Cutler Lab

2010-03-01T12:24:00.000-08:00

Research in the Cutler lab is focused on two complementary interests– the identification of new factors that regulate plant cell expansion and the dissection of natural variation using small molecules. Both of these goals have been pursued in parallel using LATCA, a collection of small molecule cell expansion inhibitors that the lab identified in several chemical genetic screens conducted over the past few years.

To harness the power of natural variation in chemical genomics, the lab's screens incorporated parallel assays on multiple ecotypes, or wild type strains. This work demonstrated that there is pervasive variation between Arabidopsis isolates in their sensitivity to small molecules. This variation can be used to identify natural drug-resistance and drug-hypersensitivity alleles, which can facilitate both mechanism of action studies and provide insight into the molecular mechanisms of pharmacogenetic variation.
By characterizing a new cell expansion inhibitor called hypost atin and naturally occurring hypostatin resistance alleles, the lab uncovered evidence for an unusual mechanism of pro-drug activation through glucosylation; we have named this process glycoactivation. This activation pathway uses enzymes that are homologous to human drug metabolism enzymes (UGTs) and the lab's work showed that natural variation in the same enzyme family can cause pharmacogenetic variation across biological kingdoms.

The lab’s efforts have recently focused on understanding the mechanism of action of pyrabactin (an ACS "Molecule of the Week"). Pyrabactin is a new growth inhibitor that we discovered in our chemical genetic screens. We have shown that it is an agonist of the abscisic acid (ABA) signal transduction pathway. ABA is a stress hormone and growth inhibitor that plays important roles in many aspects of plant physiology. A combination of genetic, transcriptomic and physiological evidence has demonstrated that pyrabactin activates the ABA pathway in a manner very similar to ABA. As such, pyrabactin is the first ABA agonist that is not an ABA analog and may ultimately lead to the development of a new family of synthetic plant growth regulators. In addition, pyrabactin shows intriguing selectivity for the seed ABA signaling pathway and has relatively modest effects on the vegetative pathway. Our characterization of pyrabactin has provided new insights into the mechanisms of ABA signal transduction, an in particular led to the identfication of a new family of ABA receptors called the PYR/PYL proteins.
Our work on pyrabactin was published online in ScienceExpress April 30. Check out the Tierney Lab post about how this paper exposed some of my, err, unusual proclivities. A discussion with my colleague, friend, ethicist and big brain, Coleen Macnamara, helped my hone my argument and enable a second round at Tierney lab.
More recently, our receptor work has led to a series of papers that provide detailed insight into the structural mechanism of ABA perception and PP2C inhibition. Of additional note, the recent work on ABA perception was selected as a 2009 "Breakthrough of the Year" by Science magazine. Our current efforts in ABA signaling are focused on 3 interrelated areas: (1) Understanding the structural mechanism of pyrabactin action and selectivity, (2) Designing, synthesizing and characterizing new synthetic ABA agonists and (3) Characterizing new endogenous plant agonists of the the PYR/PYL receptors.

The Cutler lab has also been developing new small molecule screening libraries designed specifically for chemical genetic initiatives as part of an IIGB funded project. These projects exploit click chemistry and a clickable library of ~4000 terminal acetlyene compounds assembled by the Cutler lab to capitalize on the ease and power of the "biologist-friendly" click chemistry reactions. One goal of this project is to generate new chemical probes that facilitate target identification, a current bottleneck in chemical genomic initiatives.

We're in the Sigma catalog!

2009-12-21T11:13:00.000-08:00

Pyrabactin can now be purchased from Sigma!

Ouch!

2009-05-19T11:37:00.000-07:00

Yup- it was inevitable, and I knew that ;-) What fun!

Finally, the word is out that I am a crackpot! What took so long?

Some great quotes for the back of my next book, titled "I Am A Crackpot And So Can You":

"Cutler sounds like a crackpot to me" physioprof@gmail.com

"This kind of breathless obsession with collaboration-cum-politics makes me want to barf. It's like TMZ for science geeks." Luigi

"You have to wonder how thick/naive/silly as Cutler ever got a Ph.D. (even in biology), much less a faculty position." SRC
[Agreed- My committee at Stanford was very easily fooled!]

"Sean Cutler’s report of his “little experiment” is junk science and the gullible reporter believed it without performing a simple fact check." Robert

"I’m going to gag on all this fuzzy talk about cooperation." Patricia

"This is news? ... Where has Dr. Cutler been? Up in an ivory tower?" anonymous Grad student [Can't resist an honest answer here: YES!!! I do live in an ivory tower. It is quite beautiful and I recently had my tower gold plated using my daddy's trust fund money. Awesome view!!! Peace out- Sean]

"Congratulations to Dr. Cutler for getting recognition for convincing others to collaborate with him. " Lilia
[Lilia- I agree, can you believe I pulled this off? Thanks for the recognition. Rock on! -- Sean]

Friends of the show

2009-05-17T17:31:00.001-07:00

Here are some blogs that just rock...

In the Pipeline is as addictive as the drugs that I wish my lab was discovering.

If you have not yet seen Broken Science, then I think you must. It answers the age old question: What if Britney Spears had got a PhD in Molecular Biology?

Adventures in Ethics and Science. Ever wondered about what to do? Dr. Free-Ride can help.

Pubmouth

2009-04-30T13:57:00.000-07:00

Pubmouth is a common genetic disorder that I, here for the first time, will admit to suffering from. Those who have known me long enough will not be surprised by this admission. One common struggle of pubmouthers is that they can be quite tiring to those nearby. The pubmouth allele apparently serves some adaptive value, but I am still trying to sort this out. A major symptom of pubmouth is the uncontrolled spewing of partially formed philosophical ideas, systems or epistemologies on to anyone within earshot of your 3rd to 10th pint. I took a brave step recently. With some help from the New York Times, I showcased my particularly glorious (vainglorious?) case of pubmouth disease on John Tierney's blog. I would love to continue the discussion, after I get back from The Getaway, the closest pub. Pubmouther scientists: post here. Tell me why I am wrong, I love hearing that.

Pubmouth status update, May 13 2009: A discussion with my colleague, friend, ethicist and big brain, Coleen Macnamara, helped my hone my argument and facilitate a second round at Tierney lab. My next goal- to get him to post the modestly titled "Sean Cutler's Miraculous 3 Button Solution To All Of The World's Problems", which I believe will solve science's ethical problems and more!

