TINS and STD and SPR – oh my!

Following up on our last post on the use of the NMR technique TINS applied to a membrane protein, the same research group has now compared TINS with other techniques on a more conventional target. In addition to TINS, they conducted fragment screens using another commonly used NMR technique as well as surface plasmon resonance (SPR) and high-concentration screening; the results appear in the Journal of Biomolecular Screening.

TINS involves immobilizing a target protein onto a resin, then flowing fragments across the resin and determining whether they bind to the target as assessed by a reduction in their NMR amplitudes. A reference protein is evaluated at the same time; only fragments that bind to the target protein and not the reference are considered hits. In this case, the researchers chose the viral protein RNA-dependent RNA Polymerase (RdRP) as the target and the PH domain from the human protein Akt1 as the reference. Using a total of 4 mg of RdRP, they screened a library of 1270 commercially available fragments in pools of 3 to 5 compounds each, resulting in 74 hits.

One of the most commonly used NMR techniques for fragment screening is saturation transfer difference (STD), in which the magnetization of the protein target is saturated, and so magnetization transfers to any ligands bound to the protein. The researchers tested 133 fragments (both hits and non-hits from the TINS experiments) and found a total of 49 hits, of which 40 had also been found by TINS.

The 83 fragment hits from both TINS and STD were tested for their ability to inhibit polymerase activity at concentrations up to 2 millimolar; 70 of them showed some activity, and a few of these seemed to actually activate the enzyme.

Finally, a selected set of 62 fragments (all of which were hits in at least one of the three assays) were tested in an SPR assay at concentrations up to 0.2 millimolar. Of these, around half showed binding, and these tended to be the fragments that showed the greatest activity in the enzymatic assay.

The authors conclude that TINS picks up more hits than the other assays, though high-concentration screening comes close. This may be true, but it would have been nice if they had run the entire set of 1270 fragments through each of the different methods; it is possible that there were false negatives in the TINS experiments that could have been picked up by the other techniques. Moreover, some of the TINS hits that didn’t confirm in other assays may well have been false positives. Still, there are lots of useful data in this paper, and it demonstrates yet again the importance of using multiple, orthogonal techniques to discover and properly validate fragments.

Fragments vs membrane proteins with TINS

Fragment-based ligand discovery owes much of its success to the rise of biophysical techniques such as NMR, crystallography, and – more recently – surface plasmon resonance. These have allowed the discovery of fragments against a wide range of proteins, but one notable exception has been membrane proteins, the targets of more than half of marketed drugs. In a recent issue of Chemistry and Biology, Gregg Siegal and colleagues take a crack at this diverse group of proteins.

The researchers, from Leiden University, ZoBio, and elsewhere, use an NMR-based technique called target immobilized NMR screening, or TINS. In this method, a protein is immobilized onto a solid support. A reference protein is also immobilized; this reference is usually a well-characterized protein that does not bind to many small molecules. Each protein is then put into its own compartment of a two-compartment flow-cell, and this is inserted into an NMR spectrometer. Mixtures of fragments are then flowed through both chambers: those that interact with protein show a reduction in the amplitudes of their NMR spectra. By choosing fragments that show such a reduction for the target protein and not the reference protein, fragments that bind to the target can be differentiated from those that bind to proteins in general. After each NMR experiment, the fragments are washed away and replaced with a new set of fragments. TINS has been applied to a number of soluble proteins, as reviewed here. Remarkably, the immobilized protein samples often remain stable through hundreds of screening cycles.

Membrane proteins are notoriously difficult to crystallize or characterize by NMR. Moreover, it is often difficult to obtain enough protein to work with. However, since TINS relies on a decrease in signal from the fragment rather than a signal from the protein itself, Siegal and colleagues tested whether they could use the technology to discover fragments that bind to membrane proteins.

The researchers chose a protein called disulphide bond forming protein B (DsbB), which is found on the inner membrane of E. coli and other Gram-negative bacteria and may be important in virulence factor folding. One of the challenges of membrane proteins is keeping them properly folded, and the researchers used two different approaches to do this, either detergent micelles or “nanodiscs,” lipid bilyaers surrounded by a scaffold protein. Using less than 2 milligrams of DsbB, the researchers used TINS to screen a set of 1071 fragments in groups of about 5 each, with each fragment present at 500 micromolar concentration, a process that took 5 and a half days.

The TINS process led to 93 hits, a respectable hit rate of 8.7%. Each of these was then tested in a functional assay at 250 micromolar concentration, and more than half of the hits inhibited DsbB activity by at least 30%. Eight of these were subsequently characterized using full IC50 curves and kinetic analysis. The potencies were impressive, ranging from 7 micromolar to 193 micromolar, with ligand efficiencies as high as 0.45 kcal/mol/atom. DsbB has the advantage that it has been characterized structurally, and the researchers used chemical shift information from 2-dimensional NMR experiments to show that the fragments could be divided into two groups, with one set competing with a quinone cofactor and the other binding at a different site.

This paper demonstrates that it is possible to find fragments that bind to membrane proteins. Of course, the next question is, what can you do with the fragments? In this case there were structural data about the target, but this will not generally be true for membrane proteins, and in the absence of structure, advancing fragments to leads can be challenging. On the other hand, medicinal chemists have been developing drugs against membrane targets for decades without knowing their precise structures, so perhaps the challenge is as much psychological as scientific.

Docking vs TINS on a GPCR

Practical Fragments has featured a number of posts comparing various fragment-finding methods. In some cases there is good agreement, while in others – not so much. Computational methods can in theory sample the greatest swath of diversity space: a virtual library can be orders of magnitude larger than any physical library. In a recent paper in J. Chem. Inf. Model. Gregg Siegal at ZoBio and Leiden University and Jens Carlsson at Stockhom University and their colleagues compare the performance of virtual screening with a biophysical method.

