LLE vs LELP

Besides ligand efficiency (LE), a slew of other metrics has been proposed to help evaluate what compounds to take forward in drug discovery. As seen in our poll and a recent round-table discussion, lipophilic ligand efficiency (LLE) is quite popular:

LLE = pIC₅₀ (or pK_i) – ClogP (or logD)

However, because this metric is not size-adjusted, it is not particularly useful for evaluating fragments, which often have low potency. In contrast, the metric LELP accounts for size:

LELP = logP / LE (where LE = ligand efficiency)

In a recent issue of J. Med. Chem., György Keserű and colleagues evaluate how these two metrics compare in a variety of settings.

The authors examine eight different compound sets: fragment hits and derived leads, HTS hits and derived leads, leads that subsequently became drugs (ie, “successful leads”), development candidates, compounds that entered phase II trials, and drugs on the market. Not surprisingly, drugs and phase II compounds had better LLE and LELP scores than other molecules. Also not surprisingly, fragments scored misleadingly poorly on the basis of LLE but well on the basis of LELP. What is perhaps unexpected, though, is that LELP was better at identifying successful leads than was LLE. Moreover, when compounds were evaluated for pharmacokinetic and safety parameters, LELP was more effective at predicting problems than was LLE. The authors state:

In summary, evaluation of pharmacokinetic and safety parameters revealed that LELP has benefits over LLE, as compounds with acceptable in vitro ADMET profiles are discriminated from compounds with significant liabilities.

Despite these potential advantages, LELP doesn’t seem to be widely used, perhaps because it is less intuitive than some of the other metrics. Indeed, it would be interesting to see these studies repeated using LLE_AT, which also takes lipophilicity into account but has the same scale as LE.

Fragment-based drug discovery and X-ray crystallography

I’m holding in my hands a book of this title, edited by Thomas Davies and Marko Hyvönen and published this year as part of Springer’s Topics in Current Chemistry series. I believe this is the fourth book entirely devoted to fragment-based drug discovery, which shows both the vitality and rapid development of the field.

The book starts with an introduction to fragment-based drug discovery by me. If you’re new to the field, this chapter should serve as a self-contained summary.

In the next chapter Thomas Davies and Ian Tickle describe how FBDD is practiced at Astex, paying particular attention to the use of X-ray crystallography. Notably, researchers from this company “do not consider a fragment hit to be ‘validated’ and suitable as a starting point for medicinal chemistry until it has been observed to bind by crystallography.” This chapter also contains a nice analysis of fragment library design and a couple case studies, including the discovery of the clinical-stage CDK2 inhibitor AT7519.

Rod Hubbard and colleagues at Vernalis and the University of York next describe their efforts to discover Hsp90 inhibitors using a combination of virtual and fragment screening. We’ve covered some of this before (here and here), but it’s nice to see the full story.

The next chapter also focuses heavily on a single target: Daniel Wyss and colleagues at Merck describe their success in discovering BACE inhibitors. This chapter also includes an excellent review of NMR methods for finding fragments.

Michael Hennig and colleagues at Roche (Basel) contrast the various biophysical methods used to discover fragments, with a heavy emphasis on SPR. Crystallography is also covered, in particular co-crystallization of fragments with protein. Co-crystallization is more time-consuming than soaking fragments into preformed crystals, so compound prioritization techniques such as SPR are especially useful.

One of the most promising applications of fragment-based methods is tackling tough targets such as protein-protein interactions, the subject of a chapter by Marko Hyvönen and colleagues at the University of Cambridge. The chapter contains a nice discussion of energetics and hot spots as well as a detailed analysis of methods to find fragments which complements some of the other chapters.

Eddy Arnold and colleagues at Rutgers discuss the use of crystallographic fragment screening against two HIV-1 targets, HIV protease and HIV reverse transcriptase (RT). We’ve previously discussed the former here. In the case of RT, fragments were soaked into crystals in the presence of a high affinity inhibitor, effectively blocking its binding site from fragments. More than 30 fragments were identified binding to multiple other sites on the protein – one fragment bound at 11 distinct sites! Interestingly, the fragments were enriched for halogen-containing molecules. Several also had functional activity with respectable ligand efficiencies. The authors also discuss other published fragment work on HIV RT.

