Ninth Annual Fragment-based Drug Discovery Meeting, Part 2

The first major fragment event of 2014 drew around 500 people to San Diego last week. This is part of CHI’s three-day Drug Discovery Chemistry conference, and although the official FBDD track was only one of six, it is a testimony to the vitality of the field that fragments made appearances in most of the other sessions. With 17 talks in the FBDD track alone this post will not attempt to be comprehensive; Teddy has already shared some impressions here.

Jim Wells (UCSF) gave a magisterial keynote address that emphasized how useful fragments can be for tackling difficult targets such as protein-protein interactions (PPIs). In fact, many of the talks in the protein-protein interaction track relied on fragments. That’s not to say it’s easy. Rod Hubbard (University of York and Vernalis) emphasized that advancing fragments to leads against such targets can take a long time and often requires patience that strains the management of many organizations. Fragment hits against PPIs usually have lower ligand efficiencies (0.23-0.25 kcal/mol/HA if you’re lucky), and improving potency can be a bear. Rhian Holvey (University of Cambridge) presented a nice example of how she was able to find millimolar fragments that bind to the anti-mitotic target TPX2, potentially blocking its interaction with importin-alpha, but even structural information was not enough to get to potent inhibitors.

G-protein coupled receptors (GPCRs) were thought to be unsuitable for fragments until recently, but both Iwan de Esch (whose work has been profiled several times, including here and here) and Jan Steyaert (Vrije University) presented success stories. In fact, Jan has only been working with the Maybridge fragment library for a few months, but has found agonists, antagonists, and inverse agonists for several GPCRs.

Another example of a difficult target is lactate dehydrogenase A (LDHA). We’ve previously highlighted cases where fragment linking was used to get to nanomolar binders (here and here); Mark Elban (GlaxoSmithKline) presented an example of fragment growing and using information from a high-throughput screen (HTS) to get to nanomolar binders. Mark also discussed a particularly disturbing false positive: HTS had generated dozens of confirmed hits spanning 7 chemotypes, but upon closer inspection it turned out that all of them came from a single vendor, and that – unreported by the vendor – they were all oxalate salts. Oxalate is a low micromolar inhibitor of LDHA, and is invisible in proton NMR, so I’m sure this was not fun to track down.

Ben Davis (Vernalis) also presented great examples of false positives and false negatives, and how to avoid them. In particular, the WaterLOGSY NMR technique is great for weeding out aggregators when run in the absence of protein.

A common theme throughout the conference was the integration of fragments with other methods, such as HTS. Nick Skelton (Genentech) actually titled his presentation “Fragment vs. HTS hits: does it have to be a competition?” Kate Ashton (Amgen) discussed how using information from a fragment screen helped solve pharmacokinetic issues with an HTS-derived hit. And Steven Taylor (Boehringer Ingelheim) presented a similar example (also covered here) of using fragments to fix a more advanced lead. Steven noted that fragment-based methods are now fully integrated into the organization, which marks a significant change from Sandy Farmer’s presentation at this meeting four years ago.

The roundtables are great opportunities to swap ideas and get feedback; Teddy already mentioned the excellent roundtable he chaired, but I wanted to also give a shout-out to one organized by Derek Cole (Takeda) focused on "practical aspects of fragment screening." We recently discussed discussed fragments that destabilize proteins in thermal shift assays, and it turns out that folks from both the Broad Institute and Takeda have also crystallographically characterized such fragments. There was the sense that either stabilizers or destabilizers should be considered hits, though the latter were less likely to lead to crystal structures than the former.

Finally, on the subject of library design, Damian Young (Baylor College of Medicine) described using diversity-oriented synthesis (DOS) to generate more “three-dimensional” fragments. He is planning to build a library of roughly 3000 fragments which he hopes to make widely available to the community; these should help answer the question of whether the third dimension is really an advantage.

The importance of library design was also emphasized by Valerio Berdini (Astex); they are currently on their seventh generation library, about 40% of which is non-commercial, and half of whose members have been solved in one or more of 6000+ crystal structures. Relevant to the rule of three, Astex is moving to ever smaller fragments, with an average of 12.6 non-hydrogen atoms, ClogP = 0.6, and MW = 179. Indeed, despite assertions that PPIs may require larger fragments, Rod noted that at Vernalis the average fragments hits against PPIs are only slightly larger (MW = 202 vs 189 against all targets) and more lipophilic (ClogP 1.2 vs 0.8).