The target they chose, the A_2A adenosine receptor (A_2AAR) is a GPCR implicated in a variety of diseases. It also has the advantage of multiple published co-crystal structures with either agonists or antagonists bound, making it a good candidate for computational screening.

The researchers began by conducting a computational screen of 500 fragments using DOCK 3.6 against the crystal structure of an antagonist-bound A_2AAR and ranked these according to how well they scored. Next, the researchers physically screened the same library of 500 fragments against A_2AAR using an NMR-based screening method called TINS (see also here). This resulted in a whopping 94 primary hits, which were followed up in a radioligand displacement assay to yield 5 confirmed hits with K_i values ranging from 14-600 micromolar. Happily, 4 of the 5 hits from the TINS screen were within the top 5% scoring hits identified in silico.

This is satisfying at first glance, but what does it say about the other top-scoring computational hits? Computational screening virtually docks fragments in many possible positions, or poses, which are automatically evaluated. Manual inspection of the top 50 in silico hits showed that, in some cases, the best poses had desolvated polar groups, which would presumably be energetically unfavorable. Indeed, identifying the “correct” pose seems to be a general problem with docking fragments.

But some of the top-scoring fragments looked fine by visual inspection, so 5 of these were tested in a radioligand displacement assay. Surprisingly, 3 of these were active, with K_i values ranging from 18-128 micromolar. In other words, these were false negatives in the primary TINS assay.

Having found hits that had been missed using a biophysical screen, the researchers then docked 328,000 commercially available fragments against the target – an exercise that took only seven hours on a computer cluster. Of the top hits, 22 were purchased and tested in the radioligand displacement assay, and a remarkable 14 of these were active, with K_i values ranging from 2-240 micromolar. (I do wonder how much chemical intuition played a role in choosing hits to purchase.)

Interestingly, all of the 14 hits from docking had respectable ligand efficiencies (LE > 0.3 kcal/mol/atom, with a single exception). This is consistent with previous fragment docking studies that show that the best results are obtained with the most ligand-efficient fragments. It’s also a nice feature; after all, these are exactly the kind of hits you would hope to find, though of course you want to first filter out any garbage from your virtual library.

This paper provides more evidence that computational approaches can find fragment hits for GPCRs, at least relatively “druggable” ones with good structural characterization. It is also a useful reminder of the importance of using multiple methods, to avoid both false positives and false negatives.

Finally, if you haven't already voted on your fragment-finding methods, please do so on the right side of the page!

30% of all Posts...

NOTE: Blogger blew up my post when I published it.  I have fixed what I can.  Blogger keeps on blowing up this post after I edit it.   I have removed what I think may have been causing some of the problems.  This post should be considered in "Wiki-ese" as a fragment.   Thankfully, the summary was unaffected. 

As I recently said, GPCRs are a theme around here, so this post will talk about work published last year by the folks at ZoBio and Heptares.  [In terms of full disclosure, I had a business relationship with ZoBio until recently.]  This work is also on STaRs, the stabilized GPCRs developed by Heptares.  I noted my concerns with this approach here.  These two papers focus on A2A GPCRs, while previous posts here were on A1A, A3A, and B1A GPCRs.

In the ACS Chemical Biology paper, the authors are using TINS to screen an antagonist-stabilized A2AR StAR.   The immobilized protein showed a ~50% greater retention in activity after 5 days at 4C compared to the native protein in membranes (60% vs. 30% binding competency).  So, immobilized stabilized protein is more stable than non-immobilized, non-stabilized protein.  They then took a subset (531 compounds) of the ZoBio fragment library picked for maximal chemical and shape diversity and screened using OmpA as the reference protein. As shown in the bucketing below the vast majority of the compounds cluster around a T/R of 1.  This indicates that they have a slight preference for the target or the reference. The used a T/R cutoff of less than 0 e.g

. aromatic and aliphatics.  Additionally, the use of the logarithmic plot for the bucketing obscures the spread around T/R=1.  I have never seen a discussion from the creators of TINS discussing the relative error of the method and how to best evaluate the screening data.  In this case, they chose a 0.7 cutoff because there appears to be a discontinuity in the data there.
They followed up on these (see Table 1 in the paper) as orthosteric hits by observing if they can inhibit binding of an inverse orthosteric agonist in a radiolabeled assay using WT A2AR in HEK membranes; five fragments inhibited binding by >30% at 500uM (see below). 

This data in conjunction with the TINS data shows the compounds bind reversibly with a 1:1 stoichiometry.  These fragments also inhibited A1AR, which would not be unexpected for such small molecules.  However, 3 of these compounds have poor LEAN values >0.3.  This is particularly poor for GPCR targeting compounds.

Four additional fragments either one or the other of the inverse agonist or agonist used.  The two most potent AM appear to have some subtype specificity (A2AR over A1AR).  When they tried to test these compounds in a cell-based assay, toxicity was observed so no data could be collected.

In summary, the authors show that TINS is productive in finding fragments that bind to GPCRs.  However, they have to rely on standard biochemical assays for follow up.  It would have been nice to see at least one other method used to verify the active fragments, like SPR.  What I really like is that they did the biochemical assays against WT, which does not necessarily alleviate my concerns about screening against a mutant.  I would have really liked to see a comparison of the biochemical data in the STaR and WT.

So, while people say 30% of marketed drugs target GPCRs, I can assure you 30% of all of our posts are not about GPCRs.

Weak affinity chromatography (WAC)

There are many ways to find fragments: NMR and X-ray crystallography are old favorites, but SPR is quickly catching on. There are also more specialized approaches, such as ITC, MS, TINS, and biochemical screening. Now another 3-letter abbreviation has joined the list: a paper published online by Sten Ohlson and colleagues at Linnaeus University in Sweden in Analytical Biochemistry describes weak affinity chromatography, or WAC.

