both compounds 13 and 72 decreased the maximum rate of 15-PGDH in titrations of PGE2 (Table 1). Applied at a concentration of 10 nM, compound 13 decreased Vmax by 25% (20 nM of the compound reduced Vmax to less than half; Figure 4D), while compound 72 caused a loss of approximately 50% in maximum activity. Compound 13 additionally also reduced the apparent Km by half at a concentration of 10 nM, suggesting that the affinity of the enzyme for the substrate was increased by the inhibitor, consistent with a mode of uncompetitive or mixed-type inhibition. Because both Vmax and Km were reduced, almost no change in the catalytic efficiency, kcat/Km, was observed with this compound. In contrast, Compound 72 showed no effect on the Km, and consequently also caused a reduction in the catalytic efficiency of the enzyme [37]. Comparative data fitting made a noncompetitive or mixed-type mode most likely for compound 72, while the data for compound 13 are consistent with noncompetitive, uncompetitive or mixed-modes (Table 2).
The effects of compounds 13 and 61 on the rate constant and catalytic efficiency of NAD+ reduction were investigated: The Km for NAD+ was lower than for PGE2 (15.8±0.9 µM), comparing favorably with published values [21]; the catalytic efficiency, kcat/Km, was determined as 0.7·106 (Table 1). The decrease in the maximum rate of NAD+ reduction by compound 13 at 20 nM was similar to its effect on the conversion of the substrate PGE2: the Vmax was decreased by approximately 60% and the Km for NAD+ by ∼45%. Taken together, these data suggest that compound 13 acts noncompetitively or in a mixed-type mode with respect to the substrate while uncompetitively with respect to the cofactor. For compound 61, on the other hand, a difference between the influence on PGE2 oxidation and on NAD+ reduction was observed: for the latter, this compound caused an effect similar to that of compound 13, also suggesting an uncompetitive mechanism of action with respect to the cofactor (i.e., increasing the affinity of the enzyme for the cofactor). This interpretation was confirmed by the results of comparative fitting (Table 2) that showed uncompetitive inhibition with respect to the cofactor NAD+ as the most likely mode of inhibition for both compounds.
Crystal structure of the 15-PGDH homodimer
We were interested to explore whether the structure of 15-PGDH could provide insights into the observed mechanisms of the newly discovered inhibitors. After several unsuccessful crystallization attempts with constructs bearing different tags fused to the N- or C-terminus, respectively, it was possible to grow crystals in presence of NADH from a construct purified with a C-terminal His6 tag and to determine for the first time the structure of the homodimer of 15-PGDH in a complex with its cofactor at a resolution of 1.65 Å (Figure 5 and Supplemental Table S2). This resolution is not high enough to distinguish the reduced state of the cofactor from its oxidized form [39]. It is, however, unlikely that the structure of 15-PGDH contained NADH considering the time necessary to generate the crystals and the known short half-life of NADH in solution, despite the addition of the cofactor in its reduced state. The protein shows the α/β folding pattern that is highly conserved among the short-chain dehydrogenases/reductases (SDR), where a central β-sheet consisting of 8 strands is flanked on either side by two arrays of α-helices (Rossmann fold) [18], [40]. In the crystallized construct the last ten amino acids from the 15-PGDH sequence are missing, leaving Tyr256 as the last native amino acid. Instead, the cloning procedure resulted in the fusion of nine non-natural amino acids (Gly-Ser-Lys-Glu-Asn-Leu-Tyr-Phe-Gln) to the C-terminus remaining after cleavage of the tag. In the structure of the dimer the artificial C-terminus of one protomer is folded over the top of the opposing protomer (highlighted in orange in Figure 5), with a number of residues engaging in interactions with side chains of the other protomer, notably between Phe264 and Tyr217. One cofactor molecule per protomer is located in the center of the molecule (in ball-and-stick representation in Figure 5). The acidic residue Asp36 forms hydrogen bonds to the 2′- and 3′-hydroxyl groups of the adenine ribose moiety, while Asp64 forms a hydrogen bond to the amino group of the adenine moiety (supplementary information Figure S2). The nicotinamide moiety of NAD+ is positioned close to the catalytic tetrad (Asn107, Ser138, Tyr151 and Lys155), competent for the hydride transfer from the S-side of C4 to the substrate following the reaction mechanism conserved among all SDRs [31], [40]–[42].
Displayed are the two protomers with their backbones in grey and blue, respectively. The cofactor is depicted in a ball-and-stick representation and color-coded by atom type (green, carbon; red, oxygen; blue, nitrogen; orange, phosphate). The C-terminus of each protomer whose residues originate from the cloning procedure (see text) is highlighted in orange.