CHI has already announced that next year’s meeting will be held in San Diego from April 21-23. As it will be the ten year anniversary, they’re planning something big, so put it on your calendar now!

Drug Discovery Chemistry Conference Round Up, Pt 1

As many of you know, Dan and I were at the CHI Drug Discovery Conference.  Over the next few posts, we will be posting notes, thoughts, and some comments on what happened. First off, I live-tweeted the sessions I was at.  Day 1's highlights are here , Day 2 is here, Day 3 here.  Overall, I really liked the conference and thought the agenda was very high quality (one session was poor, read my tweets and guess which one).  CHI had Dan and me hopping: chairing sessions, moderating round tables, and co-teaching our FBDD course.  I was very lucky to moderate a breakfast roundtable  on Thursday morning on PPIs and Fragments (Small Solutions for Big Problems: Fragments and PPI).  

The table was a huge hit; there were more than 30 people spread over 5 or so tables.  I am pretty sure it was because Rod Hubbard and Dan were there.  I am going to try to capture the discussion, but I am pretty sure I am missing key points, so if people where there and remember things, add them in the comments, or email me and I will edit the post.  The discussion initiated with a reference to a comment that Rod made in his talk the day before: NMR is the preferred method for screening PPIs (over SPR).  He cited two main reasons: NMR is more sensitive to very weak binders and you can do QC on the protein in every samples.  With SPR, once the complex is put on the chip, you have no idea if it is still intact/amenable to screening.  Jan Steyaert asked a question: most of the PPIs we see targeted are stable complexes, while most of the PPIs in nature are transient, why?  This led to the question of whether people are working to stabilize complexes, rather than disrupt?  People agreed that you would need to have a kinetically resolved assay, like a TR-FRET.  I raised the point that you would need a enzymologist to do this, and most biologists these days are pharmacologists.  [As an aside, this is why we focus so strongly on IC50 without knowing really what it means.  For further discussion of this, go ask Pete Kenny about this.]  It was brought up that you could possibly due this by SPR if you  had a a very special "group"[my handwriting sucks under the best of cases, rushing while moderating makes it even worse].  I think the idea is you could go to lower temperatures and start observing kinetics of fragment binding (vs. the normal square sensorgram). 

Someone from the Broad Institute said that they use Thermal Shift for PPIs, no matter what.  However, most of the people don't trust TS, no matter what.  It is cheap and fast, but so full of artifacts and errors.  It's a paradox, a quick, cheap assay for PPIs that everyone uses and no one trusts.  Seems like a classic case of the herd mentality.  

The discussion then moved on to a key concept for fragment/PPIs: how do you follow up on 3D fragments?  Most people agree that fragments with higher 3D content are better for targeting PPIs.  However, I think, in contrast to 2D fragments, exactly how you prosecute hits in this target class is less straight forward.  I think an important distinction between 2D and 3D fragments in terms of follow up is that linking 3D fragments may actually be relevant and productive, in contrast to how most people view linking 2D fragments.  

As I said above, I am sure I am missing some points, so add them in the comments or email me.  Dan and I will be posting more round up notes over the next week or so, so stay tuned.

Fragments vs soluble epoxide hydrolase – hundreds of them!

Soluble epoxide hydrolase (sEH) is a potential target for cardiovascular and immune disorders. The enzyme catalyzes the hydrolysis of long chain, lipophilic epoxyeicosatrienoic acids. These bind in a largely hydrophobic “L-shaped” pocket, with the catalytic machinery at the point of the L. Although it is relatively easy to find potent inhibitors of this enzyme, these tend to be greasy, insoluble, and non-druglike. In a recent paper in Bioorg. Med. Chem. Yasushi Amano and colleagues at Astellas describe a fragment-based approach to find better leads.

The researchers started with a high-concentration (up to 2 mM) enzymatic inhibition assay of 4200 fragments, resulting in 307 hits with IC₅₀ values between 700 nM and 1.7 mM. All of these were taken into co-crystallography trials, yielding crystals for about half of them. The other fragments were soaked into apo-crystals of sEH (that is, crystals without bound ligand) to get as many structures as possible. All together 126 crystal structures of fragments bound to sEH were solved, which is all the more impressive considering that there are only three authors on the paper!

Most of the fragments (83) bound at the catalytic site (example shown in green), while 29 bound to one of the lipophilic branches of the L and 9 bound to the other (cyan and magenta). Five fragments bound at two different sites within the enzyme. The researchers discuss ten of the fragments in some detail.