The principle is remarkably simple. First, a protein of interest is covalently immobilized onto a chromatography column packed with modified silica gel. This can be done on a standard high-performance liquid chromatograph (HPLC). Then each fragment to be tested is injected in buffer; those that have affinity for the immobilized protein will stay on the column longer than they would if they lacked affinity. The fragments can be detected with either UV spectrometry or mass spectrometry.

To demonstrate the technique, the researchers used two model enzymes, thrombin and trypsin, and a couple dozen fragments ranging in mass from 93 to 307 Da. Most of these fragments contained an amidine, a moiety known to bind to both proteins. Columns without any immobilized protein served as controls. Of course, fragments may associate non-specifically with proteins, so the researchers also treated protein-containing columns with irreversible or potent reversible inhibitors; a fragment that comes out later from a column containing an active protein than from a column containing an inactive protein is presumably binding specifically to the active site.

Remarkably, the technique appears to work: most of the amidine-containing fragments were retarded in columns containing active protein compared to columns containing inhibited protein. Moreover, the relative affinity ranking correlated with the inhibitory activity of fragments in enzymatic assays. Some of the fragments were quite weak, with calculated dissociation constants around 1 mM.

The researchers also demonstrated that they could screen a mixture of 11 fragments, using mass-spectrometry to follow each fragment, and that the change in retention time was comparable to that observed when running each fragment individually. In this case it was important to use low fragment concentrations so as to avoid saturating the protein active sites.

As with any technique, there are bound to be limitations. The immobilized protein needs to be stable for an extended time; in the current case there was some degradation in performance, albeit over the course of months and more than 200 injections. A more serious constraint is the need for a proper reference. Inactivating an enzyme provides an ideal solution, but one that won’t be so easily generalized to all targets.

In some ways WAC could be seen as a low-price cousin of TINS: both methods rely on an immobilized protein, but while TINS uses a custom modified NMR spectrometer, WAC can get by with a much less pricey HPLC (though a mass-spectrometer seems nearly indispensible). It will be fun to see how WAC develops, and in particular whether it can be used to discover novel fragments against more challenging targets.

Microscale Thermophoresis (MST)

Practical Fragments has a soft spot for new biophysical methods to identify fragments, many of which are given unfortunately non-descriptive initialisms. To a list that includes SPR, ITC, STD, MS, TINS, CEfrag, and WAC, we can now add Microscale Thermophoresis (MST), described in a new paper in Angew. Chem. Int. Ed. by Philippe Baaske and colleagues at NanoTemper Technologies as well as academic collaborators.

Thermophoresis, also referred to as the Soret effect, occurs when particles move in response to a temperature gradient. In this case, the “particles” are proteins, whose movements depend on size, charge, conformation, and solvation, and can be altered by factors such as ligand-binding.

In MST, a fixed concentration of protein is incubated with varying concentrations of ligand in small capillaries. An infrared laser rapidly heats a spot on the capillary, and an ultraviolet light source excites aromatic residues within the protein. The fluorescence in the heated spot changes as the protein moves along the temperature gradient. This movement is affected by ligand binding, and so measurements at different ligand concentrations can be used to construct a binding curve.

The researchers used MST to study the binding of ligands to several proteins, including ionotropic glutamate receptors (iGluRs), p38-alpha MAP kinase, thrombin, and even the calcium sensor Syt1. The dissociation values determined by MST were mostly comparable to literature values, and the researchers could also perform competition studies in which adding an excess of one ligand blocked a different ligand for the same site.

A nice feature of the technology is that, since it uses native protein, one doesn’t need to worry about the effects of immobilization or conjugation, factors that researchers using SPR, TINS, and WAC must consider. On the other hand, the fluorescence signal relies on native amino acid residues (tryptophan in the examples here), which can be obscured by many compounds. Also, in its current incarnation MST doesn’t appear particularly high-throughput, though it also doesn’t use much protein

Still, this seems like a pretty cool approach. I’ve started seeing NanoTemper at more conferences (such as FBLD 2012), so hopefully you will have a chance to check them out and let us know what you think.

Fragments deliver a chemical probe for CBP and EP300

As we mentioned last week, July is bromodomain month at Practical Fragments. Today we’ll start by looking at two closely related bromodomains, one found in cyclic-AMP response element binding protein (CBP) and another from adenoviral E1A binding protein of 300 kDa (EP300). Both proteins have been implicated in a variety of diseases, particularly cancer, so a chemical probe would be very valuable.

Alexander Taylor and collaborators at Constellation Pharmaceuticals, Genentech, and WuXi, describe such a probe in a recent paper in ACS Med. Chem. Lett. The researchers screened about 2000 fragments in a thermal shift assay using 0.8 mM of each fragment. Compounds that increased the melting temperature of the CBP bromodomain by at least 1° C were validated first by time-resolved fluorescence resonance energy transfer and then by ¹⁵N HSQC NMR, ITC, and X-ray crystallography. Compound 1 was one of the more attractive hits, in particular because it was considerably less active against BRD4, whose inhibition causes all sorts of changes to cells.

Crystallography of the racemic compound clearly showed that only one of the enantiomers bound, and this was confirmed in functional assays when both enantiomers were tested separately. The active enantiomer makes some of the same interactions typical of all bromodomains with the natural ligand (N-acetylated lysine). Fragment growing was attempted off the aromatic ring, and although several vectors were tolerated, most decreased selectivity against BRD4. However, close examination of the structures revealed a promising vector that led to compound 14, with good selectivity against BRD4. Further optimization ultimately led to CPI-637, with low nanomolar activity against both CBP and EP300 as well as good cell-based activity. Crystallography revealed that this compound binds in a similar manner as the initial fragment.