Sturctural Dissection of ABA Receptor Function

2009-04-26T11:36:00.000-07:00

Our work on the PYR/PYL protein family has shown that these proteins bind to PP2Cs in response to (+)-ABA and synthetic ABA agonists, such as pyrabactin and (-)-ABA. The PYR/PYLs play a major role in controling which ligands induce an ABA response in this new receptor system. For example, multiple PYR/PYLs respond to (+)-ABA, the naturally occuring form of ABA but it appears that only PYR1 is necessary for pyrabactin action in vivo. Thus, our work shows that PYR/PYLs play critical roles in controling the selectivity of agonist action. In this context, we are determining the structure of ABA agonists bound to PYR1 and other PYLs to understand the mechanims of ABA perception and to design improved agonists and receptors for agricultural uses. This work is the result of a very productive collaboration with the laboratory of Dr. Brian Volkman. Brian's lab solved the structure of the Arabidopsis protein MLP28, a START protein that is related to PYR1. We are also collaborating with Dr. Chia-en Chang's lab. Chia-en is an expert in computational studies of ligand-protein interactions and will soon be a neighbor in the new IIGB genomics building. I will update this post as more data becomes available, but check out the image above. If you would like more information about the status of this project, contact Sean for more details.

Sidestepping Functional Redundancy with Small Molecules

2009-02-01T15:57:00.000-08:00

This is a modified version of a presentation I have been giving lately on the research in my lab. The main focus is on pyrabactin, an agonist of the ABA signaling pathway that we have used as a tool for identifying a new family of ABA response factors called the PYR/PYLs.

Random Pictures

2008-07-04T13:49:00.000-07:00

In June I attended the 2008 Canadian Plant Genomics Workshop, which was a wonderful and intimate meeting. Here's a picture of me and Ralph Quatrano talking ABA. The photo was taken by either Erin Haminishi and Julia Romano, not sure exactly who, but either way- thanks!

19th International Conference on Arabidopsis Research (July 23 - 27, 2008). At the bar, with the usual suspects, (from left, Nick Provart. Marika Cooper, Sean Cutler, Dario Bonetta).

Screening Tips and Advice

2008-06-17T11:12:00.001-07:00

Tips, stories and advice about phenotype-based small molecule screens
A few questions are frequently asked by new small molecule screeners. Below are some bits of advice that I wish someone had given me before I got started. I have learned a lot of things the hard way; in fact that is how I learn most things, but then again, I enjoy the steep parts on the learning curve.

What concentration should I screen at?
There is obviously no correct answer to this question, but here a few things to consider. Most chemical libraries are sold with compounds dissolved as 10 mM stocks in DMSO. If you are performing phenotype based screens in living organisms, the effects of DMSO can put a ceiling on the highest concentration of compound you can screen at. With Arabidopsis, we have found that DMSO needs to be kept at or below 1% to prevent growth inhibitory effects; that is, there are minimal effects of DMSO in a quantitative cell expansion assay in etiolated hypocotyls at 1%, but above this, you can start to see reductions in hypocotyl length. Given that, the highest concentration we'd consider screening at is 100 uM, (stock solutions are usually 10 mM). We perform our screens at 25 uM and do not get too excited about hits if they require higher concentrations to induce robust phenotypes. This will obviously vary from case-to-case, and I can imagine scenarios where our 25 uM rule would miss interesting compounds, but that's our general baseline.

A comment on compound solubility.
On the topic of concentration, an additional consideration is compound solubility. For the LATCA project, my lab has done 1000s of dose curves (usually at concentrations between 1 and 100 uM). In doing these, we saw numerous cases where compounds crash out, usually at concentrations above 50 - 75 uM. The evidence of this can be seen indirectly from oddly shaped dose curves (potency decreases at high concentrations), and more directly, and obviously, from the formation of crystals or precipitates in screening wells (this is very obvious with colored compounds, but can also be seen with most compounds as cloudiness or speckled plates, if you look closely). Keep in mind that the compounds present in screening libraries are usually "Rule of 5" compliant, which means that, in short, they are designed or selected to have properties that help promote passive diffusion across membranes. The average CLogP for most library members is around 3, which means they are predicted to partition about 1000 times more into the organic phase of an octanol-water mixture. Many compounds in a library will have a CLogP near 5. As a result, aqueous solubility is always a factor to consider when designing screens and characterizing compounds. For most libraries out there being sold, solubility will not be a major concern at concentrations at or below 25 uM (this will depend on the compounds present, though).

What’s this Lipinski “Rule of 5” all about?
Lipinski extracted and analyzed a set of ~2000 small molecule drugs or late-stage trial compounds from the World Drug Index (where all compounds and formulations of compounds are registered). This dataset of validated bioactive small molecule drugs were examined for 4 characters: LogP, MW, number of H-Bond donors (HDon) and number of H-Bond acceptors (HAcc). The major factor that motivated Lipinski’s famous study was the desire to eliminate compounds with poor solubility / permeability from entering and / or moving down the drug-discovery pipelines at Pfizer. The paper and his famous Rule of 5 is often credited with the goal of identifying “drug-like” compounds, but it really came from the other side of things- eliminating bad compounds from further consideration. The reason it worked so beautifully is that the vast majority of drug uptake is via passive diffusion, which means that most small molecule drugs / perturbagens need to share certain properties to make uptake as good as possible. Too big (MW>500), you get stuck in the membrane and cross more slowly. To greasy (LogP>5), you have a harder time going into solution in the first place. Lipinski showed that a combined Mass > 500 and CLogP > 5 was rarely observed in small molecule drugs, and these are the two properties that Lipinski identified as contributing most to the goal of spotting “bad” compounds. Additionally, more than 5 hydrogen-bond donors or 10 H-bond acceptors are also viewed as problematic and were not commonly observed in small molecule drugs. Since my lab is a plant lab, you may ask if the Rule of 5 is applies to agrichemicals? The answer is yes, pretty much, with a few differences probably not worth making too much of a big deal about when it comes to the academic lab contemplating which libraries to purchase. Again- the name of the game is getting across cellular membranes: physical properties that interfere with that process are likely to cause problems in almost any organism examined.
Lipinski’s paper (which is highly readable and recommended) spawned an explosion in chemical informatics and a great number of other metrics have been enlisted identifying compounds unlikely to make good drug candidates including: number of rotatable bonds, predictions of aqueous solubility, total polar surface area and others. Often times, the newer metrics examined are highly correlated with MW and / or LogP, so it can be hard to tease the importance of these various factors apart. An excellent review appeared in Nature Reviews Drug Discovery recently for those with more of an interest in this topic. If you are a bioinformaticist reading this and thinking – hey I’d love to look at X’s dataset and computational tools – be forewarned. The same open source models that dominate biology were not adopted by chemical informaticists in the early days and the tools and datasets are not easy to obtain and / or are costly. One recently released software package that I highly reccomend is Thomas Girke's ChemMineR, an R package that contains many of the most commonly used routines for analysing and clustering compounds.