Finally, Didier Rognan at the University of Strasbourg discusses computational approaches to library design, binding site determination, and predicting druggability. Fragment docking is extensively covered, along with a discussion of what factors contribute to success. It seems that docking is particularly good at identifying negatively charged, relatively buried fragments that make similar hydrogen bonds as the substrate. De novo ligand design, both the successes and challenges, is also covered.

Like last year’s book, all the chapters in this one are published online, but it is worth getting a bound copy as it is nicely put together, with color figures liberally integrated throughout rather than banished to plates at the back.

Molecular Medicine Tri-Con 2012

Molecular Medicine Tri-Con has just ended in San Francisco, and although it is one of those massive conferences with a very wide scope, from bioinformatics to stem cells, there were some talks relevant to FBLD.

Teddy recently summarized how fragment-based approaches have been used to develop bromodomain inhibitors at GlaxoSmithKline, and they’re certainly not alone: Mark Bunnage of Pfizer described how they used fragment-based methods to discover nanomolar inhibitors of BRD4 that are active in cells. This work was done in collaboration with the Structural Genomics Consortium (SGC), and one well-characterized compound, PFI-1, is being released as a chemical probe to the worldwide research community with no intellectual property entanglements.

There was a heavy emphasis on metrics, and as we saw in the poll last year ligand efficiency and LLE (sometimes also called LipE) seem to be dominant. However, the latter metric is used more for advanced leads than for fragments. Siegfried Reich from the Lilly Biotechnology Center (neé SGX) said of LLE that it is less important where you start than where you end up, though starting from a very polar fragment gives you the luxury of adding lipophilicity during optimization.

Slight changes to fragments can often cause them to bind in different orientations, and the same fragment may bind differently in closely related proteins, but Siegfried argued these multiple orientations could be advantageous by providing multiple opportunities for optimization. Siegfried also mentioned that deconstruction of HTS hits to fragments has been successful at identifying fragments with high ligand-efficiency that could subsequently be optimized to new series.

Richard Law of Evotec gave as clear account as possible of fragment molecular orbital (FMO) calculations, a high-level quantum mechanical method for understanding protein-ligand interactions. Although quantum mechanical calculations are notorious for taking hours, days, or even weeks to run, the calculations can be done much more efficiently by breaking larger molecules into fragments.

Richard also kept thorough notes at a break-out discussion I moderated, and was kind enough to share them; I’ve also made a few additions. There were nine people from both large and small organizations, all but one from industry.

Screening technologies

• As we’ve seen here, SPR seems to be the most common fragment-finding technique; in his presentation, Walter Huber said that it is the primary screening method at Roche.
• "Reverse SPR" - the Graffinity/Novalix technology we’ve discussed previously, has been applied to over 100 targets. It is reversed because the small molecules are immobilised on the chip, in multiple orientations to present different moieties to the target protein. This is also a larger library (~25,000).
• NMR was not being used as much as SPR, and no one at the table was using 19F NMR, though its use does appear to be growing, and compound suppliers are coming out with 19F fragment libraries.
• FCS++ is being used at Evotec; it has the advantage of being high-throughout but still highly sensitive and therefore accommodates a larger than average fragment library (~20,000).

3-D fragments

• There is increasing desire for sp3 (3-dimensional) fragments and a move away from planar fragments, though one participant had seen chemists shy away from too many chiral centers.
• Advantage of more vectors for SBDD, and additional solubility versus otherwise equivalent flat compounds.
• Despite these advantages, often planar fragments yield more hits - likely because planar compounds are more likely to form dispersive/non-specific interactions, whereas sp3-fragments must form very specific H-bond interactions in order to bind. Does sp3 therefore also enrich for enthalpic binders?

Use of indices
LE was used by everyone at the table, whereas LipE/LLE were not used until lead compound stage. BEI/SEI and other metrics were not really used.

FBLD vs HTS
Fragment screening is being used on most programs at many companies, in parallel with HTS and virtual screens. A subset of targets is addressed only with fragment screening either because of target-specific information or specific requirements to lower costs of screening.