Docking of inhibitors to the 15-PGDH•NAD+ complex
All attempts to co-crystallize the inhibitors with 15-PGDH were unsuccessful. Therefore, we explored the inhibitor binding modes by computational docking of the compounds to the structure of the 15-PGDH•NAD+ complex. Docking experiments were carried out using the structure of a single protomer, to avoid the artifactual occupancy of the substrate pocket by the C-terminus of the opposing protomer (see above). This structure offers a large volume for binding of the inhibitors, notably a hydrophobic pocket close to the nicotinamide moiety of the NAD+, flanked by Ile195, Leu192, Ile211 and Ile215 (indicated by a green mesh in Figures 6A and 6B). An elongated tunnel leads to the exterior of the protein, formed by Phe185, Met213, Leu139, Met143 and Val145.
The view shows a cut-through into the substrate pocket of 15-PGDH, with the volume of the pocket indicated by a green mesh. Key amino acid residues (see text) are labeled. Compounds and amino acid sidechains are in ball and stick representation, with the atoms color-coded: blue, nitrogen; red, oxygen; silver, bromide; white, carbon atoms in side chains; yellow, carbon atoms in compounds; green, carbon atoms in the cofactor. The figures were created using ICM (Molsoft, LLC).
We identified plausible binding modes with well-defined side chain interactions which, interestingly, were similar for all three inhibitors, despite their structural differences (Figures 6A and 6B, supplementary information Figure S3). Binding of compound 61 appears to be driven by interaction of the imidazo-pyridine ring with the catalytic Ser138 and Tyr151 residues, while the bromo-substituent projects into the hydrophobic pocket. The cyanobenzyl group is accommodated in the substrate tunnel (Figure 6A). For compound 13, the amide carbonyl also interacts with the catalytic residues Ser138 and Tyr151, while the piperidine ring fits snugly into the adjacent hydrophobic pocket. The remainder of the molecule is accommodated along the tunnel towards solvent, with the side chain of Phe185 providing a stacking opportunity for the central benzimidazole moiety of 13 (Figure 6B). The binding mode found for compound 72 is similar, with the triazole moiety mimicking the amide carbonyl of compound 13 in accepting hydrogen bonds from both Ser138 and Tyr151 (supplementary information Figure S3). The fused azepine ring occupies the proximal hydrophobic pocket, and the remaining biaryl moiety extends into the substrate tunnel, again with the possibility for stacking between the pyrrole ring of 72 and Phe185.
Selective, high-affinity inhibitors for human 15-hydroxyprostaglandin dehydrogenase (15-PGDH) are desirable as tools to facilitate the mapping of prostaglandin signaling pathways in vitro and in vivo. Using a quantitative high-throughput screen approach we have found several new chemotypes that inhibit 15-PGDH with high affinity. Detailed biophysical analyses have demonstrated the strong stabilizing effect of these molecules on the enzyme. Based on the results from our detailed kinetic analyses a number of inhibition modes appeared possible, but the data are consistent with compounds 13 and 72 not inhibiting competitively, while a competitive mechanism of inhibition appeared likely for compound 61.
In order to gain better understanding of the distinct mechanisms of action of the inhibitors, it is helpful to examine the catalytic mechanism of 15-PGDH. The SDR enzymes have been shown to follow a common sequential ordered bi-bi-reaction mechanism, involving sequential cofactor binding, substrate binding, catalysis, product release, and finally release of co-product [31], [40], as shown in the reaction coordinate (Figure 7). In analogy with other SDRs, binding of the NAD+ cofactor is expected to alter the local electrostatic environment of the catalytic Tyr151 residue, favoring the deprotonated state. After substrate binding, the reaction proceeds by deprotonation of the substrate 15-OH group by Tyr151, facilitating hydride transfer from the substrate to NAD+, and forming the product complex consisting of protonated Tyr151, NADH, and ketone.
Compound 61 mimics the substrate (left panels), consistent with experimental data showing favored binding to the NAD+-bound form of 15-PGDH, and its competitive mechanism of inhibition with respect to PGE2. Compound 13 mimics the product of oxidation (right panels), consistent with its favored binding to the NADH-bound form of 15-PGDH, and its uncompetitive mechanism of inhibition with respect to PGE2.
One of the inhibitor chemotypes identified from the high throughput screen is a series of imidazopyridines, represented by the most potent analogue 61, with an IC50 of 25 nM and an estimated Ki of 5 nM. The biophysical data show a strong preference for 61 in stabilizing 15-PGDH complexed with NAD+ as compared with NADH.
The results of the docking experiments provide support for a competitive mechanism of inhibition by compound 61. The docking directs the imidazopyridine group into hydrogen bonding interactions with the catalytic residues Tyr151 and Ser138. In the co-complex with NAD+, as discussed above, the deprotonated form of Tyr151 is favored: the resultant oxyanion is able to interact with the protonated form of the pyridine ring, while the hydroxy group of Ser138 is able to donate a hydrogen bond to the adjacent imidazole nitrogen. The much weaker stabilization observed with NADH implies that the protonated form of Tyr151 interacts considerably more weakly with the pyridyl nitrogen.
