Poll results: affiliation and fragment-finding methods in 2019

The fourth iteration of our fragment-finding methods poll has just closed. If you want to jump right to the results feel free to skip the next paragraph, which focuses on methods.

The poll was run using Crowdsignal, the successor to Polldaddy, and ran from 20 October through 30 November. This free polling software tabulates total number of votes for a question but not the number of individual respondents. To determine individual respondents, we included a question on “workplace and practice.” Of the 137 individual respondents to this question, 116 identified themselves as practicing FBLD, and we assumed they also answered the second question. The overall number of responses is slightly higher than in 2013 but a bit lower than in 2016.

Readership demographics have shifted from previous years, with about two thirds of respondents hailing from industry, up from just over half historically. The fraction of respondents who actively practice FBLD is also up modestly, to 85%.

But the question probably of most interest is on screening methods, summarized here.

As we also saw in 2013 and 2016, nearly all fragment-finding techniques are being used more, with the average respondent employing 6 methods today compared with 4.1 in 2016, 3.6 in 2013, and 2.4 in 2011.

X-ray crystallography has leapt to first place, likely driven in part by increasing speed and automation as well as by studies suggesting that crystallography can give impressively high hit rates.

As in 2016, ligand-detected NMR, SPR, and thermal shift assays are all very popular. Use of computational approaches has increased, though perhaps not as much as might be expected given recent advances. Functional screening is the only technique for which use has remained constant, or perhaps even declined very slightly from 2013.

For the first time we asked about use of literature to identify fragments, and nearly a third of respondents said they incorporate previously published fragments into their work. As the amount of publicly available information continues to increase it will be interesting to see whether this number grows.

More niche methods such as mass spectrometry, MST, affinity selection, and biolayer interferometry are gaining adherents; 30 respondents reported using mass spectrometry, for example. While fewer than 20% of respondents are using affinity chromatography (including WAC), CE, or ultrafiltration, that proportion has nearly quadrupled from our previous three polls, though we can’t say which of these related methods accounts for the increase.

Finally, only four respondents reported using “other” methods, such as SHG. Perhaps we’ll ask about this and other emerging methods explicitly next time.

Do the results surprise you, or are they consistent with what you are using at your organization?

New poll: affiliations and methods

It has been three years since we last asked about fragment-finding methods, and a lot can change in that time – just compare the world today to the world in 2016. Our new poll has two questions (right-hand side, under Links of Utility). Please answer the first, and answer the second if you practice FBLD.

The first question asks your affiliation and whether you actively practice FBLD or whether you are interested in the topic (though hopefully the latter also applies to the former!) We’ve simplified the question from prior years to include just four categories: For-profit practice, For-profit interest, Non-profit practice, and Non-profit interest. For-profit includes pharma and biotech as well as venture capital and consulting. Non-profit includes academia, government labs, disease foundations, and retirement.

Please answer this question as it is the only way we can count the number of respondents, which is essential for determining how many fragment-finding methods people are using on average.

If you do practice FBLD, the next question asks which method(s) you use to find and validate fragments. Please click every method you use, whether as a primary screening technique or for validation. You can read about these methods below, and if you select “other” please describe in the comments.

• Affinity chromatography, capillary electrophoresis, or ultrafiltration
• BLI (biolayer interfermotry)
• Computational screening
• Functional screening (high concentration biochemical, FRET, cell-based, etc.)
• ITC (isothermal titration calorimetry)
• Literature or known fragments
• MS (mass spectrometry)
• MST (microscale thermophoresis)
• NMR – ligand detected
• NMR – protein detected
• SPR (surface plasmon resonance)
• Thermal shift (or DSF)
• X-ray crystallography
• Other

Please forward this so we can get as many responses as possible.

Let the voting begin!