Fragment screening of GPCRs
There was very limited experience using FBLD on G-protein coupled receptors, though one company is trying to use nanodiscs to stabilize GPCRs for fragment screening. As more crystal structures are solved people may become more comfortable tackling this target class.

Finally, there was widespread agreement that fragments are ideal for designing in the desired physico-chemical properties of molecules as fragments are developed. The parent fragment is not biasing, and could often be med-chemed away. Richard Law offered the analogy of a rock-climber:

An HTS gets you halfway up the cliff, but the route to the top may not be from where you are, or may just be too difficult to find from that position. Whereas a fragment hit is at the base of cliff but enables you to see and select the exact route to the top that you need.

If you attended the break-out discussion or the meeting please comment on your impressions, and if you missed this conference don’t worry – there are many more great events coming up throughout the year.

Upcoming Webinar on NMR in DD

I wanted to make everyone aware of an upcoming webinar: "You are smarter than you think: Applications of NMR in Hit-to-Lead Discovery". Here are the key learning points:

• Application of NMR in Fragment Screening using both target and ligand based methods
• Utilization of NMR Data, e.g. epitope mapping, in Hit to Lead efforts
• Structure determination of compounds by NMR

What I would love to have from the readers of this blog is suggestions as to what particular topics they feel would be most important to learn about. Obviously, the first two topics are broad and I wanted to give the readers here a chance to help set the specifics that will be discussed. Leave a comment or send me an email directly.

Slow-off, albeit tight, fragments

Practical Fragments recently discussed binding kinetics, and that got me wondering whether any fragments have slow off-rates. Turns out some do: a January 2012 review of protein-ligand energetics and kinetics in Drug Discovery Today by Sara Núñez and colleagues at Abbott summarized a paper published last October in Eur. J. Med. Chem. by Jos Lange et al. In it, Lange and colleagues extensively characterize six inhibitors of the enzyme D-amino acid oxidase (DAAO), a potential target for schizophrenia.

The researchers use biochemical assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to characterize the thermodynamics and kinetics of their inhibitors binding to DAAO. Although all six molecules are fragment-sized, these are not your typical fragments: the weakest has a Kd better than 1 micromolar, and all have ligand efficiencies of 0.79 kcal/mol/atom or better! Three of them are shown below, along with their dissociation constants (determined by ITC) and their dissociation rate constants (determined by SPR).

One interesting aspect of the kinetics is that compound 6 dissociates from the enzyme roughly 50-fold more slowly than compound 3, even though it binds only about 3-fold more tightly. As an interesting aside, compound 1 has a slower on-rate than any of the other molecules, a phenomenon the researchers attribute to tautomerization around the pyrazole.

The researchers go on to measure a number of other properties of these molecules, including Log P, pKa, thermodynamic and kinetic solubility, cell membrane permeability, and in vivo pharmacokinetics. There is a tremendous amount of data here, and it’s a lot of fun to dig into.

With a predicted half-life of more than 2 hours, compound 6 certainly classifies as a slow-off fragment. So is it a better drug? Well, it’s not orally bioavailable, in contrast to some of the other compounds, and it has faster clearance in rodents. Unfortunately the researchers do not report in vivo target modulation, but one has to assume that a schizophrenia drug would need to be oral. Chemists can optimize affinity, thermodynamics, kinetics, and drug-like properties all we want, but the body still has the final say.

Who's Regulating the Regulators?

In two recent ASAP papers in J. Med Chem. Chung et al. from GSK in Stevenage report on their recent efforts in discovering bromodomain inhibitors. Bromodomains are part of the "hot" target class largely lumped together in epigenetic targets. Bromodomains are the sole reader for Acetyl-lysine (AcK) and are thus important components of maintaining the "histone code". These domains are small (~110 aa) and have a common fold of four anti-parallel helices with the peptide recognition site in loops at one end of the helices. There are at least 56 bromodomains encoded in 42 proteins in the human genome.