In addition to providing a convincing basis for interaction of the heterocyclic motif with the enzyme's catalytic machinery, this binding orientation also favorably directs the bromo-substituent into the nearby hydrophobic pocket, and places the S-linked cyanobenzyl group into the substrate tunnel leading towards the exterior of the protein, which contains side chains available for stacking interactions (Phe185, Tyr217). Taken together, the data argue that the position for binding of compound 61 along the reaction coordinate of 15-PGDH is at step 2, i.e., occurs to the complex of 15-PGDH•NAD+ (left panel in Figure 7). This interpretation is consistent with the observed uncompetitive mechanism of action with respect to NAD+, since the higher affinity of the inhibitor compared to the substrate is expected to increase the affinity for NAD+.
The majority of the remaining inhibitor hits fall into three structural clusters (clusters 1, 2 and 4; supplementary information Table S1), all containing an amide moiety as the common feature (Figure 2). Structure-activity relationships indicate that activity is favored when the nitrogen of the amide forms part of a saturated heterocycle, most commonly piperidine. In all of the active amides identified, the carbonyl is linked to a ring, which is aromatic in the most potent analogues. The highest potency is achieved when this ring is linked to a further aryl ring, with a variety of linkers being tolerated. In compound 13, this linker is incorporated into the central fused benzimidazole ring system.
The results of the docking experiments are consistent with the observed structure-activity relationships and provide help with the decision about which of the mechanisms of inhibition that are possible according to the kinetic analyses would be most likely for the inhibitors. In the model of compound 13 docked into 15-PGDH, the oxygen of the amide carbonyl presents two lone pairs that can accept hydrogen bonds from both the catalytic Ser138 and Tyr151 residues, mimicking the ketone product of oxidation of the PGE2 substrate. Tyr151 is only able to donate this hydrogen bond when protonated, which is favored by binding of NADH, while binding of NAD+ favors the deprotonated state. Hence, compound 13 acts as a product analogue in the sequential ordered bi-bi catalysis mechanism, and a noncompetitive mode of inhibition with respect to PGE2 and an uncompetitive mode with respect to NAD+ would be expected [43]. Our kinetic analysis is consistent with this mode, identifying uncompetitive inhibition with respect to the cofactor, while equal probability was found for noncompetitive, uncompetitive and mixed-type inhibition with respect to the substrate (Table 2). Uncompetitive inhibition by the compounds of the series with respect to NAD+ is, furthermore, consistent with the observed enhanced stabilization of 15-PGDH when the latter is bound to NADH as compared with NAD+; this stabilization of product-bound 15-PGDH ultimately results in a suppression of enzymatic turnover.
The preference for small alicyclic amides is also explained by the docking model: the piperidine moiety of compound 13 is relatively tightly accommodated in the hydrophobic pocket adjacent to the catalytic residues (Figure 6B). Consistent with this binding mode, inhibitory potency is weaker for secondary or tertiary straight-chain amides, and further diminished or abolished with bicyclic amides that can clearly not be accommodated in this pocket (see Figure 2). As described above, the structure-activity relationships indicate that activity is retained with a variety of groups attached to the amide carbonyl carbon atom, although there is a preference for aromatic groups, and in general these groups are substituted in such a way as to be able to adopt an extended conformation. Such a conformation is consistent with the docking mode, placing the extended group into the substrate tunnel leading towards the exterior of the protein. The availability of side chain groups capable of stacking interactions (Phe185, Tyr217) adds further support to this proposed binding mode (Figure 6).
Compound 72 shares aspects of its mechanism with compound 13, notably a strong preference for NADH in co-stabilization of 15-PGDH (Figures 3 and 4A) and a cofactor-uncompetitive inhibition mechanism. With respect to the substrate PGE2 the kinetic analysis favored the noncompetitive mode, consistent with the expectation for a product-analogous binding mode (see above). Although compound 72 does not contain an amide group, the fused 1,2,4-triazole is capable of acting as a bioisostere, with a lone pair of electrons on each of the adjacent nitrogen atoms mimicking the two lone pairs of the carbonyl oxygen in 13 (Supplementary Information Figure S3). Thus, in the docking experiments, the triazole moiety accepts two hydrogen bonds from Ser138 and the protonated form of Tyr151; the remaining hydrophobic and stacking interactions are remarkably similar to those modeled for compound 13. Therefore, both compounds 13 and 72 bind at step 4 along the 15-PGDH reaction coordinate (Figure 7), mimicking the product, favoring co-complex formation with NADH and behaving in an uncompetitive manner with respect to the PGE2 substrate.