The selectivity of CPI-637 against other bromodomains is also good (> 700-fold less active against BRD4), though it does hit BRD9 with sub-micromolar activity. Just as with the initial fragment, the opposite enantiomer of CPI-637 is considerably less active. Although no pharmacokinetic data are provided, at the very least this should be a useful probe for cell-based studies.

Switching gears to another aspect of CBP, the multidomain protein p300/CBP-associated factor (PCAF) has a bromodomain that may bind to CBP, though the biology is not entirely clear. PCAF is known to bind an acetylated HIV protein, and has been proposed as a target for AIDS. Obviously this is another opportunity for a chemical probe! The first steps are reported in a paper by Stefan Knapp and collaborators at Goethe University Frankfurt, University of Oxford, Leiden University, ZoBio, and University of Cambridge, published in J. Med. Chem (and open-access).

The researchers screened two separate fragment libraries using either thermal shift assays (at 1 mM fragment) or TINS. Hits were confirmed using SPR and crystallography, resulting in seven structures. As expected, all the fragments bound at the site where N-acetylated lysine normally binds. The PCAF bromodomain appears to be quite rigid, with little movement in structures with the different bound fragments. A few elaborated molecules were tested, with the best showing low micromolar affinity as assessed by ITC; crystal structures with these molecules are also reported and deposited in the protein data bank. It will be fun to see whether their potency can be improved.

We’ll have another post on bromodomains next week, but first stay tuned later this week for an updated list of fragment-derived drugs that have entered the clinic.

Current Topics in Medicinal Chemistry Special FBDD Issue

This year ends with an entire issue of Current Topics in Medicinal Chemistry devoted to FBDD. Rob van Montfort and Ian Collins provide a brief editorial overview of the six papers. Collins and colleagues also describe their application of fragment-based methods to develop inhibitors of the anti-cancer target protein kinase B (AKT).

Half the papers cover various forms of fragment screening: the Medical Structural Genomics of Pathogenic Protozoa Consortium describes their crystallographic approach, Helena Danielson discusses the use of SPR, and Gregg Siegal and Johan Hollander present their Target Immobilized NMR Screening (TINS) methodology.

Charles Reynolds and colleagues review metrics, such as ligand efficiency and fit quality, for evaluating fragment hits.

Finally, in the longest article in the collection, Vicki Nienaber discusses how fragment-based methods may be particularly useful for discovering compounds that will cross the blood-brain-barrier to target the central nervous system.

As 2009 comes to a close, Practical Fragments would like to thank our readers, old and new. May you all have a happy and productive 2010!

Fragments vs GPCRs

G protein-coupled receptors, or GPCRs, have been one of the most fruitful areas of drug discovery. Humans have 390 of them throughout the body (plus many more in the nose, where they are essential for smelling), and almost a quarter of new drugs approved in the past decade target GPCRs. Despite these successes, there are plenty of “difficult” GPCRs that have resisted drug-discovery efforts. Since GPCRs are membrane-bound proteins, crystallography has until very recently been all but impossible, making structure-based design and fragment approaches correspondingly more difficult. In a recent issue of J. Med. Chem., John Christopher and colleagues at Heptares Therapeutics describe their success against one member of this target class (see also here for In The Pipeline’s coverage).

Fragments have been screened against other types of membrane proteins using surface plasmon resonance (SPR) and TINS, but one of the particular challenges of GPCRs is that they are generally quite unstable and conformationally flexible. Heptares solves this problem by making a small number of targeted mutations to increase receptor stability and lock the conformation. In this case, the researchers targeted the human β₁-adrenergic receptor (β₁AR); both agonists and antagonists of β-adrenergic receptors are approved drugs.

Approximately 650 fragments were screened using SPR against the stabilized human β₁AR as well as another GPCR, the adenosine A_2A receptor. Selective hits were identified against both targets; among the β₁AR-selective hits were compounds 7 and 8, both with impressive affinities and ligand efficiencies.

Co-crystal structures of various ligands bound to turkey β₁AR (which is identical to its human counterpart in the ligand binding domain) had previously been solved, and molecular modeling of the fragment hits led to the purchase of a set of analogs, which were then tested in a radioligand membrane binding assay. Happily, compounds 19 and 20 both bound with improved affinity over the parent fragments. Crystal structures of these new molecules in complex with turkey β₁AR were also determined, revealing that they do not completely fill the ligand-binding pocket, and suggesting additional modifications to further improve potency and alter their pharmacology.

There are still many unanswered questions. Phenylpiperazines such as these are unusual ligands for β-adrenergic receptors, but they are known to bind other GPCRs, so selectivity will need to be investigated thoroughly. Also, the researchers don’t say whether their molecules are agonists or antagonists, though they suggest the later. Some of this work was publicly presented as early as 2010, so presumably there is plenty more data beyond what’s reported here.

All that said, this is a nice milestone in fragment-based ligand discovery, and it will be fun to see how crystal structures play a role in understanding (and drugging!) this important class of targets.

Fragments in vivo

The number of ways to find fragments just keeps growing. A few weeks ago we discussed WAC, which takes its place alongside ITC, SPR, MS, TINS, and more traditional methods such as NMR, X-ray and biochemical screening. However, all of these approaches are somewhat reductionist, relying on isolated target proteins. In an effort to bring the whole organism into the picture, our friends at the University of Shutka, Russia, have come up with an approach they call “Fragments in Bodies,” or FIB.

The researchers have assembled a collection of very small fragments, purchased for the most part from Lilliput Pharmaceuticals. These are then screened in mouse models to look for positive phenotypic effects.