The GSK group assembled a library that mimicked AcK: compounds had hydrogen bonding functionality and a small alkyl group. 1376 compounds were tested in a fluorescence anisotropy assay. Of these, 132 (~10%) showed >30% displacement of the fluorogenic ligand. After all actives were fully titrated, compounds were soaked into apo crystals. 40 structures were then analyzed by X-ray. The figure below shows how the native peptide is bound, analysis of the crystals showed that the H-bond interaction with the bridging water dominates that with N156, but both influence ligand positioning.
Below the structure of one bromodomain is shown, the yellow spheres are AcK sites. The other figure shows the preferred binding surface and low energy waters involved in AcK mimetic binding. These interacting residues tend to be conserved among bromodomains. The authors conclude the first paper by stating that their fragments are unlikely to discriminate between bromodomains. But, the real question is: is it possible to create compounds from these fragments which can?

And the answer is....[I did say there were two papers] maybe.

People have found compounds which work against bromodomains before (Cpds 1 and 2).
As can be seen they are relatively potent (nM) and decently ligand efficient. Cpd 3 and 4 are fragments found in the screen from GSK. Cpd 3 had ~30 % activity against two bromodomains. The authors admit it was far from the best fragment, but "it was small, efficient, novel, and chemically attractive." Who can argue with that.

Following up with this cpd, they found this compound binds in a manner consistent with its selection: one methyl of 3 mimics the terminal methyl of AcK, the second one overlaps the e-CH2 of AcK, and isoxazole N and O mimic the carbonyl. At 2A resolution, they couldn't differentiate which heteroatom was where, so they made 4 to prove their placement: it was.

As a start to the hit expansion, they used a 3D pharmacophore model to search for commerically avaailable analogs: analog by catalog. The found a series of phenyl-isoxazole with meta-sulfonamides on the phenyl ring. Compound 5a is ~100x more potent than the parent fragment and is very efficient (0.39). The xtal structure of 5a shows that it binds exactly as expected. SAR around this compound showed an increase in affinity, but with the expected loss of LE. This series also suffered from poor solubility. They could not add functionality to the sulfonamide (its pocket is rather hydrophobic), but instead where able to increase solubility through para-phenols (this points towards the solvent). They were able to increase solubility via this route, while keeping potency, but losing LE. This series also had cellular activity.
















This work shows that not all protein-protein interactions are flat and featureless. This work also shows that you can target PPI without having to change the rules: no need to relax the Lipinski Rules. These exciting new results show that the hot new targets in drug discovery play by the same rules as the same old targets.

Fragment linking: flexible rules

Linking two fragments together to achieve a boost in potency has been done a number of times (see here, here, here, and here), though it often doesn’t work as well as might be hoped (see here). To better understand the energetics of fragment-linking, Marc Nazaré, Hans Matter, and colleagues at Sanofi-Aventis Deutschland have analyzed ligands for the blood coagulation enzyme factor Xa (fXa) and published their results in a recent issue of Angew. Chem. Int. Ed.

The researchers “deconstructed” potent fXa inhibitors into component fragments, measured their inhibition constants (and thereby inferred their binding energies), and compared these binding energies with those of the original linked molecules. One of the first observations was that many of the component fragments bound so weakly as to show no measurable activity, a phenomenon that has been observed previously.

In an exemplary case, cleaving a single bond connecting the two component fragments of a 2 nM ligand (1a, below) yielded one fragment (1g) with 58 micromolar activity and another (1d) whose activity was worse than 10 millimolar. Because the second fragment has such low affinity, the binding energy of linking is really just a lower estimate, but it seems to be at least 3.3 kcal/mol, which is greater than the binding energy of fragment 1d itself. In other words, the affinity brought about by linking is greater than the affinity of the weakly binding fragment. The superadditivity provided by the linker in this case is about 300-fold, a similar value to that observed in the unrelated MMP-12 system. This is perhaps all the more remarkable given the fact that the fragments are connected by a linker containing several rotatable bonds, the entropy of which should partially counter the advantages of linking.