Do stock solution concentrations matter?
Short answer: Yes, at least, sometimes.
Long answer: I am now going to start sounding like the anal-retentive chef, but this one is not so obvious to most new screeners. The concentration of a stock solution matters (not just the final assay concentration) and changing the stock concentration can affect assay results. This is particularly true for compounds with aqueous solubility problems. For many compounds it may not matter at all- but it can occasionally make a huge difference in results and it is wise to keep track of stock solution concentrations used to make assay plates.
I will relate the importance of this with the experience Yang Zhao and I had with one of our compounds of interest, hypostatin. Hypostatin is quite a greasy compound and shows solubility problems above 50 uM (this is evidenced by the formation of small crystals / aggregates in plates after they have solidified). When we first isolated hypostatin, the screening stock used was 2.5 mM (in DMSO). Hypostatin was the first really nice hit in our natural variation project- and I was quite excited when we saw it, asking the students involved for details almost hourly (I have calmed down a bit since then). Hypostatin's effects retested several times in independent replicates in our 96-well assay system, so I was satisfied that it was a "real" hit (or more appropriately a "well-behaved" hit). When it came time to do more characterization, we purchased mg quantities of the compound and observed that the original phenotype was not reproducing.
HPLC-DAD analysis of the new and old stocks showed that they looked the same, so everyone in the lab was baffled. Yang, the main student on the project, mentioned that he noticed "milkiness" when adding the compound to agar that he had not seen before. The new and original assay plates both contained 25 uM hypostatin, but it turned out Yang was using a new, more concentrated stock (I think it was 20 mM, instead of 2.5 mM). Changing the stock solution back to 2.5 mM fixed everything, and the project was resuscitated from a potentially very frustrating end point. Admittedly, this particular problem is not going to be an issue for most compounds. Nonetheless, it is important to keep track of details so that the source(s) of problems can be located when bizarre things pop up. Stock solution concentrations can matter, so keep track. Pay attention. I credit Yang with that particular save because he is very attentive to details.

Should I bother with replicates?
Yes, definitely! But there is one thing I suggest you consider. Just because a hit does not appear in both replicates does not mean it is not real. A number of factors can contribute to poor behavior of an individual compound in the primary screen, so if a hit looks golden, but you only see it in one rep, you might consider following it up to see if you can get it to behave better. This is true even if you have a very reproducible and consistent assay. In some cases the issue is the chemical and not the assay.

Do I need robotics / liquid handling equipment to start screening?
Short answer: No.
Long answer: It can be very helpful, but it is not necessary and it can even slow you down a bit in the beginning because of the learning curve for operating and programming liquid handling. My advice is this: run a pilot screen using a small library like LOPAC, Spectrum or LATCA. For this pilot screen, you can use multi-channel pipetters, or more preferably, pin tools. Pin tools are cheaper on a per-screen basis because there are no tip costs, but they have an initial cost that is high. Pin tools will increase your throughput and reduce the possibility for error, but if you are trying to be as cheap as possible, a multichannel pipetter is the way to go. After your pilot screen, you will have developed a sense for ways to optimize the screening process and learned about molecules that are active in your assay. Once you have this information, you can decide if you want to scale things up. For larger screens, investing in automation (through a screening center or collaborator or whatever) is probably worthwhile. My lab does a lot of screening and at this point I cannot imagine working without our liquid handling facilities. Having said that, all the screens that went into developing LATCA and our natural variation project (a screen of ~14K compounds against 8 strains) were all done without liquid handling (not by choice, though)- so don't let the absence of a liquid handling station nearby deter you from initiating a project. I should point out though, that there is a belief by some that robotics are necessary for screening. I had an early grant that tried (and failed) to get the LATCA project funded. One major criticism of the proposal was that we had a screening section that included the use of pin tools and this indicated that we were not equipped properly for the research.

How do you make your screening plates?
Using a pin tool or pipette tip, we spot 1 ul of a DMSO stock solution into the well of a 96-well polypropylene plate. DMSO is greasy and likes to spread out over the surface of polystyrene. We then add 100 ul of molten agar, that is kept at 55 oC with a stirring heat block with an attached temperature probe. The the molten agar is applied with rapid expulsion force at the side of the well to promote compound mixing. Plates are allowed to solidify at 4 oC for several hours to overnight.

Who should I buy a screening library from?
There are dozens of companies that sell individual compounds or "diversity" sets that are subsets from a larger compound collection in plated formats. If you want a list of all of the vendors out there, a great collection of commercially available compounds can be found on the Shoichet lab's ZINC website. The number of commercially available compounds is in the millions (>8 million purchasable according to ZINC, but I don't know if that is non-redundant or not). A not-too-old publication by Baurin et al. that I found useful found 1.6 million unique compounds from 2.7 million compounds listed for sale from 23 vendors (this paper has a table with all of the vendors, which is quite handy). So there is no shortage of compounds available, and a great number of them are Rule of 5 compliant (again see the Baurin et al. publication). My lab has used pre-plated diversity libraries from Chembridge, Tripos and Sigma/TimTec. We have additionally made our own cherry-picked libraries using compounds from Life Chemicals, Asinex, Chembridge and Vitas M (more on “cherry picking” later).
OK, so now here is what I wish knew and or thought of before I got into this:
Before you buy a library, get a clear statement from the vendor about restocking policies, resupply pricing and the prices charged for resynthesis of compounds. These issues are critical, because you don't want to be stuck with a great hit you cannot follow up. In general, for any given diversity library you are considering, the vendor will know how many of the compounds are currently still in stock. You and others may end up screening the library purchased for several years, so if 10% are out of stock now, what will it be in 5 years? Some vendors will guarantee resupply even if a compound goes out of stock, which is a great policy (i.e. they will cover the resynthesis costs in the event the original stock dries up). When pressed, the vendor may try to side track the issue or say that it varies from case to case. Have none of that. It is in your best interest to push them for a firm statement and policy. You may find it acceptable if 5% of their compounds are out of stock if resynthesis costs are low. But if their resynthesis costs are ridiculously high (as I experienced in one case), then you may be in for unexpected headaches. One other thing to keep in mind is that a great number of compounds are available from multiple vendors, so if you are caught in a bind, search before you contract the synthesis of a hit compound. Having said all of that, it has generally been my experience that I can usually get enough of a hit compound from the original vendor or another source for follow up studies. So you shouldn’t waste months agonizing over the "perfect" decision, but try to get all the relevant information up front. Unfortunately, the one time I did have trouble with resupply was also with a vendor that had high resynthesis costs. This is why I wish I had had this particular advice earlier!