The compound library used for screening of 15-PGDH in this study has been, so far, tested in screens against over 320 targets (http://pubchem.ncbi.nlm.nih.gov/). This list includes two other proteins that are involved in prostaglandin signaling, the EP2 prostaglandin-E2 receptor and the M1-muscarinic receptor. The list also includes two SDR enzymes, hydroxyacyl dehydrogenase (HADH2, also known as type-10 hydroxysteroid dehydrogenase, HSD17β10; PubChem Assay ID 886) and type-4 hydroxysteroid dehydrogenase (HSD17β4; PubChem Assay ID 893), and a medium-chain dehydrogenase/reductase (MDR), aldehyde dehydrogenase 1 (ALDH1A1; PubChem Assay ID 1030), each of which was tested using a similar experimental protocol to that used for 15-PGDH. As the results in Table 3 show, all lead inhibitors identified in this study were largely inactive against ALDH1A1: compounds 61 and 72 showed a flat response, while compound 13 displayed only a shallow curve with an approximate IC50 of 36 µM. Against the two hydroxysteroid dehydrogenases (HSD17β10 and HSD17β4), both compounds 13 and 72 were inactive up to the top concentration of 57.5 µM.
In summary, the new inhibitor chemotypes identified in this study provide a set of tools, with complementary mechanisms of inhibition, for functional studies on the role of 15-PGDH in prostaglandin signaling pathways. The lead compounds that have been characterized in detail exhibit nanomolar affinities. The existence of disease scenarios involving over-activation of 15-PGDH and in which selective inhibition of the enzyme would be beneficial, is currently unclear. Work published by Tai and co-workers suggested that certain prostate cancers might involve higher-than-normal activity of 15-PGDH [44], and for such cases application of inhibitors based on the chemotypes identified in this study may prove useful. The potential utility of the probes is reinforced by the low cross-reactivity of these compounds in screens against multiple targets, among them receptors with a role in prostaglandin signaling and dehydrogenase enzymes utilizing the same cofactors, suggesting selectivity of the inhibitors for 15-PGDH. The availability of multiple inhibitors with diverse chemical structures for cellular studies enhances confidence in any response observed being due to on-target effects; the likelihood for such diverse compounds having an identical off-target activity profile is very small. While it is difficult to anticipate differences in the cellular response between inhibitors showing contrasting modes of action, one might expect the noncompetitive compounds to demonstrate inhibition independently of the substrate concentration. This may help to avoid problems similar to those encountered, for example, with ATP-competitive protein kinase inhibitors, where high inhibitor concentrations are required in cells to compete against the endogenous co-factor. Finally, in this work we present the novel crystal structure of human 15-PGDH, which will serve as a basis for further studies on the function, mechanism and inhibitor design for this enzyme.
Materials and Methods
Cloning, Expression and Purification
The protein was expressed in E. coli BL21(DE3)-R3 cells, from a synthetic DNA sequence comprising residues 3–256 of the human HPGD gene (GenBank identifier: 1203982). Cloning into the p15 vector added a TEV-cleavable (*) C-terminal His6 tag (gskenlyfq*ghhhhhh) and four extra amino acids, MAHM, at the N-terminus. The protein was purified using immobilized Ni-affinity chromatography followed by proteolytic cleavage of the His6 tag and size exclusion chromatography. As judged by SDS-PAGE, the sample was >95% pure; the correct molecular weight was confirmed using electrospray mass spectrometry. The protein was frozen in aliquots in liquid nitrogen and stored at −80°C until use.
Quantitative high-throughput screening (qHTS)
The 160,182-member library consisted of compounds from the National Institutes of Health Molecular Libraries Small Molecule repository (NIH MLSMR), prepared as 10 mM stock solutions in 384-well plates and delivered by Galapagos Biofocus DPI (South San Francisco, CA, http://mlsmr.glpg.com), from the NCGC internal exploratory collection comprising several commercially available libraries of known bioactives, as well as from libraries provided by commercial and academic collaborators. Details on the library composition and its formatting for qHTS have been published elsewhere [27], [29], [45].
The enzymatic reaction was performed in 50 mM Tris-HCl pH 8.0, containing 0.01% Tween 20. Final reactions comprised 20 nM 15-PGDH, 1 mM NAD+ and 30 µM PGE2. The high-throughput screen was performed on a fully integrated robotic system (Kalypsys Inc, San Diego, CA) as described elsewhere [45]. Library compounds, arrayed as seven-point inter-plate concentration series [29], were screened from the lowest (3.5 nM) to the highest (57.5 µM) concentration, with plates containing DMSO only, serving to note any systematic drifts in the signal, interspersed evenly throughout the screen (approximately every 50 plates). Briefly, 3 µL of reagents were dispensed into 1536-well Greiner black solid-bottom assay plates using integrated nanoliter solenoid-technology dispensers [45]. Compounds and controls (23 nL) were transferred via Kalypsys pintool equipped with 1,536-pin array (10-nL slotted pins; V&P Scientific, Palo Alto, CA). After a 15-minute incubation (to allow for interaction between compound and enzyme), a 1-µL substrate addition step initiated the reaction. Kinetic data were collected on a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with standard UV fluorescence optics (exc. 340 nm, em. 450 nm).