In fact, a common strategy to improve the potency of two linked fragments is to rigidify the linker. Often this doesn’t work: in a second case, the Sanofi-Aventis researchers cleaved one bond of a 3 nM ligand (2a, below) to yield two fragments with roughly equal potency. However, even though the linker is more rigid than in the previous example, the binding energy due to linking is less – just 2.0 kcal/mol, representing a boost of about 30-fold.

As the authors note:

The introduction of rigid aromatic moieties as a common approach to increase affinity does not necessarily maximize the benefit from the linker effect as detrimental affinity contributions might originate from suboptimal orientation and accommodation of specific binding elements.

There are many more examples in this paper than can be covered in a blog post; the authors dissect compounds 1a and 2a at a number of different points, and while the component fragments typically bind less tightly than simple additivity would suggest, there are lots of interesting details.

Finally, it is interesting to note that ligands 1a and 2a consist of a relatively hydrophobic fragment (1g or 2g) connected to a more polar fragment (1d or 2h). The fact that these show superadditivity is consistent with Mark Whittaker and colleagues' proposal last year that linking such fragments is likely to maximize additivity, although given the precise interactions made by both parts of the molecules the details get a bit messy. We’re not yet at the point where the universe of molecular interactions can be distilled to rigid rules.

Fragment events in 2012

2012

February 19-23: Molecular Medicine Tri-Con 2012 will be held in San Francisco, with a section on fragments on February 21.

March 13-14: Select Biosciences is holding its Discovery Chemistry Congress in Munich, Germany, with a full two days devoted to fragment-based lead discovery.

March 19-23: Keystone Symposium: Addressing the Challenges of Drug Discovery – Novel Targets, New Chemical Space and Emerging Approaches will be held in Tahoe City, CA. Although not exclusively devoted to fragments, there are many speakers I look forward to hearing.

March 22: Dr. Teddy Z will be giving a webinar on NMR in hit-to-lead discovery.

April 17-18: Cambridge Healthtech Institute’s Seventh Annual Fragment-Based Drug Discovery will be held in San Diego, with short courses on SPR and FBLD taking place on April 16. This is a chance to meet both editors of Practical Fragments! You can read impressions of last year’s meeting here and 2010’s here.

May 13-17: The 30th Noordwijkerhout-Camerino-Cyprus Symposium Trends in Drug Research will be held in Amsterdam, including a session on fragment-based drug discovery.

June 6-8: Cambridge Healthtech Institute’s Twelth Annual Structure-Based Drug Design will be held in Boston, with a session on FBLD.

August 19-23: The Fall 2012 ACS meeting will take place in Philadelphia, PA, and has at least one section on fragment-based drug discovery.

September 23-26: Finally, FBLD 2012, the fourth in an illustrious series of conferences, will be held in my fair city of San Francisco. This should be a biggy – the first such event in the Bay Area (and the weather in September is usually decent too). You can read impressions of FBLD 2010 and FBLD 2009.

2013

March 4-5: Fragments 2013, the 4th RSC-BMCS Fragment-based Drug Discovery meeting will be held at the Harwell Science and Innovation Campus near Oxford, UK.

Know of anything else? Organizing a fragment event? Let us know and we’ll get the word out.

Enthalpy Arrays

As regular readers know, there are lots of ways to find fragments, each with its own strengths and weaknesses. Isothermal titration calorimetry (ITC) is useful for being able to extract thermodynamic values from an experiment, but it tends to be low-throughput and is thus used more as a secondary rather than a primary assay. To make calorimetry more convenient, Michael Recht and colleagues at Palo Alto Research Center have constructed nanocalorimeters. They describe using these “enthalpy arrays” for fragment screening in a paper just published online in the Journal of Biomolecular Screening.

In a typical ITC experiment, a protein is mixed with a ligand and the tiny temperature change that occurs upon binding is detected. In the case of nanocalorimeters, up to 96 detectors are arranged on a plate, and sample volumes are typically a few hundred nanoliters. At this scale, the enthalpy of binding becomes very challenging to measure, but it is possible to measure the heat generated during the course of a reaction as an enzyme processes its substrate. This allows one to follow the reaction in real time without any dyes, labels, or artificial substrates. Also, since an inhibitor will change the reaction profile in a predictable manner, one can determine its mechanism of action.