Poll results: affiliation, metrics, and fragment-finding methods

The latest poll has just closed, and the results are quite interesting – I’ll get to these in the next paragraph. First, a quick note on methodology. The poll ran from August 27 through September 30. Due to issues with polling in Blogger, we began running polls in Polldaddy in 2013; its interface gives the total number of votes for a question but not the number of individual respondents. Thus, for the questions on metrics and methods, I assumed that the number of respondents was equal to the number of people who identified themselves as practicing FBLD in the first question, or 123 out of a total of 154. The true percentages for the metrics and methods that people use could be higher or lower if not everyone answered all the questions.

Readership demographics have been remarkably stable since 2010 and 2013, with just over half of respondents from industry, and around 80% of all respondents actively practicing FBLD.

The next question asked about screening methods, and here things get more interesting.

The first thing to notice is that, as we also saw in 2013, nearly all fragment-finding techniques are being used more, with the average user employing 4.1 distinct methods today compared with 3.6 in 2013 and 2.4 in 2011. Ligand-detected NMR has jumped to first place in terms of popularity, with SPR and X-ray crystallography tied for second, followed closely by thermal shift. MST, while still in the minority, has had the largest percentage increase. The use of crystallography has certainly jumped since 2011, which fits with recent publications.

Finally, with regards to metrics, ligand efficiency (LE) continues to dominate, followed by LLE (or LipE), though overall usage of both is down compared with 2014. Only one of the other metrics broke the 10% mark. 

Again, if some practitioners answered the first question of the poll, but not the next two, the use of all methods and metrics could be underestimated. Still, these results seem to fit with what I’ve heard talking with folks – any surprises?

2016 polls!

We're heading into election season here in the United States, which reminds us that we haven't run any polls recently at Practical Fragments. How has the community changed in the past few years? To find out, please answer the three questions in the poll on the right-hand side of the page, under "Editors." Also, please note that you need to hit "vote" for each question separately.

The first question asks whether you are in academia or industry and whether you practice FBLD.

The second question asks what methods you use to find fragments. For purposes of this poll please choose all that apply, whether primary or secondary screens. You can read about these methods in the following links.

Affinity chromatography, capillary electrophoresis, or ultrafiltration
BLI (biolayer interfermotry)
Computational screening
Functional screening (high concentration biochemical, FRET, etc.)
ITC (isothermal titration calorimetry)
MS (mass spectrometry)
MST (microscale thermophoresis)
NMR – ligand detected
NMR – protein detected
SPR (surface plasmon resonance)
Thermal shift assay (or DSF)
X-ray crystallography
Other – please specify in comments

The third question asks what metrics (listed below) you use. Again, you can choose multiple answers.

Antibacterial efficiency
BEI (binding efficiency index)
Enthalpic efficiency 
FQ (fit quality)
Fsp3
GE (group efficiency)
LE (ligand efficiency)
LELP (ligand-efficiency-dependent lipophilicity)
LLE or LipE (ligand lipophilic efficiency)
LLEAT
%LE
PEI (percentage efficiency index)
SEI (surface-binding efficiency index)
SILE (size-independent ligand efficiency)
Other
None

Finally, are there other topics you'd like to see polled? Please let us know in the comments.

Fragments as enzyme activators

About half of all approved drugs are small molecules that inhibit enzymes. This makes sense intuitively: an enzyme is like a complicated little machine, and there are lots of ways you can wreck a machine. But there are times when you might want to activate an enzyme, and this is conceptually more difficult. In a new paper in Angew. Chem. Int. Ed., Rod Hubbard and colleagues at the University of York show how fragments can help.

The researchers were interested in the enzyme O-GlcNAc hydrolase (OGA), which removes N-acetylglucosamine from proteins. They were looking for inhibitors of a bacterial version of this enzyme, so they screened it against 100 fragments using three ligand-observed NMR methods (STD NMR, PO-WaterLOGSY, and T_1ρ-filter). This resulted in a very high hit rate: 22 fragments showed binding in all three assays, and 18 of these were competitive with a known substrate-like inhibitor, PUGNAc. Some of these were also active in an enzymatic assay.