Starting fluorescence intensity and the change in fluorescence over the two-minute initial rate period were recorded for every well. Screening data were normalized against no-enzyme (64 wells, columns 3 and 4) and no-compound (32 wells located in column 1) controls, and concentration-response relationships were derived as described previously [46]. A four-parameter Hill equation [47] was fitted to the concentration-response data using publicly available algorithms (http://www.ncgc.nih.gov/pub/openhts/curvefit/). Similarity clustering of active compounds and SAR (structure-activity relationship) derivation were performed as described previously [46].
Kinetic analysis and inhibitor mechanism of action studies
Kinetic studies were performed in 100 mM Tris buffer pH 8.0, containing 0.01% Tween-20 and 10 nM 15-PGDH enzyme. NAD+ and PGE2 were applied from stock concentrations of 100 or 2 mM, respectively, in 500 mM Tris/HCl, pH 8.0. All inhibitor compounds were dissolved in 10% (v/v) DMSO at varying concentrations from which they were applied at 1∶5 ratio (resulting in a final concentration of 2% (v/v) DMSO in all assays). For each experiment an aliquot of purified enzyme was thawed freshly and pre-diluted to 10 µM in buffer I (5 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.01% Tween-20). All experiments were performed in white 384-well PCR microplates (Bio-Rad Laboratories, Inc.) in final assay volumes of 20 µL. Twelve-point compound titrations were set up from serially diluted stock solutions in 10% (v/v) DMSO. Before starting the reactions by addition of 30 µM PGE2 substrate and 200 µM NAD+ in buffer I (from injectors built into the spectrometer, PolarStar Omega, BMG Labtech GmbH), the compounds were incubated in the wells of the plate with enzyme for ten minutes at the experimental temperature of 25°C. Experiments comprising substrate titrations, from serially diluted stock solutions in buffer I, were started by injection of buffer I containing enzyme and NAD+, while cofactor titrations were set up in assay buffer containing 30 µM PGE2 substrate and started by injection of enzyme in buffer I. The reduction of NAD+ was followed by monitoring fluorescence emission at 460 nm, excited at 355 nm (10 nm bandwidth filters, PolarStar Omega, BMG Labtech GmbH), at a reading frequency of 22 s/point or 12 s/point for compound- or substrate/cofactor titrations, respectively. The initial rates recorded in compound titrations were plotted against -log[compound], and non-linear regression (Levenberg-Marquardt) fitting of the dose-response curve to the Hill equation [47] was performed to derive the IC50, using PRISM 5.0 (GraphPad Software, Inc.). For substrate/cofactor titrations, curves with initial rates plotted against the concentration of substrate or cofactor were fitted to the Michaelis-Menten equation, to derive maximal activities, Vmax, and Michaelis constants, Km. Fitting qualities were judged from the goodness of fit (R2>0.98).
Differential Scanning Fluorimetry (DSF)
Studies to assess the thermal stability of human 15-PGDH in presence of compounds were performed as described previously [30], with variations. Briefly, the protein was diluted to a final assay concentration of 1 µM in 100 mM Tris buffer pH 8.0, containing 0.01% Tween-20 and 1∶1000 SYPRO orange dye (Invitrogen). The final assay volume was 10 µL, with or without 200 µM of either NAD+ or NADH. Compounds were added at a ratio of 1∶5 from appropriate stock solutions in 10% (v/v) DMSO to comprise either 50 or 250 µM final concentrations. Heat denaturation curves were recorded using a RT-PCR instrument (Mx3005p, Agilent) applying a temperature gradient of 2°C/min. Analysis of the data was performed using DSF ANALYSIS 2.5 (ftp://ftp.sgc.ox.ac.uk/pub/biophysics) and PRISM 5.0 (GraphPad Software, Inc.).
Chemical Synthesis of compounds
Compounds were synthesized following established procedures, from starting materials and reagents purchased from Aldrich Chemical Co. (supplementary information Text S1). The identity of the compounds was confirmed by analytical thin layer chromatography (TLC), nuclear magnetic resonance (NMR) and high-resolution mass spectrometry.