I got a hit, now what?
The very first thing is to make sure it is real. If you can dip back into the original screening plates, do another set of tests to confirm activity (for good reasons, many centers and / or drug czars will not let you cherry pick from their wells). Whatever the case, you should next restock your hits to confirm their activity again. This is because what is in the well of the screening plate may no longer be what the vendor originally sold you (more about this later- but the vendor keeps solids which are much, much more stable than compounds in DMSO solutions). As a parenthetical comment- you should be wary of publications or colleagues that make big claims based only on assays from library screening plates. The data is frequently reliable, but there are enough examples of problems with the stock solutions that compounds must be restocked QC’d before any sound (i.e. publishable) claims can be made (more on the QC later). Before you do any restocking though, consider the following. Most vendors have a graded pricing scheme where the cost per mg compound goes down substantially as the number of compounds purchased increase. It is therefore in your best interest to be organized about what compounds you want and to order as many as you need at the same time.
If your screen yielded many hits, you should inspect them to identify trends, or use similarity-based clustering tools (ChemMine, JKlustor) to identify clusters of similar compounds. From this preliminary analysis, you may identify potential structure-activity relationships that could help guide you in the selection of analogs, which is my next point. Depending on how many hits compounds you have, and your budget, you may want to purchase analogs as well. Analogs may, in some cases, be more potent / active than the parent hit, their main value is usually that they can provide closely related, but inactive, negative control compounds.

OK, I've still got a hit, and I still need to know: now what?
My next bit of advice is what I consider generating a null hypothesis about mechanism. It is easy to think you have discovered a molecule with amazing biological properties that targets a protein one else has ever "drugged" before, but the reality is that medicinal chemists and agricultural chemists have been doing this kind of stuff for a very long time, and you cannot ignore their impressive and voluminous "prior art". Additionally, the scaffolds used in many library syntheses are often inspired (stolen?) from well characterized and easily synthesized classes with validated bioactivity (as one example the dihydropyridines, which are best known as anti-hypertensives / calcium channel antagonists). So there is a good chance that a little, or maybe even a lot, may be known about a compound that is similar to your hit.
In my opinion, if there is a compound reasonably similar to yours and its mechanism is known, the null (or skeptics?) hypothesis should be that your compound has a mechanism of action similar to the known compound. I do not mean to imply that this is generally likely to be true (obviously varies from case to case)- but before a “new mechanism” hypothesis is chased down, you might consider some simple experiments to rule out the null hypothesis, if you can. The flipside to my advice here is that there are many examples of chemically related compounds that work by different mechanisms (the sulfonamides are a classic example, but there are others too). So the observation of strong similarity between a hit and a published molecule is not going to prove anything. Nonetheless- you should examine existing similarities and consider if the mechanisms of related molecules might explain the phenotypes you are observing in your assay. Given this long-winded preamble, the real question to be answered is:

How do I identify what is known about my hit and closely related compounds?
The answer is Scifinder Scholar. In an ideal world, chemists would have built their databases in the open source model that drives biology (but they are working on this, for example the PubChem project). But at the moment, the definitive repository of all knowledge about all small molecules resides in the hands of the American Chemical Society and their cash cow: Scifinder Scholar. Scifinder is really an amazing thing, because it keeps track of the compounds listed or used in all published literature (including patents). So, once you have a molecule of interest, you HAVE to search Scifinder to learn if anything is known about it or related compounds. To not search Scifinder after discovering a new small molecule of interest would be like cloning a gene and not performing a BLAST search. Most campuses have a site license for Scifinder, so for academics, getting access to this tool is usually not a problem. In spite of that, it is still very frustrating that you can't just download all the data (or even small chunks of it) and mine it like you might mine whole genome data with perl scripts and the like, but I'm off topic.

More to follow...

Blogging your lab?

2008-06-11T18:19:00.001-07:00

Welcome to the Cutler lab blog. Yes, a blog. I know, a bit odd and unconventional for a lab's web face, yet I am kind of growing attached to it. There are a number of reasons I chose to blog the lab. No, it's not because the lab is so poor that we can't afford a server- the funding milieu is not that bad. The reasons are quite simple: ease of posting new content, ease of maintenance and most importantly- interactivity (there are a few others too...). I am very much attracted to the idea of a continuously growing structure fed by a wider group of interested individuals, which makes blogs so much more organic than the conventional website. Yes, I do realize that comments are currently a whopping total of 0. So, if the organic thing I'm envisioning never happens, then I guess I'll just have a plain old web site hosted by google with annoying " O COMMENTS:" tags at the bottom.

If you are looking for the lab's main areas of interest at the moment, we have been focusing our efforts on: a new family of candidate ABA receptor proteins, a large target identification project, and LATCA, which is a library of small molecule perturbagens that we share with other labs for their chemical genomics initiatives. Visit the links under the "PROJECTS" and "RESOURCES" headings.

Chemical Genomic Facilities at UCR

2008-06-10T11:18:00.000-07:00

UCR has an impressive array of instrumentation and Academic Coordinators that together help make ideas (as opposed to trouble shooting technical problems) the limiting factors to success in your science. Through the guidance of leadership at the IIGB, CEPCEB and the UCR research community, UCR has invested heavily in infrastructure that is invaluable for chemical genomic research. The tools available are diverse and include: screening libraries (~70K compounds), liquid handling, automated microscopy, chemical informatics, LC-MS, proteomics and new deep sequencing technologies. In addition, tools for chemical analyses (NMR, MS, IR etc.) are available through UCR's analytical chemistry facility, which also has a battery of impressive equipment.
One critical feature of the IIGB / CEPCEB infrastructure for chemical genomics is its organizational structure. Each core facility is run by an Academic Coordinator, a specialist who is a talented PhD research scientist that can collaborate with scientists on method development and experimental design. For example, if a lab develops an interest in automating an experiment using liquid handling, the organizational structure ensures that it does not need to reinvent the wheel. An Academic Coordinator (in this particular case, David Carter) is on hand who has done this before and can guide students and post-docs reach their goals. This creates a level of flexibility in the design of experiments, and is also valuable for dealing with the loss of expertise that occurs when experienced post-docs or graduate students leave a lab for the next stages of their careers.
As one recent example, the Academic Coordinator Sonquin Pan in the Proteomics facility has
helped the Cutler lab tremendously with the development of methods for small molecule LC-MS/MS, which we use to study drug uptake and metabolism in Arabidopsis and other organisms. On our target identification project, we anticipate working closely with Glenn Hicks to help identify causal mutations in drug-resistant mutant strains using whole-genome resequencing methods. Glenn is also the most experienced member on campus when it comes to automated microscopy-based chemical genomic screens. Chemical informatics is another strong area of expertise that has been invaluable to the lab. Thomas Girke has developed a number of tools for chemical informatics including ChemMine, which is useful for many things including clustering, and a related and very nice R-package (ChemMineR).

Post-doc positions

2008-06-08T16:08:00.000-07:00

The lab is looking for a post doctoral researcher to join its ABA signal transduction project. This project has opportunities for genetic, biochemical and chemical dissection of a newly discovered family of candidate ABA receptors. Extensive experience in either genetic analysis, protein biochemistry or synthetic organic chemistry /medicinal chemistry is desired. The ideal post doctoral applicant will also have an eagerness to learn methods and approaches complementary to their primary area of training. An impressive array of instrumentation supported by talented Academic Coordinators helps to ensure that post-docs can efficiently do experiments of wide breadth with ease.