Many of the fragments that bind at the catalytic site contain amides or ureas – moieties in known inhibitors – but some of them (such as compound 3 above) are more unusual. Also, despite the generally lipophilic nature of the binding pocket, many of the fragments – even those that bind in the hydrophobic branches of the L – make hydrogen bonds to the protein or to bound water molecules. This suggests that it should be possible to find potent inhibitors that are less hydrophobic than previously reported molecules. Fragment growing, linking, and merging approaches could all work, and indeed the researchers hint that results of these studies will be reported in future papers.

More importantly, this paper provides a great set of crystallographically validated fragments binding to distinct sites on a well-characterized protein. Helpfully, the researchers have deposited the coordinates of the 10 co-crystal structures discussed in the protein data bank. Moreover, all of the fragments are commercially available. This seems like an ideal model system for validating computational docking and scoring approaches as well as for better understanding the energetics of protein-ligand interactions. If I were an academician working in these areas, I’d jump in feet first!

What we do in life, echoes in eternity (or the life of the patent)

Next week is the Drug Discovery Chemistry conference where Dan and I will be co-teaching our award-winning FBDD short course (or at least our mom's think it is great). We look forward to seeing any/all of you next week.  Blogging may be light next week, but we promise to give an update of the going-ons at the conference soon after.  

Kinases are fun, and those of us who have worked in them have probably all worked on the same ones.  I always loved the MAP family.  Why would I have a favorite kinase family?  Because of the cascading MAP kinases, like the one in this paper, Mitogen-activated protein kinase kinase kinase kinase 4 (that's a lot of kinase!).  But, unlike a lot of other kinases, there is no good tool compound.  So, using SPR, they decided to generate one. This paper is not particular interesting in terms of what they did, but rather it raises interesting questions. While the approach they describe is not novel, it is nice to see the data supporting them. 

They screened their 2500 fragment library against immobilized protein at 100 uM (single point).  225 hits were found with Kd ranging from 10 to 2000 uM (LE =0.24 to 0.59) for a 9% hit rate.This paper is about progressing this oxazole fragment 1: 

Based upon its structure and the wealth of kinase structure knowledge extant, they surmised it would be ATP-competitive and a hinge binder.  Based upon a binding model, the attempted to prosecute this fragment by "close-in" analogs and looking for groups that would extend farther into the hydrophobic pocket, but with MW less than 350 Da and clogP less than 3.5.  Exploring bi-aryl space resulted in 8:  

This compound had an activity of 143 nM and it was at this point that they decided to switch to the biochemical assay as their primary assay.  In the end, using X-ray focusing on LLE, they ended up deliveringa low molecular weight compound with favorable in vivo PK.  It also demonstrated a pathway functional response. 

This raises an excellent point, something I get asked frequently.  When do you switch from a biophysical assay to a biochemical one?  This maybe arguing semantics, but I think as more and more companies enter this arena these are exactly the things we need to discuss.  I think the switch happens when you feel comfortable, there is no hard and fast rule.  There is a difference in the SPR Kd and biochemical IC50 by more than 10x.  It is very important to note that they relied heavily on LE (-RTlnKd/HA) and LLE (pKd-cLogP), or pIC50 for biochemical assays.  But, it also raises the issue of correlation between SPR Kd and IC50.  I raise these socratically, and as maybe as a topics for discussion next week (or in July and September).   

Can selectivity of fragments be maintained?

In 2011 we highlighted an analysis of kinase inhibitors that demonstrated that non-selective fragments could produce selective leads, and vice versa. However, that study was based on hundreds of compounds not necessarily chosen from the same projects. Are the results the same within individual fragment-to-lead programs? This is the question that Ian Collins and colleagues at the Institute of Cancer Research address in a recent paper in MedChemComm.

The researchers examined three fragment-to-lead efforts: two targeted the kinase PKB and the other targeted the kinase CHK1. In all three cases they started with fragments and used structure-based design and fragment growing to obtain low nanomolar inhibitors. Importantly, they also obtained crystal structures of key compounds along the way, demonstrating that the initial fragment – a hinge-binding element – maintained its position and orientation throughout the process.