The researchers face some unique challenges. For example, it is difficult to measure changes in body mass as the animals need to consume such large amounts of fragments that they can become somewhat bloated. Still, if the animals can be safely dosed with massive amounts of micro-molecules, "FIB"ing could provide very good starting points for further work!

Review of 2014 reviews

The year is spinning to an end, and as we did in 2013 and 2012, Practical Fragments is looking back on notable events as well as reviews we didn’t cover previously.

2014 was full of conferences, starting with the CHI meeting in San Diego (here and here), moving to the Zing conference in the Dominican Republic, on to the Fall ACS meeting in beautiful San Francisco, and ending with FBLD 2014 in Basel.

In terms of reviews relevant to the fragment community, John Christopher and colleagues at Heptares published an extensive analysis of “Structure-based and fragment-based GPCR drug discovery” in ChemMedChem early in 2014. The last few years have seen an efflorescence of new structural information on G protein-coupled receptors, and this paper provides a thorough compilation of crystal structures and small molecule ligands. The review also discusses methods that have been used to discover fragments that bind to GPCRs, including TINS, SPR, CEfrag, radioligand binding, and fluorescence assays, and ends with case studies on A_2A antagonists and β₁AR ligands.

In contrast to GPCRs, kinases represent a well-established target class for fragment-based drug discovery, as exemplified by the first approved drug, vemurafenib. Structural biology has played a major role in this success; more than 200 of the 518 human kinases have had their X-ray crystal structures determined, and more than 3000 protein kinase structures have been deposited in the protein data bank. Astex has put several kinase inhibitors into the clinic, and in Methods in Enzymology Paul Mortenson and colleagues from the company discuss the state of the art. This is a clear and concise review of fragment-based drug discovery in general and as specifically applied to kinases. It serves as an excellent introduction to the topic.

Any chemist who has worked on kinases will be familiar with azaindoles, and in Molecules, Sylvain Routier and colleagues at Université d’Orléans discuss “the azaindole framework in the design of kinase inhibitors.” This provides a thorough compilation of azaindole inhibitors against ALK, Aurora, Cdc7, CHK1, C-Met, DYRK1A, FAK, IKK2, JAK2, KIT/FMS, PAK1, p38α, PIM1, B-Raf, ROCK, m-TOR, and TrkA, replete with synthetic methods. The paper also includes a nice analysis of binding modes. Of the 58 crystal structures of azaindoles bound to kinases in the protein data bank, the majority (48) are with 7-azaindole rather than the three other positional isomers. This isomer (found in vemurafenib) is also over-represented in the patent literature and among commercial compounds.

Another target that has yielded to FBLD is BACE1, a hot but still controversial target for Alzheimer’s disease, and in Bioorg. Med. Chem. Lett. Daniel Oehlrich and colleagues at Janssen review “the evolution of amidine-based brain penetrant BACE1 inhibitors”. This is very much a medicinal chemist’s review, with over 100 chemical structures, including a nice summary of the various chemotypes used by different companies. The authors do an excellent job synthesizing a tremendous amount of data, much of it reported only in the patent literature, and engage in some intriguing chemical sleuthing to guess at the identity of clinical candidates whose structures have not been publicly disclosed, such as MK-8931.

Jia Zhou and collaborators at the University of Texas Galveston and Fuzhou University discuss “Evolutions in fragment-based drug design: the deconstruction-reconstruction approach” in Drug Discovery Today. After briefly describing fragment-finding methods and library design, the review focuses on deconstruction of known ligands to generate “privileged” fragments that are then reassembled into new molecules. Although this approach can be productive, if one doesn’t exclude PAINS the result can be garbage-in, garbage-out.

Finally, in Methods in Enzymology, Katherine Warner (National Heart, Lung and Blood Institute) and Adrian Ferré-D’Amaré (University of Cambridge) review the crystallographic analysis of fragments binding to the TPP riboswitch. This is a concise how-to guide, and the methodology could be applicable to other RNA targets.

And with that, Practical Fragments says farewell to 2014. Thanks for reading, and may the New Year bring wonderful new discoveries!

Methods in Enzymology: Fragment-Based Drug Design

In addition to dozens of reviews on fragment-based drug discovery, entire books have been published, the first in 2006 and the second in 2008. Now a third has joined the list: Volume 493 of the venerable Methods in Enzymology series, edited by Lawrence Kuo at Johnson & Johnson, is titled Fragment-Based Drug Discovery: Tools, Practical Approaches, and Examples. With 21 chapters and roughly 600 pages, it is a comprehensive addition to the field. Whether you’re just starting out in the field of fragments or already an expert, this is an invaluable resource.

As the subtitle suggests, the book is divided into three sections. The first, Tools, is the shortest, consisting of just 6 chapters. Chapter 1, by Brett Tounge and Michael Parker, describes how they assembled a 900 fragment library at Johnson & Johnson for use in crystallography-based screening. Chapter 2, by Gaetano Montelione and colleagues, discusses the high-throughput protein preparation approach that the Northeast Structural Genomics Consortium has taken, including advice on what to do when problems arise. The next two chapters are devoted to crystallography: Jark Böttcher and colleagues from Proteros Biostructures provide a general overview including practical tips in Chapter 3, while Doug Davies and colleagues from Emerald BioStructures analyze the results of 18 in-house campaigns to try to draw general conclusions of why some targets are more successful than others in Chapter 4. On the subject of difficult targets, Chapter 5 is devoted to GPCRs, specifically the stabilized versions being generated by Miles Congreve and coworkers at Hepatares Therapeutics and analyzed by SPR and TINS NMR in collaboration with researchers at the University of Utah and ZoBio. Finishing out this section, Renee DesJarlais of Johnson & Johnson provides an overview and practical tips for applying computational approaches to FBDD.