X-ray crystallography
For crystallization of human 15-PGDH, purified protein was concentrated by ultrafiltration to 17 mg/mL (Vivaspin, cut-off: 30 kDa). Prior to crystallization NADH (Sigma) was added to a concentration of 5 mM to the concentrated protein in a volume of 100 nL. The solution was then mixed with 100 nL of crystallization solution containing 30% (w/v) PEG-1000 and crystals were obtained at 4°C using the sitting drop vapour diffusion technique. Crystals were transferred to a cryo-protectant consisting of a fifth of 100% (v/v) glycerol and four fifths of well solution, and flash-frozen in liquid nitrogen. A dataset extending to a resolution of 1.65 Å was collected on an R-AXIS HTC imaging plate area detector mounted on an F-RE SuperBright rotating anode generator (Rigaku MSC) operating at 45 kV and 45 mA (supplementary information Table S2). Data were indexed, integrated and scaled using MOSFLM v6.2.5 [48] and SCALA v5.0 (implemented in the CCP4 suite [49]). Initial phases were calculated by molecular replacement implemented by PHASER v1.3.1 [50] using an ensemble of three SDR structures (PDB codes 1WMB [51], 1IY8 [52] and 1ZK3 [53]) as search models over 50 cycles of automated model building using ARP/wARP [54]. Water molecules were automatically picked by ARP/wARP and later checked manually for appropriate density and hydrogen-bonding pattern. The final rounds of manual model building and refinement were carried out in COOT [55] and REFMAC v5.02.0005 [56], respectively (supplementary information Table S2). The coordinates of the final model and structure factors were deposited with the PDB accession ID 2GDZ. The structural information in this article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions, available on the PLoS website (Datapack S1). A web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in supplementary information Text S2.
Molecular Docking
Docking was performed using GLIDE (Schrodinger, LLC) [57] and the structure of 15-PGDH was prepared using the software's default minimization protocols. Docking grids were generated using a box around Ser138 in the catalytic site. For docking to the NAD+ complex, Tyr151 was kept de-protonated; for docking to the NADH complex NAD+ was changed to NADH with a OPLS-2005 force field [58] and a hydrogen constraint to Tyr151 was used. Ligands (substrate and inhibitors) were prepared with the LIGPREP and IONIZER tools in GLIDE, at pH 7.0+/−2.0, to determine the ionization states of the ligands. All inhibitors were docked using GLIDE-SP and the top 5 scored poses were kept. The binding poses were visually inspected and hydrogen bond patterns were analyzed in order to determine the best pose.
Supporting Information
Table S1.
Structural clustering of 87 compounds re-tested after qHTS, with their potency in 24-point titrations compared to their effects on 15-PGDH thermal stability.
Table S2.
Data collection and structural refinement statistics for human 15-PGDH.
Figure S1.
Miniaturized screening assay for 15-PGDH. A. Activity heatmaps showing the assay miniaturization to a 4 µl volume in 1536-well format (increasing concentrations to the right). B. Plot of the Z' factor associated with each plate of the pilot concentration-response screen of the LOPAC1280 library performed to validate hit reproducibility. C. Molecular structure of the control inhibitor GW5074 that was added as a 16-point dilution series in duplicate between 57.5 µM and 1.75 nM into the second column of every assay plate (panel D). The average IC50 for the compound was 10.4 µM and the associated minimum significant ratio was approx. 1.5. E. Example of a fluorescent inhibitor (applied at between 3.5 nM and 57.5 µM) on the time course of NAD+-reduction upon injection of PGE2.
Figure S2.
Binding of the cofactor NAD+ in the active site of human 15-PGDH. The acidic residue Asp36 forms hydrogen bonds to the 2′- and 3′-hydroxyl groups of the adenine ribose moiety, while Asp64 forms a hydrogen bond to the amino group of the adenine moiety. The nicotinamide moiety of NAD+ is positioned close to the catalytic tetrad (Asn107, Ser138, Tyr151 and Lys155).
Figure S3.
Binding of inhibitor 72 in the active site of 15-PGDH as predicted by docking studies. The view shows a cut-through into the substrate pocket of 15 PGDH, with the volume of the pocket indicated by a green mesh. Key amino acid residues are labelled. The figures were created using ICM (Molsoft, LLC).
Text S1.
Methods used in the chemical syntheses of the three lead compounds.
Text S2.
Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).
Datapack S1.
Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at http://www.molsoft.com/icm_browser.html.
Author Contributions
Conceived and designed the experiments: FHN DM UO TDH AS. Performed the experiments: FHN LS CB KG DM ESP. Analyzed the data: FHN LS AJ CB DM ESP MW UO TDH AS. Contributed reagents/materials/analysis tools: FHN AJ DM. Wrote the paper: FHN AJ DM UO TDH AS.
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10/08/2019 Facility Closures
Dear Citizens
**UPDATE** – October 29, 2019 Legacy Park, Chelsie Meadows, and Vae View tennis/pickleball nets have been taken down, due to current temperatures and upcoming weather. Each year the City closes down the facility amenities and restrooms due to freezing temperatures so the pipes and toilet bowls do not break. Thank you for your patience and we apologize for any inconvenience this may cause. Please contact the Parks and Recreation Department at 801-336-3900 with any questions.