Each fragment, lead compound, and intermediate molecule was tested for selectivity in a panel of 91 kinases using a microfluidic mobility-shift peptide phosphorylation assay. The concentration of ATP in each assay was at the K_M,ATP, and each compound was tested at 10-fold above its IC₅₀ for the target kinase (so for example fragment 1 below was screened at 1000 μM, and fragment 5 was screened at 8 µM). For each compound a selectivity score was calculated based on the number of kinases inhibited at a certain threshold. For example, if S_(30%) = 1, this would mean that all of the kinases were inhibited by at least 30% at the concentration tested, whereas if S_(30%) = 0.03 this would mean that only 3 kinases (3/91= 0.03) were inhibited.

It's worth noting that selectivity is tough to define specifically. Although the selectivity score makes sense intuitively – each compound is tested at a concentration relevant to the intended target – I am concerned that it will make potent compounds appear more selective than they really are. Indeed, a plot of S_(30%) versus -log[concentration tested] is fairly linear. (Compounds discussed below are labeled by number on the plot.)

Accepting this definition of selectivity, though, it appears that nonselective fragments, such as 7-azaindole (fragment 1) could be progressed to nonselective leads such as compound 4, which was an early milepost en route to AZD5363, currently in Phase 2 clinical trials.

Fragment 1 was also modified to slightly less promiscuous fragment 5. A slight tweak to this molecule produced selective fragment 6, which was then optimized to the selective compound 8 (closer to AZD5363). In the case of fragment 6, even though the structural change was minor (removal of a single methylene) this was enough to make a specific interaction with a residue in PKB not found in other kinases. The CHK1 story is similar in taking a nonselective fragment to a selective lead.

So what’s the conclusion? The authors suggest that:

Broad kinase selectivity screens of fragments could be predictive of the lead, provided strategies to conserve the profile are followed in the elaboration, avoiding introducing new interactions with target-specific residues. Conversely, the initial fragment selectivity patterns are unlikely to reflect those of developed leads if the fragment does not already encode the anticipated target-specific interactions.

In other words, it depends. This is not meant as a criticism: I think this conclusion is about as decisive as possible when generalizing about fragment-to-lead strategies. At the very least, the work suggests that effort spent optimizing a fragment before growing or linking could be worthwhile. And even a promiscuous fragment may be only one atom away from something quite specific.

Covalent, destabilizing fragments against TB target BioA

Differential scanning fluorimetry (DSF) is a hit-finding technique in which a protein is incubated with a small molecule and heated until the protein reaches its “melting temperature” and unfolds. If a small molecule binds, in theory it should stabilize the protein towards thermal denaturation and thus raise the melting temperature, giving a positive thermal shift. However, most folks who have performed these types of experiments have also found (and usually ignored) molecules that lower the melting temperature of the protein. In a new paper in ChemBioChem, Barry Finzel and coworkers at the University of Minnesota follow up on one of these with very interesting results.

The researchers were interested in the enzyme 7,8-diaminopelargonic acid synthase (BioA) from Mycobacterium tuberculosis, the organism that causes its eponymous disease. This enzyme – which is not found in mammals – is involved in the synthesis of the essential cofactor biotin. DSF was used to screen 1000 compounds from the Maybridge Ro3 Diversity Fragment Library at 5 mM concentration, resulting in 21 hits which changed the denaturation temperature (T_m) by more than 2 °C. A dozen of these decreased the T_m, but although all of these were taken into crystallography trials, only compound 1 yielded a structure. STD NMR was also used to confirm that the compound binds to BioA in solution.

Next, the researchers used the classic “SAR-by-catalog” approach and purchased analogs of compound 1. Compound 2 turned out to be particularly interesting: it decreased the T_m by a whopping 18 °C! Weirder still, when soaked into crystals of BioA, they turned from yellow to red.

BioA is a transaminase: it takes a nitrogen from one molecule (called SAM) and transfers it to another molecule (called KAPA). En route to its final destination, the nitrogen is transferred to a co-factor, pyridoxal phosphate (PLP), which contains an aldehyde. Compound 2 contains a hydrazine, which is known to react with aldehydes, and in fact a co-crystal structure of compound 2 bound to BioA shows that this is exactly what happens. Interestingly, compound 2 binds in a somewhat different manner than compound 1 despite their similar chemical structures.

Compound 2 turns out to be a reversible inhibitor of BioA, and the researchers were able to demonstrate that it is a moderately potent and competitive inhibitor with respect to SAM and a less potent uncompetitive inhibitor with respect to KAPA. This is exactly what you would expect, since it competes with SAM for binding to PLP but does not compete with KAPA.