The second section, Practical Approaches, begins with a chapter by Lawrence Kuo on how to ensure that hits from fragment screens will be distinct from those coming out of HTS. Chapter 8, by Tony Gianetti at Genentech, is a valuable and comprehensive tutorial on using SPR for fragment screening. The next two chapters are devoted to NMR: Chapter 9, by Christopher Lepre at Vertex, discusses such practicalities as library preparation and NMR screening conditions, while Chapter 10, by Joshua Ziarek and colleagues at the Medical College of Wisconsin provides more detail on specific NMR techniques. The following two chapters discuss a couple less common techniques. Chapter 11, by James Kranz at GlaxoSmithKline and Celine Schalk-Hihi at Johnson & Johnson, gives an excellent description of the protein thermal shift technique for identifying and characterizing fragments, complete with all the mathematics. And Chapter 12, by Lars Neumann and colleagues at Proteros Biostructures, describes the reporter displacement assay and its use to select for fragments with desirable kinetic and thermodynamic profiles. In Chapter 13, John Spurlino of Johnson & Johnson describes crystallography-based fragment screening and advancement without collecting affinity data; this was recently discussed here, but the chapter provides additional details and examples. Finally, the last chapter in this section, by Zenon Konteatis and colleagues at Ansaris, discusses the incorporation of protein flexibility into computational fragment-based screening.

The last section, Examples, opens with a chapter by Masaya Orita and colleagues at Astellas that covers ligand efficiency and related indices and an overview of strategies to advance fragments, with a particular emphasis on “anchor-based drug discovery.” Chapter 16, by James Lanter and colleagues at Johnson & Johnson, emphasizes the importance of engaging medicinal chemists in fragment projects from an early stage. In Chapter 17, Hugh Eaton and Daniel Wyss at Merck describe using NMR in FBDD both in general terms and with respect to their BACE1 program, which has entered clinical trials. Chapter 18, by Till Maurer at Genentech, is also devoted to NMR, focusing on the specific steps involved. Chapter 19, by Marta Abad and colleagues at Johnson & Johnson, returns to crystallography, further discussing the electron-density guided FBDD approach in the context of the target ketohexokinase. In Chapter 20, Rod Hubbard and James Murray discuss a decade of fragment work at Vernalis, integrating many of the techniques discussed in the rest of the volume (as well as a few new ones!) and providing a valuable discussion on why orthogonal biophysical fragment-finding methods are necessary. The book closes with a chapter by Darren Begley and colleagues at Emerald BioStructures and collaborators at the University of Washington on a structural genomics initiative to use crystallography-guided methods to tackle proteins important in infectious diseases.

Although each of these chapters can be downloaded individually, it is well worth getting a copy of the book itself. Like other members of the series, it is beautifully put together. Having all the papers in one place is extremely convenient, and simply paging through the volume is bound to generate new ideas.

NMR poll results

The results of our latest poll are in – thanks to all who participated! Of the 119 people who responded to the first question, 87% said they use NMR for finding or validating fragments. Even if we assume that responses were biased towards NMR aficionados, big magnets are clearly popular.

The second question asked about specific NMR techniques. If everyone who said they used NMR in the first question also answered the second, this means the average user applies more than 3 different techniques; I’ll let Teddy weigh in to see whether this matches his experience.

One surprise for me was that, although many techniques are widely used, none are nearly universal; even the most popular methods seem to be used by just over half of respondents.

Among ligand-detected methods (blue in the figure), STD ranks at the top, with line-broadening, WaterLOGSY, and fluorine-based techniques all tied for second place.

Protein-detected methods (red in the figure) also appear quite healthy, with nearly as many respondents using ¹⁵N-HSQC/HMQC as STD.

Finally, 11 of you said you use "other" techniques. We didn't include TINS, even though it seems quite useful, because it is only available through the services of ZoBio. But what else is out there?

Fragments vs membrane proteins with SPR

Membrane proteins such as GPCRs account for something like half of all drug targets, but they present a serious challenge for fragment-based approaches. This is partly because the biophysical methods usually used for fragment screening often don’t work for membrane proteins, and partly because, in the absence of structural information, it’s hard to know what to do with fragment hits. But there is progress. We’ve highlighted a couple papers that use functional screens or TINS to find fragments against membrane proteins, and in a recent issue of Biochem. Pharmacol. U. Helena Danielson and colleagues at Beactica and two academic institutes show how surface-plasmon resonance (SPR) can also be applied.

The researchers were interested in GABA_A receptors, a class of ion channels involved in multiple physiological functions. The receptors normally form hetero-pentamers, but for simplicity the researchers used a homo-oligomeric receptor, consisting of five β3 subunits. Each β3 subunit carried a tag containing eight histidine residues that could be recognized by antibodies immobilized to the surface of the SPR chip. The GABA_A receptors were detergent-solubilized; control channels contained antibodies and detergent with no receptors. The resulting GABA_A-modified chips were quite stable; the researchers report being able to run roughly 200 samples over the course of 20 hours with a single chip. (The specific detergents and conditions are critical, so if you’re interested in pursuing this yourself the experimental section is invaluable.)

A set of 51 histaminergic and 15 GABAergic ligands were tested for binding, resulting in nearly two dozen hits with dissociation constants (K_Ds) between 13 and 300 micromolar. Some of these are exceptionally small: for example, histamine, with a molecular weight of 111 g/mol and just 8 heavy atoms showed a K_D of 98 micromolar, which is consistent with published results using different methods. A number of other ligands were also identified, some for the first time, though other previously reported ligands did not repeat in this system.

It will be fun to see the screening results of a larger, unbiased library. Of course, finding fragment hits against a membrane protein is only the first step to developing drugs – one still needs to figure out how to improve potency, most likely in the absence of structure. But, to paraphrase Churchill, at least this paper and related research represent the end of the beginning.