Contact Sean for more information.

It's a boy!!!

2008-06-07T21:41:00.000-07:00

PR

2008-06-05T16:09:00.000-07:00

My old friend from grad school Jonathan Eisen, properly calls this part of the website Ego-surfing.

UCR press release about NCB paper
Radio interview w/ Paul Raeburn about NCB paper

Blogosphere:
Dude, I joined the Tierney lab!!! What if Scientists Didn't Compete?
Let's clarify that Tierney post a tad, shall we? (Many thanks to Coleen Macnamara!)
Dude, I'm like, totally on that Broken Science blog and, like, totally loving it, because like, he gets what I did, you know, and like that makes me feel good, you know?
You know that totally addictive drug discovery blog, "In the Pipeline"- Derek get's it too
Taking out the trash: an interview with ethicist Janet Stemwedel; part one.
Policy Statement about Priorities for Research on improving plant water use America.gov

Mainstream Media:
Interview with Patt Morrison @ NPR (realplayer file)
Chemistry World
Chemistry and Engineering News
San Diego Union Tribune article
CBC (go Canada!)
Press Enterprise story (go Inland Empire!)

F1000

UCR Press release about Pyrabactin and PYR/PYLs

The Group

2008-06-02T18:40:00.001-07:00

The UC-Riverside Lab, June 2008.

From left to right: Sang Park, Simon Alfred, Andrew Defries and Jayati Mandal.

Cigar Disclaimer.

The University of Toronto Lab, June 2006.

From Left to Right: Freeman Chow, Simon Alfred, Sean Cutler, Pauline Fung, Rachel Puckrin and Yang Zhao

Smoking Disclaimer

2008-06-02T15:22:00.000-07:00

The images of lab members smoking delicious Romeo y Julieta cigarillos were captured during a recent "Cutler Lab Smoke Out" organized to celebrate karyotyping and ultrasound results that indicate the impending arrival of an XY, 46 chromosome child. Despite appearances, smoking is not fun. Smoking is very bad for your health, particularly when practiced near volatile solvents. Non-smoking lab members are invited to smoke outs and are provided with unlit novelty cigars. Cigars for "Cutler Lab Smoke Outs" are generous donations from the humidor of Paul Cutler, MD.

Update: Maxwell Finnegan Cutler-Long was born November 25, 2008. He is healthy, cute and unbelievably happy (unlike either of his parents).

The genetic approach

2008-06-01T22:33:00.000-07:00

The Genetic Approach
If the molecule is active in a model system for which appropriate tools are available, genetic strategies can be used to isolate drug resistant mutant organisms. The mutations causing resistance often define targets because they disrupt critical side-chain drug interactions.
In the case of an antagonist, target-site mutations may present themselves with dominant inheritance patterns, agonist target-site mutations often behave recessively, and if a target is dosage sensitive, semi-dominant or haplo-insufficient behavior can be observed for both agonists and antagonists. Since other resistance mechanisms are possible, such as defects in pro-drug activation or defects in the pathways that may be needed for bioavailability of a given molecule under normal circumstances, careful analysis of mutant phenotypes, inheritance patterns, in vivo drug levels and metabolism are used to assess which mutations isolated are most likely to uncover target proteins.
Unlike the biochemical approached outlined below, the genetic approach does not require knowledge of structure-activity relationships for a given molecule, which simplifies its application. With sequencing technologies evolving at their current rates, whole genome resequencing is likely to be affordable to most biologists relatively soon. This will greatly accelerate the genetic identification of targets by removing time consuming map-based cloning steps from the process.
However, as appealing as the genetic approach is, it is clear from systematic analyses of herbicides in plants, that resistance mutations may not be isolated for every molecule analyzed (given a finite, but large population size). Like all methods the genetic approach is not guaranteed to work all of the time. Additionally, because of the rarity of target-site mutations, the genetic approach usually requires drug-induced phenotypes that are coupled to easily scored traits such as growth. In spite of these significant limitations, the genetic approach is a simple, inexpensive and general approach to target identification.

LATCA

2008-05-30T13:36:00.000-07:00

This presentation is modified from one originally given by Freeman Chow at the 17th International Conference of Arabidopsis Research. For more information about LATCA visit here, or contact Sean.

test

2008-05-29T23:52:00.000-07:00

test

Click Chemistry Reading List

2008-05-29T12:40:00.001-07:00

Alumni

2008-05-29T08:43:00.000-07:00

This post will be updated shortly. Well, one day.

Graduate training opportunities in the Cutler lab

2008-05-29T08:38:00.001-07:00

The lab is currently looking for both graduate students and post-docs. Graduate students interested in the Cutler lab should consider the UCR IGERT fellowship program in plant chemical genomics. In addition to course work and group dynamics designed to promote interdisciplinary learning, IGERT fellowships are NSF funded and include additional benefits above a standard admissions package including stipend supplements, research funds, travel allowances and internship opportunities.
Most thesis projects in the lab involve the discovery and characterization of new small molecules that affect plant growth. Projects often span a wide breadth of terrain, from genetics, biochemistry and cell biology to organic chemistry. Although knowledge of organic chemistry (particularly synthetic / analytical) is beneficial for students in the lab, it is not a prerequisite for success and productivity in the lab and the relevant background can be picked up over time. Additionally, UCR's impressive infrastructure and talented Academic Coordinators ensure that a wide array of technical expertise is always available to students to help them realize their research goals. Students with an interest or background in chemistry are encouraged to apply to the UCR chemistry graduate program and students with a background or interest in cell biology / genetics can apply to either UCR's Plant Biology graduate program or its Genetics, Genomics and Bioinformatics graduate program.

Contact Sean if you are interested in a position or research in the lab.

Publications

2008-05-29T08:37:00.000-07:00

Pubmed search.

Google Scholar Search.

Contact Us

2008-05-29T08:36:00.002-07:00

sean.cutler@ucr.edu
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Sean Cutler
Assistant Professor
University of California, Riverside
Department of Botany and Plant Sciences
Center for Plant Cell Biology
Department of Chemistry (CFM)
5451 Boyce Hall
Riverside, CA 92521
(office) 951.827.6990
(lab) 951.827.6991
(fax) 951.827.4437

Cigar Disclaimer.

Protocols

2008-05-29T08:36:00.001-07:00

Phenotype Gallery

2008-05-29T08:35:00.000-07:00

Jayati Mandal

2008-05-28T11:25:00.000-07:00

Cigar Disclaimer.
Junior Specialist and lab's fully automated high-throughput map-based gene cloning instrument.