Now you may think that hydrazines aren’t exactly drug-like, but it turns out that a commonly used drug against tuberculosis is isoniazid, which contains an analogous acyl hydrazide. The researchers found that isoniazid also decreases the T_m of BioA, though less dramatically than compound 2. Though isoniazid works through an entirely different mechanism, the researchers were able to obtain a co-crystal structure of this binding to PLP in BioA (magenta; PLP is on the left, and protein is not shown), showing that it binds differently than either compound 1 (green) or 2 (cyan). Nonetheless, it did not show any inhibition of the enzyme, demonstrating that covalent binding alone is not sufficient for disrupting enzymatic activity.

This is a very nice paper, and it will be fascinating to try to understand how the fragments so effectively destabilize the protein despite binding tightly, and how this translates into inhibition. The researchers suggest that finding ligands that destabilize proteins could be generally useful for turning off proteins. Are there other well-characterized examples out there?

It's A Start

As the readers of this blog know, I tend to be harsh on academic "Drug discovery" papers.  Sometimes, there is a really worthwhile academic paper, but by and large I find that they tend to publish things that are barely "drug discovery" and more the For Dummies...of what they think drug discovery is.  Which way will I swing on this paper from researchers Down Under?  

The bacterial Sliding Clamp, aka polymerase 3beta, is a key player in bacterial replication and is an "emerging" target. It interacts with other proteins via LM (Linear Motifs): 4-10 amino acid disordered regions.  This is typically a weak interaction (1-100 uM). These LM exist at termini, but sometimes in loops.  A consensus sequence for the LM that interacts with the Sliding Clamp has been identified: QLx1Lx2F/L (S/D preferred at x1; x2 may be absent).  Two classes of compounds have been identified previously but with >10 uM affinity and no -cidal activity. 

So, these authors went after this target using X-ray as the primary screen.  The Zenobia Fragment Library was used (352 molecules) to soak into crystal in pools of 4 fragments.  Four fragments (below) were found to bind to Subsite I on Chain A.  However, no changes in the main chain density were observed.

They also found several other fragments with weak density, and several that were deemed crystallographic artifacts.  None of these compounds showed significant activity below 1 mM in their competition assay.  So, the story then continues that they "sought to improve binding affinity by identifying fragments that could more completely occupy" the binding site.  

[An aside:  To me, this brings up an important point about the choice of fragment collection.  Fragments that are designed for X-ray soaking tend to be small (10-12 HAC).  Just from a theoretical standpoint, those fragment would have to have an affinity in the 250 uM range (LEAN 0.3).   This was covered in a poll and most most people are happy going < 10 HAC.  My question is how often is a very small fragment found as an active?]

To do this, they noted that the fluoro-phenyl group in 1 was previously reported, leading to investigations with compound 5. It was found to fully occupy the binding site.

They searched ZINC for compounds similar to 1-5.  Their initial purchases failed to find any compounds with activity < 1mM.  Eventually, they landed on the hypothesis that chlorocarbazoles were "promising", leading to compound 6.  At this point, I think Dan's head exploded, Scanners-style.  Yes, that is an epoxide.  The co-crystal structure showed that it was binding in the active, albeit with weak electron density.  Their SAR, wisely, did not include the N-alkyl epoxide. 

Both 7 and 8 show good LE and LLEAT.  Only the R enantiomer of 8 caused movement in the main chain.  It also was the most potent in the replication inhibition assay (64 uM).  It was also the most potent in terms of -cidal activity against both Gram positive and negative microbes. 

So, is this a good or bad paper?  I would say it is a start, but if I had been a reviewer I would have made them change the title "Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp
Using a Fragment-Based Approach" to "Discovery of ACTIVE Compounds Targeting the Bacterial Sliding Clamp Using a Fragment-Based Approach".

Funky fragments

Natural products have led to many approved drugs, and there is an increasing appreciation that Nature often knows best. Indeed, several published fragment libraries incorporate natural products or natural product-like molecules (see for example here, here, and here). With all this attention, it was inevitable that commercial fragment suppliers would spot this market need.

SerpentesOleum, Inc. has just launched a library they call FUNK: Fragments Uncovered in Natural Kompounds. This set consists of several hundred natural products and derived fragments carefully selected to maximize hit rates. For example:

The company has screened their library against targets such as PTP1B and falcipain-1 and obtained remarkably high hit rates in functional assays. In fact, SerpentesOleum is so confident that they’re offering a money-back guarantee if you don’t obtain at least one active against your target, no matter what it is. Looking at the structures of their compounds, I have no reason to doubt their claim.