You Probably Already Knew This...

Academics can spend time and resources doing, and publishing, things that people in the industry already "know".  This keeps the grants, the students, the invitations to speak rolling in.  It also allows you to cite their work when proposing something.  This is key for the FBHG community.  There are many luminaries in the FBHG field, and we highlight their work here all the time. Sometimes, they work together as a supergroup.  Sometimes, Cream is the result.

Brian Shoichet and Gregg Siegal/ZoBio have combined to work together.  In this work, they propose to combine empirical screening (TINS and SPR) with in silico screening against AmpC (a well studied target).  They ran a portion of the ZoBio 1281 fragment library against AmpC.  They got a 3.2% active rate, 41 fragments bound.  6 of these were competitive in the active site against a known inhibitor.  35 of 41 NMR actives were studied by NMR; 19 could have Kds determined (0.4 to 5.8 mM).  13 fragments had weak, but uncharacterizable binding; 3 were true non-binders. That's a 90% confirmation rate.  34 of 35 were then tested in a biochemical assay.  9 fragments had Ki below 10 mM.  Of the 25 with Ki > 10mM, one was found to bind to target by X-ray, but 25A from the active site.  They then did an in silico screen with 300,000 fragments and tested 18 of the top ranked ones in a biochemical assay.  

So, what did they find? 

"The correspondence of the ZoBio inhibitor structures with the predicted docking poses was spotty. "  and "There was better correspondence between the crystal structures of the docking-derived fragments and their predicted poses."

So, this isn't shocking, but it is good to know.  This is also consistent with this comment.  So, the take home from this paper is that in silico screening can help explore chemical space that the experimentally much smaller libraries miss.  To that end, the authors then do a a virtual experiment to determine how big a fragment library you would need to cover the "biorelevant" fragment space [I'll save my ranting on this for some other forum].  Their answer is here [Link currently not working, so the answer is 32,000.]

Fragments in the Caribbean

Last week saw the inaugural Zing FBDD conference in Punta Cana, Dominican Republic. Zing has been around only since 2007, and seems to focus on small conferences in exotic locales. The benefit is that they are able to attract high-profile speakers, as illustrated by the group photo below. However, in an era of shrinking travel budgets, getting approval to attend a conference at a resort is becoming a bit more challenging. That said, participants enjoyed nearly 30 presentations and great discussion – think of a Gordon Conference without the dorms, and breaks on the beach.

My favorite “equation” from the conference comes from Mike Serrano-Wu of the Broad Institute:

Undruggable = Undone

This was supported by some nice work on the anti-cancer target MCL-1, which makes a protein-protein interaction that was widely consider undruggable just a few years ago. An ¹⁹F NMR fragment screen gave a hit-rate of around 10%, leading eventually to low nanomolar leads. Fragment optimization was facilitated by a new crystal form of the protein that allowed the team to rapidly generate over a dozen protein-ligand co-crystal structures. Rumor has it that more details on this will be disclosed at FBLD 2014 in Basel in September (there are still a few openings available, but register soon.)

MCL-1 also figured heavily in talks by Andrew Petros (AbbVie, see also here) and Steve Fesik (Vanderbilt, see also here), who described cell-permeable molecules with high picomolar activity in biochemical assays. Steve also discussed programs against Ras and RPA, both also using SAR by NMR. As Mike Shapiro (Pfizer) pointed out in his opening presentation, one of the breakthrough ideas of SAR by NMR was to screen a library more than once per target, the second time in the presence of a first ligand to identify another. It is nice to see this strategy continuing to deliver against difficult targets, though preliminary results of our current poll (right hand side of page) indicate that linking is not necessarily easy.

One of the payoffs of doing fragment screens for many years on dozens of targets is a rich internal dataset. Chris Murray (Astex) mentioned that company researchers have solved close to 7000 protein crystal structures, more than a third of them with fragment ligands. A cross-target analysis found that hits tended to be more planar (ie, less “three-dimensional”, with apologies to Pete Kenny) than non-hits. This was particularly true for kinases; for six protein-protein interactions (PPIs) there was no correlation between shape and hit rate. Although defining complexity is difficult, Chris provided evidence that 3D fragments tend to be both larger and more complex.

Rod Hubbard (University of York and Vernalis) mentioned that Vernalis has determined more than 4000 protein crystal structures. Since 2002, 2050 fragments have been screened against more than 30 targets. Based on “sphericality” – the distance from the rod-sphere principle component axis – hits against kinases are marginally less spherical, while PPI hits reflect the shape of the overall library. So, despite the current push for more three-dimensional fragments, it remains to be seen whether this will be useful.

Jonathan Mason (Heptares) described how successful fragment approaches can be against membrane proteins such as GPCRs. Anyone who has worked on these targets will know that the SAR can be razor sharp, and their surfeit of structures is helping to explain this. For example, although many of the protein-ligand interactions appear merely hydrophobic, some displace high-energy water molecules, which can be revealed by crystal structures of both the free and bound forms of the protein. Displacement of high energy water molecules also helps to explain some “magic methyl” effects.

Fragment-finding methods were not neglected. Jonathan mentioned that, for the A2A receptor, SPR identified only orthosteric ligands, while TINS identified only allosteric ligands – the orthosteric ligands were actually too potent to be detected by this technique. John Quinn (Takeda, formerly SensiQ) and Aaron Martin (SensiQ) also discussed SPR, and in particular how variable temperature SPR analyses could be used to rank ligands based on their enthalpic binding, though as Chris Murray warned, this information can be difficult to use prospectively.

I also learned that a selective BCL-2 inhibitor from Vernalis and Servier has just entered into Phase 1 clinical trials. This has been the result of a long-running collaboration that has required creativity on the part of the scientists and patience on the part of management.