Simon Alfred

2008-05-28T11:16:00.002-07:00

alfred@csb.utoronto.ca
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Simon is a Canadian, born and raised in Toronto. He completed his B. Sc. at the University of Toronto and joined the Cutler lab in 2003 as a U. of T. Ph.D. student in what is now the Department of Cell and Systems Biology. His research is focused on understanding how organelles maintain shape and structure. A small molecule approach was used to identify probes of organelle morphology and function using the lab's LATCA library in a series of microscopy based screens. He identified a number of perturbagens of the ER, Golgi apparatus, actin and microtuble cytoskelton, peroxisomes and cytokinesis. He is currently using a genetic approach to identify the target(s) of Eroonazole, a new inhibitor of ER structure. Sadly, Simon had to temporarily leave the Canadian winter behind while finishing his thesis work in sunny Southern California.

Cigar Disclaimer.

Suvadeep Nath

2008-05-28T11:16:00.001-07:00

This post will be updated shortly.

Andrew Defries

2008-05-28T11:15:00.002-07:00

andrewd@ucr.edu
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Andrew is another of the lab's wacky Canadians (they're everywhere). After receiving a B.Sc. from the University of Toronto in 2006, Andrew moved to Riverside to pursue graduate work under Sean's supervision. During a brief stint as a tech in the lab, Andrew worked on the LATCA project annotating and characterizing its compounds. During this time he participated in multiple Biomek training courses and is the lab's leading robot guy. He even has a blog about his adventures taming the Biomek.
Andrew joined UCR as a Ph.D. student and IGERT fellow in the fall of 2007. Andrew's thesis work currently has two aspects. On the ABA signal transduction project, he has conducted screens for new compounds that interact with different members of a new family of candidate ABA receptor proteins (the PYR/PYLs). These new small molecules will be used to dissect the roles of the various family members. His second area of focus is on the lab's glycoactivation project. The lab recently discovered a novel route of pro-drug activation via enzyme mediated glucosylation. Andrew is using high-throughput screening methods to search for other glycoactivated molecules. By characterizing several glycoactivated molecules, Andrew aims to build a deeper understanding of the implications of glucosylation on target site interactions.

Cigar Disclaimer.

Sang Park

2008-05-28T11:15:00.001-07:00

Cigar Disclaimer.
Senior researcher and unstoppable force.

Sean Cutler

2008-05-28T11:14:00.001-07:00

Contact Sean.
Cigar Disclaimer.

Scientific troubadour and the lab's principal investigator.

ABA signal transduction

2008-05-28T11:09:00.001-07:00

The plant hormone abscisic acid (ABA) regulates many aspects of plant growth and development, plays an important role in adaptive responses to environmental stress and is a key regulator of stomatal aperture. Genetic studies in Arabidopsis have identified several factors that participate in ABA signal transduction, including factors that act primarily in seed or vegetative responses as well as core components that affect most tissues and responses. In spite of its success for other aspects of the pathway, forward genetic approaches have failed to identify ABA receptors. Using alternate approaches, three proteins that bind ABA have recently been identified. The picture to emerge from these studies is that the multiple functions of ABA may be executed by a multiplicity of receptors. A detailed understanding of ABA perception and signal transduction is a major goal in plant biology.
As part of a wider chemical genetic initiative, members of the Cutler lab uncovered a new naphthalene sulfonamide hypocotyl cell expansion inhibitor called pyrabactin (for pyridyl containing ABA activator). A combination of physiological, genetic and transcriptomic data show that pyrabactin is an agonist of the seed ABA signaling pathway. Pyrabactin shows poor agonist activity in seedling assays, suggesting it is seed-selective. Pyrabactin is the first agonist of the ABA pathway that is not structurally related to ABA and may be useful as a template for future agrichemicals that modulate the ABA pathway.
In the Cutler lab, target identification is usually tackled using genetic strategies. A genetic screen for pyrabactin resistant mutants showed that a locus called Pyr1 Pyrabactin resistance 1) is necessary for pyrabactin agonist activity. Pyr1 was isolated by map based cloning and determined to encode a putative ligand-binding protein in the START domain super family. Structurally, members of this family are characterized by the presence of a conserved hydrophobic ligand-binding pocket, otherwise their functions in vivo are poorly defined .
One theme in plant signal transduction is that plant hormones often induce downstream effects by modulating protein-protein interactions between receptors and effectors. In this context, we hypothesized that pyrabactin might work by binding to PYR1 and inducing a protein-protein interaction that regulates ABA signaling.

To test this idea, a yeast two-hybrid (Y2H) screen using PYR1 as bait was performed with pyrabactin added to the yeast growth medium. This screen successfully identified pyrabactin induced protein-protein interactions that are being characterized in the lab. Of note, ABA is also able to regulate PYR1 protein-protein interactions in the Y2H assay, which suggests that PYR1 could be a new ABA receptor. Current experiments in the lab are focused on building a deeper understanding the role of PYR1 and related proteins in ABA signaling .

Sidestepping Functional Redundancy with Small Molecules

This project was started by Pauline Fung when she was an M.Sc. student in the U of T lab. Pauline demonstrated that pyrabactin is a seed selective agonist and isolated the pyrabactin mutants that were critical to building an understanding of pyrabactin's mechanism of action. Sean had a hand in the project and did the fine mapping of pyr1 (with Pauline's help). Yang Zhao helped identify the pyr1 locus and characterize the polymorphisms in the various pyr1 alleles Pauline isolated. Yang also assisted with complementation studies. When the lab moved to UCR, the project was picked up by Sang Park. Sang performed the Y2H screen and has been involved in the biochemical and genetic characterization of PYR1 and related genes / gene products. Most recently, Andrew Defries has identified new agonists of the PYR/PYL proteins. Since presenting on this work last summer a number of collaborations have formed. The presentation above lists the numerous labs that have played instrumental roles in helping us understand how the PYR/PYL proteins work

Hypostatin

2008-05-28T11:05:00.000-07:00

This post will be updated shortly.

Clickables: a small molecule library for click chemistry

2008-05-28T10:57:00.001-07:00

"Click chemistry" is a term introduced by Barry Sharpless to describe high yielding, "easy" chemical reactions. Although there are several potential click reactions, the Copper(I) catalyzed synthesis of triazoles from azides and acetylenes has become synonymous with the term click chemistry.
The Cutler lab has assembled a collection of ~2800 drug-like terminal acetylene small molecules from a variety of vendors. Hits from this library can be used directly in downstream click chemistry reactions. Additionally, library compounds can be clicked against azide building blocks to create new libraries with tailored properties.
We have been funded by the IIGB to use the Clickables to synthesize libraries where all members possess an amine functional group and one of several different fluorophores. The amine handle enables affinity resin synthesis and target identification via reaction with activated carboxylic acid affinity resins (i.e. Affigel) and the fluorophore enables visualization of hits in living cells or other contexts.