Starting today, October 8, 2019, due to current temperatures and the upcoming hard freeze, Layton City has closed the following Park restrooms: Grey Hawk Park, Sand Ridge Park, Andy Adams Park, Legacy Park, Chelsie Meadows Park, Wendal Starkey Bathroom (Located inside the Commons Park), Vae View Park, Chapel Park, and Oak Forest Park.
Park Amenities
Pavilion Reservations
Ellison Park
Parks and Recreation
465 N Wasatch Dr
Layton, UT 84041
(801) 336-3920 Rainout Hotline
(801) 336-3930 Museum
(801) 336-3939 Surf 'n Swim


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A burglar was arrested after he was captured on CCTV wobbling along on a stolen bicycle with his ill-gotten gains.
Leeds Crown Court heard Nathan Anthony Burton was on bail for a break-in at the Perfect Home shop in Dewsbury's Princess of Wales Precinct when he broke into a flat in Batley.
The occupier had left the flat locked when she went to work at 7.15am on November 27, but returned home to find an intruder had forced a window in the living room.
Prosecutor Rukhshanda Hussain said the woman was shaking and crying when she phoned her father for help.
Items stolen included her laptop, a camera, a Kindle and some jewellery. Police investigations led to the discovery that a £500 bike was missing from another address and CCTV footage showed Burton on the bike carrying a blue bag.
"He had very little control of the bike because of the property he was carrying," Ms Hussain said. "The footage was seen by a police officer who recognised him and he was arrested."
Burton, 36, of Whitehall Way, Dewsbury, was jailed for a total of four years last Thursday after admitting burglary and theft. He also asked for nine other offences of commercial burglary and theft to be taken into consideration.
Anastasis Tassou, mitigating, said Burton's offending stemmed from his need to finance his taking of hard drugs including crack cocaine and heroin.
But he had turned to drugs in an effort to forget an ordeal in 1995 when he was violently kidnapped and held a prisoner in a cellar. He was beaten, tied up and was only eventually rescued because an off-duty police officer had heard his screams and went to investigate.
Mr Tassou said Burton had never received any counselling for that trauma and dealt with it by abusing drugs.
He said Burton had managed to reduce his methadone prescription from 140mg to 20mg while in custody and welcomed the help he was being given.
Judge Tom Bayliss QC said the flat burglary had left his victim nervous of living in her own home and had deprived her of sentimental jewellery as well as electrical goods. He was satisfied Burton had also "cased the joint" in advance, knocking at least on one other door nearby to see if someone was in.


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Posted by Steve McKinley on Wednesday, August 01, 2012 12:04 AM
One warm July night Mrs. McKinley and I treated ourselves to a Neil Diamond concert at the local hockey-and-concert arena. We've seen Neil in action before and were quite eager to go again.
While there were some younger people there, most of the crowd was our generation, more twilight-side-of-the-hill than morning-side-of-the-mountain. But then Neil himself is 71. Nonetheless, the man radiates energy and performed for two solid hours without an intermission. Of course many of the songs were his familiar hits from over the years, so the concert became a sing- and dance-along.
As Neil directed the crowd into a third additional chorus of "Sweet Caroline," I wondered how many times he has sung "Sweet Caroline" and "Crackling Rose" and "America" and his other hits over the years. Thousands, I am sure. And yet when he sings them now in concert, it is with an energy and passion that can make them seem both familiar and new. Amazing.
Some would say that it is because he is a performer, and draws energy from the audience, and I know that is true, at least in part. But I think it is also true that the audience draws energy from him, from the performer. If he were to come out one night and "mail it in," give half-hearted performances with less energy, I am sure that the energy of the audience would not be the same. That isn't going to happen, because as an entertainer he is a professional in the best sense of the word.
So it is a Sunday in August, with the temperature in the mid-80s and headed upwards. The church air-conditioning, if in fact the church has air-conditioning, isn't working. Most of the worshippers have shown up dressed as empty pews. You had vacation in June, and that seems forever ago. The liturgy is the same old liturgy. It is a "B" year in the lectionary, you've been preaching on the Bread of Life from John 6 for weeks and weeks, and you've told every bread story you know. A new program year is peeking over the horizon. Church finances are in their typical summer slump. In other words, it is the kind of Sunday that calls on the pastor to be a professional, in the Neil Diamond sense of professional.
Forty years ago there was a debate about attaching the label of "professional" to the pastor. Some believed calling the pastor a professional de-spiritualized the calling. Whatever. I do believe that pastors can and should be professionals in the best sense of the word, delivering their best on all occasions, leading worship with as much enthusiasm and care on that dull August Sunday as on Easter morning. Like it or not, we as "professionals" bear serious responsibility for the climate of worship around us. As I look back on my own years in parish ministry, I realize that usually when I complained after worship about the congregation being dull, I was the one who was dull in the first place.