Each detector in the enthalpy array is set up such that droplets are rapidly mixed together in sets of two. In the experimental set, one droplet contains the protein of interest while the other contains a substrate and a fragment. (Each detector also incorporates a control pair – one droplet containing the substrate/fragment mixture and another containing bovine serum albumin.) In the current paper, the researchers were looking for competitive inhibitors of PDE4A10, a phosphodiesterase implicated in inflammatory disorders. The protein was present at a final concentration of 5 micromolar, substrate was at 2 mM, and each fragment was present at up to 2 mM. 160 very small fragments (average molecular weight only 154 Da) were screened individually, resulting in 11 competitive hits with Ki < 2 mM; 2 other hits displayed more complex kinetics.

The 11 competitive hits were characterized in more detail; the most potent had a Ki of 0.32 mM and the most ligand efficient had LE = 0.43 kcal/mol-atom. In collaboration with Vicki Nienaber and colleagues at Zenobia, all 11 of these were taken into crystallography experiments. This proved challenging: unliganded PDE4A10 crystals suitable for fragment soaking could not be grown, necessitating more labor-intensive co-crystallography. Unfortunately, although some crystals were obtained, they did not diffract at high enough resolution to unambiguously fit the electron density of the fragments, though there was evidence for binding in the active site. The researchers were able to crystallize PDE4A10 with pentoxifylline, a known phosphodiesterase inhibitor. Since many of the fragments have structural features reminiscent of other phosphodiesterase inhibitors, this suggests starting points for modeling.

As described in this paper, enthalpy arrays could be used as a primary screen for fragment hits with defined modes of action before follow-up by slower methods. Although in this particular case crystallography was not successful determining co-crystal structures of the novel fragments, in a recent talk Michael described a related system which did yield good crystal structures. I look forward to seeing additional applications of this approach.

19F NMR...Just Like 1H NMR

Fluorine is a powerful nucleus for NMR: it has almost the same sensitivity as 1H and is 100% abundant. 19F NMR based screening is relatively new in the pharmaceutical industry. One of the biggest proponents of this approach has been Claudio Dalvit. In 2009, Dalvit and colleagues (J. Am. Chem. Soc. (2009) 131:12949) proposed the LEF (Local Environment of Fluorine) approach which allows for efficient screening of even weak binding fragments containing fluorine and the discovery of fluorophilic protein environments on the target. Despite the obvious advantages of 19F, it still has not been taken up widely in the industry. A new paper in J. Med. Chem. from Amgen may change that.

In this paper, they demonstrate multiple practical applications of 19F in FBDD, essentially demonstrating congruence for 19F-based methods with 1H-based methods. This is a particularly important as most people are conversant with how to understand and utilize 1H-based data; there is no "re-learning" necessary to adopt 19F based data. One potential obstacle to adoption is the argument that the 19F fragments would not be "fragment-y" or would be unlike other libraries and thus have little cross-fertilization. As shown in the table below, their 19F Fragment library is based on typical Ro3 rules with similar size and diversity.

As can be seen here, this library is relatively diverse. The authors argue that ~30% of "Fragment Space" is covered by this library. But, due to congruency with 1H molecules, this is more than sufficient to give adequate screening hits.The paper demonstrates the utility of this approach on BACE (one of everybody's favorite targets). In less than 24 hours they were able to screen the entire library and identified five compounds (out of 6 hits) for follow-up. They introduce a method for Kd determination by NMR which cannot be done by 1H-based methods, differential Chemical Shift Perturbation: which utilizes changes in the 19F chemical shifts to determine the Kd. In cases where chemical shifts are not observed, the authors state you can use differences in line broadening as the measurement. This is an interesting and novel approach to Kd determination and they validated this approach by using SPR in parallel. The authors also demonstrate that the Holy Grail of FBDD (simultaneous binding fragments) can be identified and oriented based upon 19F-19F iLOE, exactly analogous to 1H-1H NOEs.

This is an excellent paper, that I hope sparks more people to investigate the utility of 19F-based screening and post-screening confirmation.