Updates
April 2009: ~1300 new terminal acetylenes purchased from Enamine. The "clickables" terminal acetylene library now contains over 4000 clickable small molecules.
March 2009: Andrew Defries has taken over the project and is in the midst of synthesizing and screening libraries made from the 8 building blocks shown below. Our target library of 8 tags clicked against 2760 acetylenes (~22K compounds) should be complete by April 2009. The first library synthesis and screen (DEAC tag) enabled us to work out a few kinks in the methodology and hit followup process, which is now running smoothly. We have isolated some compounds producing nice phenotypes and when the screens are completed we will select compounds for preparation of affinity reagents.
August 2008: The first ~2800 member tagged / fluorescent library has been synthesized using the diethylcoumarin (DEAC) building block shown at left (thank you IIGB for financial support of this pilot project!). We have found that the majority of acetylenes in the library (~70%) work extremely well when clicked against our DEAC probe (i.e. 100% dye consumption in the reactions). A pilot screen of 1040 fluorescently tagged compounds for bioactivity in Arabidopsis etiolated seedlings will be completed in the next week. We are screening for cell expansion inhibitors in etiolated Arabidopsis seedlings and also looking for enhancers of three herbicides (screened at sub-phenotype inducing concentrations). Details will be posted as available. We are sharing the tagged libraries with UCR CEPCEB members and anyone else with an interest in screening them. Contact Sean for more information.

If you are interested in obtaining an aliquot of the Clickables or our tagged libraries, contact Sean.

Natural Variation

2008-05-27T17:53:00.001-07:00

This post will be updated one day, until then check out our paper published in Nature Chemical Biology.

Enlightened Chemical Genetics

2008-05-27T17:51:00.000-07:00

Target Identification

2008-05-27T17:50:00.001-07:00

Target Identification Basics: Genetic and Biochemical Strategies
The molecules identified by forward chemical genetic screens can be powerful probes of biological processes, but their utility can be hampered by the technical challenges of target identification. Historically, both genetic and biochemical strategies have been utilized for target identification. The genetic approach is to identify and characterize drug-resistant mutants. This approach is simple and general in scope, but typically limited to model systems and does not always succeed.
The biochemical approach usually involves isolating proteins that directly bind the molecule of interest. This strategy usually requires knowledge of structure-activity relationships (SAR) so that suitable locations for the attachment of labels or linkers are known. The biochemical approach is a general one, but can be difficult for membrane and low abundance proteins. Challenges in applying the biochemical approach include the SAR studies and chemical syntheses required. New "tagged" libraries have been synthesized around templates that facilitate biochemical target identification experiments and bypass the need for SAR studies.

Target Identification Projects in the Cutler Lab
The Cutler lab has two general target identification projects underway. A drug-resistance screening pipeline has been established and utilized in screens for resistance to 33 bioactive compounds. This approach led to the successful identification of PYR1, the target of a new agonist of the ABA signaling pathway called pyrabactin. Mutations conferring resistance to 19 additional chemicals from our small molecule screens have been isolated, utilizing resistance screens. Positional cloning is being used to identify loci. Additionally, deep sequencing (2 alleles for each resistance locus) will be combined with mapping data to expedite cloning. As a complementary strategy, tagged libraries designed to facilitate biochemical target identification and in vivo imaging are being synthesized using click chemistry and a collection of ~4000 clickable building blocks. To date, ~11K tagged compounds have been screened for phenotypes in Arabidopsis by Andrew Defries, a graduate student in the Cutler lab. Andrew is characterizing his hits using using chemical, genetic and biochemical approaches.

This post will be updated with more details, eventually.

Eroonazole

2008-05-27T17:49:00.000-07:00

This post will be updated shortly.

Synthetic ABA Agonists

2008-05-27T17:48:00.000-07:00

This post will be updated shortly.

Glycoactivation

2008-05-27T17:47:00.000-07:00

The lab's genetic, chemical and biochemical characterization of hypostatin, a new cell expansion inhibitor, showed that this molecule is a pro-drug that requires in vivo glucosylation to be converted into a biologically active form. Many pharmacological and agrochemical agents are administered as pro-drugs, but what makes hypostatin unique in comparison to other pro-drugs is its activation by glucosylation, which we have named glycoactivation. There are relatively few clear precedents in the literature for glycoactivation. It is well documented that morphine-6-glucuronides, which are metabolites of morphine found in serum, possess much higher potency than morphine itself. There is debate as to whether these metabolites are the main pharmacologically active effectors of morphine action in vivo, however. Nonetheless, it is clear from examples like morphine and hypostatin that sugar modifications can increase the bioactivity of molecules after their uptake into mammals and plants.
Many natural products are glycosides and in several cases their sugar residues play important roles in mechanism of action. In spite of their pharmacological importance, glycosides are virtually non-existent in the commercial small molecule screening libraries used by both academic and industrial labs to identify new drug leads. A project underway in the lab exploits UGT-mediated glucosylation and glycoactivation as tools for the synthesis and identification of new bioactive glucosides from existing small molecule screening libraries.

Contacts

2008-05-27T17:45:00.001-07:00

LATCA

2008-05-27T16:26:00.000-07:00

LATCA is collection of ~3600 biologically active small molecules that the Cutler laboratory originally organized for its own internal screening projects. The library is arrayed in 96 well microtiter plates and has been distributed to several labs. It can be used for both assay validation and the identification of known and novel pertubagens of a process of interest. We welcome its use by other labs, and distribute it under the minimally restrictive condition that users eventually send data back to us regarding new bioactivities ascribed to LATCA compounds.

LATCA Presentation

What does LATCA stand for?
Originally, LATCA was intended as an acronym for "Library of AcTive Compounds on Arabidopsis". It could just as easily represent "Library of Annotated Compounds for Arabidopsis". Yes, Sean does like latkes, quite a bit. If anyone can figure out what collection of molecules the acronym GEFILTE (or a reasonable homonym) would be good for, please let Sean know.

How were the compounds in LATCA assembled?
LATCA was assembled in a number of ways. Whole organism, phenotype-based screens of Arabidopsis seedlings were performed using the LOPAC and Spectrum libraries. These were used to identify pharmacological agents from these libraries with activity in Arabidopsis. A screen of a 10K diverse-structure library purchased from Chembridge was additionally performed. Collectively, the hits from these three library screens amount to about 1000 of the compounds in LATCA. An additional 600 compounds were purchased from Chembridge for other projects in the lab and incorporated into LATCA. About 400 "shelf stocks" consisting of herbicides, common inhibitors, plant hormones, research chemicals and other bioactive compounds were included as well. Lastly, ~1600 compounds were obtained by exchanging our compounds for a portion of a library of yeast actives assembled by the Tyers lab. This set contains ~1600 compounds identified as growth inhibitors of S. cerevisiae in a screen against 50K compounds from Maybridge.