More interestingly, the four hits that were not competitive with PUGNAc actually appeared to bind more strongly to the protein in the presence of that inhibitor. In this case, the inhibitor can be considered a stand-in for the natural substrate, and the results suggested that the fragments might enhance binding of the enzyme to its substrate. Indeed, when these fragments were tested in the enzymatic assay, one of them (compound 2) actually activated the enzyme, with an “AC₅₀” value of 3.5 mM.

The activator was further characterized by several orthogonal methods. NMR revealed that, in the presence of PUGNAc, compound 2 bound with a dissociation constant of 3.1 mM, very close to its AC₅₀ value. In the absence of PUGNAc, the dissociation constant was too weak to be determined. Isothermal titration calorimetry experiments revealed that compound 2 increased the affinity of the enzyme for PUGNAc by more than three-fold, while Michaelis-Menten kinetics revealed that, at a concentration of 8 mM, compound 2 nearly doubled the k_cat/K_M. A crystal structure of both compound 2 and PUGNAc bound to the enzyme revealed that the two molecules bind near one another in what appears to be a catalytically active conformation of the protein.

Next, the researchers took an “SAR by catalog” approach to find more potent molecules, leading to compound 4, with an AC₅₀ value roughly ten-fold better than compound 2. Detailed enzymatic characterization revealed that the molecule acts as a “nonessential reversible activator,” and improves substrate binding roughly 7-fold while improving k_cat by a factor of 1.7.

Interestingly, differential scanning fluorimetry (DSF) revealed that all the activators destabilize the enzyme, while PUGNAc stabilizes the enzyme. This is yet more evidence that compounds can decrease the melting temperature of a protein while still binding specifically.

This is an academic study in the best sense of the phrase. The immediate utility of these molecules is tenuous, as they do not work with human OGA (which has some important active site differences), though there may be industrial applications. The more important finding of this rigorous paper is that discovering enzyme activators might be easier than expected. There aren’t that many reported, but this may just be because people tend not to look for them: if you find an activator of an enzyme you are trying to shut down you probably won’t pursue it. That said, a few companies have been founded on enzyme activators. Perhaps fragments can help discover more.

New poll: FBLD in academia

Since almost half our readers come from academia, we thought the following poll would be of interest. It is being conducted by Michelle Arkin of the University of California San Francisco, one of the powerhouses of FBLD, and should take just a couple minutes.

Please click here to answer four questions regarding your lab, whether you do FBLD, which fragment-finding techniques you use, and how you follow up on fragment hits. The results will be published in a forthcoming new edition of "Fragment-based Approaches in Drug Discovery."

Thanks!

Poll results: affiliation, fragment-finding methods, and library size

Here’s a summary of the latest poll.

Readership demographics have not changed significantly since 2010, aside from a slight shift towards industry (~58% today vs 51% in 2010).

The next question asked about screening methods, and here things get more interesting.

The first thing to notice is that, with one minor exception, most fragment-finding techniques are being used more, with SPR and ligand-detected NMR now being used by more than half of all respondents. As a consequence, the average number of techniques being used jumped from 2.4 in 2011 to 3.6 in 2013.

The overall order of popularity doesn’t seem to have changed much, the major exception being X-ray crystallography, which is way up. However, this may be an artifact; a comment on the 2011 poll suggested that some voters interpreted the question to be about primary screening methods, as opposed to all methods.

(Technical disclosure: feel free to skip this paragraph unless you’re a data geek. Due to issues with polling in Blogger, this year’s poll was run in Polldaddy, the free version of which gave total votes for this question but not the number of individual respondents. However, since the two other questions in the poll allowed only single answers, the number of responses was equal to the number of respondents: 95 for the demographic question and 97 for the library question below. I thus assumed 97 respondents for this question, which is coincidentally identical to the number in 2011. Note also that the categories BLI and MST were new for 2013.)

Finally, the question on fragment library size shows that most folks are using libraries of 1000-2000 fragments, with only ~10% of respondents using very small (≤500) or very large (≥5000) collections of fragments.