I am conservative enough to be turned off by the idea of worship as performance. When a congregation "shows its appreciation" by applauding, usually occasioned by either a spirited performance by a children's choir or a lachrymose chunk of cotton-candy spirituality presented in the cocktail-sacred manner, I'm turned off and do not join in. Nonetheless, a community at worship is influenced by the manner and style and attitude of those who lead the worship. If we want enthusiastic worship, that enthusiasm needs to start with us.
If Neil Diamond can whip up an audience on a Wednesday night with "Brother Love's Traveling Salvation Show," we should be able to do the same thing with the real thing on a Sunday morning, even a hot one when we'd rather be at our air-conditioned home enjoying a third cup of Sunday morning coffee.
To interns still stoked with the enthusiasm of the young, this might be incomprehensible. Supervisors will understand. If you are in the former camp…when that Sunday comes for you, and it will come, just remember Neil.
Tags: Steve's column, Steve McKinley
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text: Computers are just too damned hot. It's becoming a real problem. Big internet companies like Facebook have taken Intel to task over the issue and -- sensing a real opportunity -- other companies have started to build new systems using the same kinds of low-power processors you'll find in your mobile phone. In fact, 2013 is likely to be the year that we get an inkling of how far these new ARM systems are going to push old-school chipmakers like Intel and AMD. But over in Poughkeepsie, NY, IBM is studying these server power problems. And you know what? It's seen it all before. IBM started building water cooling into its System 360 computers back in the early 1960s. Fifteen years later, IBM built most of its chips using a transistor technology, called bipolar, that was being stretched to its limits. Chips were getting too hot, and IBM developed new kinds of water radiators to cool them down. Big Blue's designs were so accomplished that one of them -- a cooling unit that used dozens of spring-powered pistons to pump water off of the chip -- was featured on the July 1983 cover of Scientific American. A new generation of transistors -- they're called Complementary Metal-Oxide Semiconductor, or CMOS -- came in during the early 1990s and saved the day. Much more power-efficient than their bipolar predecessors, they ushered in an era of air-cooled servers. IBM stopped building water-cooled systems in 1995. But it's building them again and for many, the air-cooled era may be coming to an end, says Roger Schmidt, the IBM Fellow in charge of the company's energy efficiency research. In 2005, IBM took a small step back into water cooling -- piping out a heat exchanger that fits onto the back of a server rack. And now the company is looking at new ways of water-cooling its chips, just like back in the 80s. The water-cooling proposition often comes as a bit of a shock, except to the old-timers who remember the 70s and 80s. "A lot of the people out there who run data centers aren't into water because they've grown up in the CMOS era," Schmidt says. "The guys that were never in water and were just air-cooling CMOS, they say, 'Keep that away from me.'" Above: IBM Engineer Levi Campbell checks out an air chiller outside of IBM's labs in Poughkeepsie, NY. Photo: Jon Simon/Feature Photo Service for IBMHere's what the inside of a water-cooled server looks like. In this prototype, engineer Milnes David looks at copper pipes carrying the water to radiators located on the server doors. Back in the day IBM water-cooled systems used water that was a relatively cool 68 degrees Fahrenheit. Nowadays the water is warmer -- more like 113 degrees. IBM has a test system in the lab that runs at about 150 degrees. Photo: Jon Simon/Feature Photo Service for IBMBob Simons checks out the cooling system he designed for the IBM mainframe back in the 1980s. Called the Thermal Conduction Module, it used dozens of miniscule spring-powered pistons to pump water off the mainframe's bipolar chips. Update: Mr. Simons has written us to offer a better explanation of how the TCM works: "The spring-loaded pistons did not 'pump water off the mainframe's bipolar chips.' Instead, they provided a mechanical means to conduct heat from the back of the chips (as many as 100 chips in a module) to the back of the module housing. Each piston was spring-loaded to ensure that it would contact a chip after the module was assembled. A hollow copper structure called a cold-plate was attached to the back of each module. Cool water flowed through each cold-plate to carry away the heat dissipated by the module to which it was attached. He also points out that three other engineers share the TCM patent with him: Richard Chu, Un-Pah Hwang and Ohmkarnath Gupta. Photo: Jon Simon/Feature Photo Service for IBMA closer look at Bob Simons' Thermal Conduction Module heat-exchange assembly, used in the 1980s. Photo: Jon Simon/Feature Photo Service for IBMToday IBM is developing new water-cooling systems that are only a little bigger than a sugar cube. This is a test system developed by IBM researchers. Photo: IBMA model 3-D liquid cooled chip built by IBM researchers in Zurich. The 3-D chips use miniscule cooling channels to dissipate the intense amount of heat generated by these chips, which stack lots of processor cores in a very small amount of space. Photo: IBMIBM says that this water-cooled supercomputer, called SuperMUC, in Munich's Leibniz Supercomputing Center is 40 percent more power-efficient than an air-cooled system. Photo: IBMMarvel at the pipes in this closeup look at the IBM iDataPlex Direct Water Cooled dx360 M4. Photo: IBM


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