How do you define "bioactivity"?
All of the compounds, once assembled, were tested on Arabidopsis with dose curves to assess their potency. The effects of compounds on etiolated hypocotyl length were measured in a simple, quantitative assay to generate IC50 values for all library members. The Cutler lab defines a compound as "active" in this assay if it shows greater than 20% inhibition of hypocotyl length at 100 uM. In addition to the IC50 data, publication quality images were taken of the phenotypes induced by the 3600 LATCA compounds. This data has been used to classify the compound effects into common phenotypic classes such as isoxaben, auxin, oryzalin-like and other classes.

Why would I want to use LATCA?
As is often the case in experimental science, the most important experiment can be the control. In screening experiments, "control" bioactive libraries play an important role. Screening a small collection of bioactive compounds prior to a large scale screen is critical for assay validation and optimization. Additionally, a library of known bioactive compounds can be used to implicate known pathways or target proteins in the process under study. The lab has seen "awesome hits" from diversity-library screens turn out to perturb well characterized pathways through established targets. Establishing this information in advance of a large screening project gives the screener baseline information about what classes of molecules are likely to yield positives in their screen. It can prevent investment in compounds that are not likely to yield new information or targets.

Do you have any general screening tips you could offer me?
Yes, the first installment of Sean's screening tips is available here, check later for more tips.

So, why not just screen the LOPAC and / or Spectrum libraries?
These libraries are excellent screening tools, and LATCA was modeled after them. There are a few major differences to note, however. LATCA is biased towards molecules that are bioactive in plants. More than 50% of the compounds possess bioactivity in Arabidopsis. The hit rate we observed when we screened LOPAC against Arabidopsis was closer to 10%. Additionally, the pathways modulated by LOPAC compounds are biased to those with relevance in mammalian disease biology. Unlike Spectrum and LOPAC, LATCA contains 1000s of novel-structure compounds that have emerged from screens of commercially available diversity libraries. LATCA can therefore inform users about both new and well characterized molecules that affect the assay under study.

What is your lab using LATCA for?
We have used LATCA in a number of microscopy based screens looking for small molecules that affect organelle structure and function. This work was started by a PhD student Simon Alfred. Most recently, Simon has been characterizing a compound named Eroonazole which causes rapid disintegration of ER-tubule integrity. This activity is quite novel and appears to be distinct from the phenotypes caused by many classic perturbagens of the endomembrane system. In general, when a new screen is started in the lab that we ultimately intend to perform on a larger scale, we first screen LATCA to work out kinks in the methodology etc. and to identify known compounds that are active in the assay ("assay validation"). LATCA compounds were also used extensively for our natural variation in drug sensitivity project. We also have a large genetic target identification project underway where we are isolating resistant mutants for >30 compounds from LATCA. If you would like to know if we have isolated mutants for your compound of interest (highly unlikely!), please contact us. If you would like us to add a particular compound to our screening pipeline, also let us know.

How do I restock hit compounds?
The compounds in LATCA are commercially available. Chembridge compounds can be ordered through Hit2Lead, Maybridge compounds through Ryan Scientific, Microsource compounds can be ordered directly from the company. Many of the compounds are carried by multiple vendors, so if you have trouble with resupply, try looking for other vendors using a search engine such as eMolecules (free) or SciFinder Scholar (subscription, but most universities have a site license).

How do I cite my use of LATCA?
An unfinished manuscript describing the assembly and annotation of LATCA has been on Sean's desk for sometime now and will likely make its way into print sometime just before he goes up for tenure review. The main screens used to assemble LATCA are described in this recent Nature Chemical Biology article. We ask that you cite this paper until a formal LATCA publication comes out. According to anonymous sources, more than a few people find it frustrating that Sean has not published on LATCA yet. Well, the latest new is that Simon Alfred, a Cutler lab Ph. D. student from the University of Toronto, is currently writing his dissertation and one of his chapters will present his work using LATCA in microscopy based screens. Sean's current plan is to include the description of LATCA, its assembly and contents in the publication associated with that chapter from Simon's thesis. So if all goes well, summer 2009 will see a formal LATCA paper in print. Sean sincerely apologizes for any inconvenience, frustration or undue duress the delays in publishing LATCA may have caused you or your research.

What format is LATCA distributed in?
LATCA is distributed to other labs in 96 well polypropylene plates as 100X screening stocks in DMSO. The compounds (with a few minor exceptions) are 2.5 mM stocks in DMSO. Our standard distribution set is 47 plates, with each plate containing 80 compounds (columns 1 and 12 empty). 12 ul is sent per well, which is enough for 12 assays, assuming a 100 uL assay volume.

Could I please obtain a set LATCA plates?
In general, we send LATCA out under collaborative terms (at no cost, except that you cover shipping). Keep in mind that LATCA is not a formally funded project and a tremendous amount of work (and resources!) went into assembling it. There is also a substantial amount of work involved in maintaining and distributing the collection. Sean is currently looking into ways to get grant support for the LATCA project; if successful LATCA will be distributed to academic labs without collaborative strings at a very modest cost that covers materials etc. If you cannot wait until then and are not too keen on collaborating with Sean, you might consider purchasing or screening the LOPAC or Spectrum libraries, which LATCA was, in part, modeled after. These tools are present at many screening centers. Alternatively, if you would like to replicate LATCA for your own screening center without having to deal with Sean (a serious pain) you can either (A) wait for the publication on LATCA to come out or (B) deal with Sean. Scenario (B) would involve Sean sending you the contents of LATCA so that you can replicate it. Contact Sean if you are interested in obtaining LATCA.

Could I please get an SDF, DB or PDF of LATCA's contents?
Please contact Sean for this information.

Who contributed to the development of LATCA?
The LATCA project was started by Tsz-Fung (Freeman) Chow, who Sean lovingly refers to as Freebot, on account of his skill at manually doing tasks that a robot really should have performed (if we had had automation in the lab at the time...). Freeman's MSc thesis describes the assembly and characterization of the original ~2000 LATCA compounds. Since that work, the 1600 Tyers lab yeast active compounds were "assimilated" into LATCA and characterized for bioactivity and whole seedling phenotypes in Arabidopsis by Andrew Defries, at the time a technician in my lab. Andrew chooses to program experiments on the Biomek whenever possible, and consequently does not get a "bot" suffix for his name. Andrew is currently a graduate student in the Cutler lab and one of UCR's IGERT trainees. A full list of acknowledgments can be found in the online LATCA slide show.

Under construction....

2008-05-27T16:24:00.000-07:00

After years of meaning to get around to this, I am finally establishing a communication venue for my lab and its members.