This result is strikingly similar to the median of 1300 fragments that Jamie Simpson, Martin Scanlon, and colleagues found in an analysis of 22 published libraries. Teddy’s notes from FBLD 2012 put the median slightly higher: around 2500 for 17 libraries. Perhaps people with larger libraries tend to broadcast their size? Of course, in the end, it’s not the size of your library that matters; it’s what’s in it, and what you do with it.

Thanks again for participating, and if you have ideas for new polls, please let us know.

Updated polls: affiliation, methods, and library size

Practical Fragments has run polls on topics including readership, screening methods, favorite metrics (here and here), maximum and minimum fragment size, and the importance of structural information.

We’re interested in how things are changing, particularly in terms of our readership (last polled in 2010) and what fragment-finding methods are most popular (last polled in 2011).

Also, one topic that comes up repeatedly (for example here and here) but has never been actually polled is how many fragments make a good library – we’ve added a question about that too.

Please add your input for 2013; the more people who vote, the more representative the numbers will be. You can find all three questions on the right side of the page, below the "Links of Utility".

We’ll summarize the results and compare them to the previous polls in an upcoming post.

Also, please let us know if you would like us to repeat any of our other polls, and feel free to suggest new topics.

Happy voting!

Way back when I was but a young lad, I worked on dual inhibitors of 5-LO and PAF in the lab of T.Y. Shen at UVa.  They were crazy compounds, but as an undergraduate it was a great experience.  We even did our own assays requiring fresh whole blood.  I loved it and learned to HATE HPLC assays at the same time.  People still seem to be doing stuff like that.  Overall, the field of leukotriene antagonists has been pretty successful, but it is littered with debris. 

Prostaglandin E2 (PGE2) is a key mediator in inflammation, pain, fever, atherosclerosis, and tumorigenesis: a veritable collection of the key therapeutics areas for most pharmaceutical companies.  Arachidonic acid (AA) is acted upon by COX1/2, target for a whole pile of drugs, to generate PGH2.  This is then isomerized to PGE2 by microsomal Prostaglandin E2 synthase (mPGES).  Inhibition of mPGES would be expected to only preclude PGE2 and thus eliminate many of the adverse side effects of COX inhibitors.  As would be expected, mPGES inhibition is not new and several inhibitors are in the clinic.  So, we happen upon this paper: a recent academic effort at a potentially rich field.   The authors begin the explanation of their approach with this: 

To date, there is no real three-dimensional X-ray crystal structure of mPGES-1 in the apo form or with an inhibitor bound with exception of electron crystallographic structure complexed with glutathione in its closed state (PDB code:3DWW).

Their strategy is to replace glutathione with non-peptidomimetics via fragments based upon this pharmacophore model (3DWW): two negative ionizable, one HBD, and one HBA

 They hypothesized their molecule thusly:

The choice of the triazole is to leverage click chemistry to generate it, while the sulfonamide has been shown to a "privileged" fragment in other drugs. Prior to synthesis, the decided to calculate the binding energies (with three decimal place precision!).

The authors then tested the compounds in a biochemical assay.  The first thing that they noticed is that n=2 for the linker is better than n =1 or 3 (50% increase in potency).  Compound 6f (R1=R2=phenyl) was the most potent (1.1 uM).  However at 29 HAC, its LEAN is 0.21 and would be considered lousy.  It can be argued that lipases and such are going to have relatively inefficient compounds due to the highly hydrophobic nature of the active site. [I completely ignored their discussion of the calculations and modeling, because honestly, does it really matter?] Compound 6f showed ~1000x mPGES-1 selectivity over COX1 and no COX2 activity.  They then (much to Dan's delight I am sure) tested for activity with and without detergent.  Not surprisingly, in the presence of detergent, compound 6f lost significant amount (75), but not all of its activity.  The authors very honestly say:

Therefore, compound 6f may be judged as a partial nuisance inhibitor
of mPGES-1 instead of true mPGES-1 inhibitor.

 I would have like them to have tested ALL of their compounds in the presence of detergent to begin with.  This smacks of something that was added in after initial review and honestly really makes the paper uninteresting.  I would really like to know if 6f would still have the outstanding potency from the compounds made, or if some other compound would have aggregated less and thus been of interest.   I would also argue that calling this paper "fragment-based" is a stretch.  As Dan just pointed out, some target classes may just need larger compounds to hit them. 