There is much more to tell – for example Teddy's extended metaphor of the Silk Road (this one, not this one!) – but in the interest of space I’ll stop here. Feel free to comment if you were there (or even if you weren’t!)

Fragment-Based Drug Discovery

This is the straight-to-the-point title of a new book published by the Royal Society of Chemistry, edited by Steven Howard (Astex) and Chris Abell (University of Cambridge). It is the second book on the topic published so far this year, and it is a testimony to the fecundity of the field that the two volumes have very little overlap.

After a brief forward by Harren Jhoti (Astex) and a preface by the editors, the book opens with two personal essays. The first, by me, is something of an apologia for Practical Fragments and the growing role of social media in science (and vice versa). If you’ve ever wondered how this blog got started or why it keeps going, this is where to find out. The second essay is by Martin Drysdale (Beatson Institute). Martin is a long-time practitioner of FBDD, dating back to his early days at Vernalis (when it was RiboTargets) and he tells a fun tale of “adventures and experiences.”

Chapter 1, by Chris Abell and Claudio Dagostin, is entitled “Different Flavours of Fragments.” With a broad overview of the field it makes a good introduction to the book. There are sections on fragment identification, including the idea of a screening cascade, as well as several case studies, some of which we’ve covered on Practical Fragments, including pantothenate synthetase, CYPs, RAD51, and riboswitches.

The next two chapters deal with two of the key fragment-finding methods. Chapter 2, by Tony Giannetti and collaborators at Genentech, GlaxoSmithKline, and SensiQ, covers surface plasmon resonance (SPR). This includes an extensive discussion of data processing and analysis, which is critical for improving the efficiency of the technique. Competition studies are also described, as are advances in hardware, notably those from SensiQ. This is a good complement to Tony's 2011 chapter.

Chapter 3, by Isabelle Krimm (Université de Lyon), provides a thorough description of NMR methods, both ligand-based (STD, WaterLOGSY, ILOE, etc) and protein-based (mostly HSQC). The chapter does a nice job of describing techniques in terms a non-specialist can understand while also providing practical tips on matters such as optimal protein size and concentration.

Chapter 4, by Ian Wall and colleagues at GlaxoSmithKline, provides an overview of FBLD from the viewpoint of computational chemists. The chapter includes some interesting tidbits, such as the observation that fragment hits that yield crystal structures tend to be less lipophilic but also contain a smaller fraction of sp³ atoms and more aromatic rings. The researchers note that the current fashion for “3D” fragments is yet to be experimentally validated. They also include accessible sections on modeling, druggability, and integrating fragment information into a broader medicinal chemistry program.

The remaining chapters focus on specific types of targets. Chapter 5, by Miles Congreve and Robert Cooke (both at Heptares) is devoted to G protein-coupled receptors (GPCRs). This includes descriptions of how to screen fragments against these membrane proteins using SPR, TINS, CE, thermal melts, and competition binding. It also includes a detailed case study of their β1 adrenergic receptor work (summarized here). Congreve and Cooke assert that, although many of the GPCR targets screened to date have been highly ligandable, technical challenges only now being addressed have caused this area of research to lag about a decade behind other targets. They predict a bright future.

Rod Hubbard (Vernalis and University of York) turns to protein-protein interactions in Chapter 6. After describing why these tend to be more challenging than most enzymes and covering some of the methods for finding and advancing fragments, he then presents several case studies, including FKBP (one of the first targets screened using SAR by NMR), Bcl-2 family members (including Bcl-x_L and Mcl-1), Ras, and BRCA2/RAD51. He concludes with a nice section on “general lessons,” which boils down to “patience, pragmatism, and integration.” As Teddy recently noted, this can lead to substantial rewards.

Allosteric ligands have potential advantages in terms of selectivity and addressing otherwise challenging targets, and in Chapter 7 Steven Howard (Astex) describes how fragments can play a role here. This includes how to establish functionality of putative allosteric binders, as well as case studies such as HIV-1 RT, FPPS, and HCV NS3. Astex researchers have recently stated that they find on average more than two ligand binding sites per protein, and this chapter includes a table listing these (including 5 binding sites each on bPKA-PKB and PKM2).

The longest chapter, by Christina Spry (Australian National University) and Anthony Coyne (University of Cambridge) describes fragment-based discovery of antibacterial compounds. After discussing some of the challenges, the authors report several in depth case studies including DNA gyrase, DNA ligase, CTX-M, AmpC, CYP121, and pantothenate synthetase, among others. At least one fragment-derived antibacterial agent entered the clinic; hopefully more will follow.

Chapter 9, by Iwan de Esch and colleagues at VU University Amsterdam, focuses on acetylcholine-binding proteins (AChBPs), both as surrogates for membrane-bound acetylcholine receptors and as well-behaved model proteins on which to hone techniques (see for example here, here, and here). Since AChBPs have evolved to bind fragment-sized acetylcholine, these proteins can bind tightly to small ligands; 14-atom epibatidine binds with picomolar affinity, for example, with a ligand efficiency approaching 1 kcal mol^-1 atom^-1.

And Chapter 10, by Chun-wa Chung and Paul Bamborough at GlaxoSmithKline, concisely covers epigenetics. Bromodomains are well-represented, including a table of ten examples (see for example here, here, here, here, here, and here). Happily, although some of these projects started from similar or identical fragments, the final molecules are quite divergent. However, the authors note that much less has been published on histone-modifying enzymes, such as demethylases and deacetylases, perhaps reflecting the challenges of achieving specificity with what are often metalloenzymes.

Finally, this is the 500^th post since Teddy founded Practical Fragments way back in the summer of 2008. Thanks for reading, and special thanks for commenting!