Metallophilic fragments

A post earlier this month mentioned matrix-metalloproteinases (MMPs). Now, a recent issue of J. Am. Chem. Soc. carries a Communication about fragment-libraries designed for zinc proteases, of which MMPs are a subset.

Seth Cohen and coworkers at University of California San Diego and the Weizmann Institute of Science in Israel designed two libraries based on known zinc chelators: quinoline sulfonamides (QSL) and benzimidazole sulfonamides (BISL) (see Figure). They rapidly assembled 40 of the former and 37 of the later using microwave chemistry and tested these against a handful of different MMPs.

Both libraries produced hits against MMP-2, MMP-3, MMP-8, and MMP-9. Control compounds designed not to chelate zinc showed no activity, and X-ray adsorption fine structure spectroscopy experiments suggest that the molecules are indeed binding to the catalytic zinc. Selectivity is often an issue in targeting metalloproteinases, and it was thus gratifying to find that at least one fragment inhibited MMP-2 with low micromolar activity while showing no activity against the other MMPs. Molecular modeling provides some rationale for this selectivity.

One could argue that many of the library members do not meet conventional definitions of fragments, and could be seen as more scaffold-like (or worse – one has a molecular weight pushing 600 Daltons!) And of course, it is not clear that either scaffold will be suitable for drug development or even tool compounds – it is possible their propensity for zinc binding will be a problem inside cells. Still, the notion of creating custom-made fragment libraries for various classes of targets certainly makes sense; folks have done this for kinases and even RNA, and it is reasonable to see this approach extended to metalloproteinases. Cohen and colleagues described a fragment library consisting of more conventional metal chelators earlier this year in ChemMedChem.

This publication also confirms the results of our poll that fragment-based approaches are catching on in academia. But industry is already in the sandbox: at least two companies, AnCore and Viamet, are using similar strategies to target metalloproteins.

Poll results: academia/industry

Just a quick summary of our poll last month. The results (below and to right) show that just over half of you who responded (51%) are in industry and just under half (46%) are in academia, government, or other non-profit organizations. Also, over three-quarters of you (77%) are active practitioners of FBDD.

Thanks to everyone (over four score) who took the time to respond!

Poll: academia/industry

It’s been a while since we’ve done a poll, but the latest post at FBDD-Lit on fragments in academia, combined with an earlier post on this site, leads us to wonder how many of our readers are from academia and how many are from industry. (For purposes of this poll, let’s lump government and other non-profit organizations with academia).

Please respond by clicking on the right-hand side of the page – we’d like to know more about you!

“The Hidden Pool” revisited

Last year, in response to a post by Teddy on whether there is a “hidden pool” of FBDD practitioners being trained in academia, guest blogger Derren Begley suggested that for the most part fragment-based approaches are restricted to industry: in universities “there are ‘puddles’ of FBDD here and there, but not what I would call a vast resource.” I think this statement was true at the time, but may now be changing. For example, Practical Fragments' last four posts have all covered papers that came out of academia.

There also seems to be an increasing trend of industrial scientists moving to academia, driven by factors ranging from the decreasing number of jobs in industry to the increased freedom in academia. These moves span the gamut, from world-class scientists leading entire departments to folks coming in as assistant professors, staff scientists, and research associates. But they are bringing their interest in fragments with them. In fact, of the last four blog posts mentioned, at least two involved people with current or former industry ties.

Finally, there seems to be increasing academic interest in fragments. I’ve given a couple talks in the past two months at Carnegie Mellon – University of Pittsburgh and St. Jude Children’s Research Hospital, and Peter Kenny has spent the past year as an itinerant fragment evangelist at universities around the world. I know that St. Jude in particular is actively seeking someone with an interest in FBDD, and with resources comparable to what you would find in big-pharma, they make a pretty appealing destination.

What are you seeing? Is FBDD going ivory?