25 July 2016

Multiple bromodomains, multiple methods, and even more fragment hits

All this month Practical Fragments has been focused on bromodomains, highlighting chemical probes against BRD9, CBP and EP300, and family VIII bromodomains. Today’s post covers three earlier-stage programs on three different bromodomains.

In Acta Pharm. Sinica, Bing Xiong, Nai-xia Zhang, and colleagues at the Chinese Academy of Sciences discuss their work on BRD4, an anti-cancer target about which we’ve written previously. The researchers describe the construction of a fragment library designed for NMR screening; this is a good resource for people undertaking similar efforts. Interestingly, of 800 compounds purchased, only 539 were soluble to at least 100 µM in aqueous buffer. These were pooled into 56 groups of 8-10 compounds and screened at 200 µM (total fragments) using STD and T1ρ. This yielded 10 hits, of which three had measurable IC50 values from 110 to 440 µM. Five of the hits were characterized in more detail using two dimensional NMR (1H-15N HSQC), and three by X-ray crystallography. Some of these fragments are less-precedented as bromodomain ligands, and could be useful starting points for further work.

In contrast to BRD4, for which multiple ligands have been reported, the bromodomain on BRPF1 is less explored. In a recent paper in J. Med. Chem., Jian Zhu and Amedeo Caflisch (University of Zürich) provide 20 new co-crystal structures, all of which have been deposited in the protein data bank. The researchers performed a computational screen of 24,133 molecules using a program called SEED, which was able to crank through the entire set in just a day. Crystal soaking was attempted with thirteen of the top 30 hits, resulting in five structures, of which three bound in the manner predicted. Crystal structures of another 15 analogs and other bromodomain inhibitors were also determined. Some of the molecules are reasonably potent, with double-digit micromolar affinities and good ligand efficiencies.

Finally, while most bromodomains have a conserved asparagine residue that makes hydrogen bonds to the substrate (or inhibitor), 13 of the 61 known human bromodomains do not, and these tend to be more difficult targets. The second bromodomain of the pleckstrin homology domain-interacting protein (PHIP(2)), which has been implicated in melanoma, is one of these “atypical” bromodomains. Researchers at the Structural Genomics Consortium (SGC) led by Frank von Delft (Diamond Light Source) and Paul Brennan (University of Oxford) took a crystallography-first approach toward this target, as they report in an open-access paper in Chemical Science.

The researchers started by assembling what they call a “poised fragment library”. This is essentially a library designed for rapid follow-up chemistry, in which each library member can be deconstructed into individual components, which can be systematically varied. For example, a fragment might consist of two moieties connected by an amide bond, so that analogs could be easily made using parallel synthesis. The initial 2347 fragments were a subset of the 11,677 fragments available in-house or through collaborators, but the researchers also identify a set of 10,448 commercially available poised fragments. Commendably, they also provide full identities of both sets of fragments, which could be useful for folks building or adding to their own collections.

The Diamond Light Source is able to crystallographically screen 1000 fragments per week, but in this case only 406 diverse fragments were tested. Rather than using the nearly universal DMSO as a solvent, the researchers dissolved their fragments in ethylene glycol, since DMSO actually binds to bromodomains. Previous solution-phase screens of PHIP(2) at the SGC had come up empty, so the crystallographic screen was done at the very high concentration of 200 mM. Not surprisingly, this yielded just four hits.

Each of the hits bound in the acetyl-lysine recognition pocket, and three of them even showed high-micromolar activity in an AlphaScreen assay, with impressive ligand efficiency values. A few dozen analogs were made, which led to slight increases in activity in all cases, and measurable activity for analogs of the fragment which had shown no activity by itself. Although there is still a long way to go to find chemical probes for PHIP(2), at least there are now good starting points.

And that concludes bromodomain month. The number of papers and chemical probes that have come out just this year are a testament to the power of fragments to tackle this class of targets, perhaps equaled only by kinases. And while I'm not aware of any clinical candidates targeting bromodomains that started as fragments, I'm sure these will be coming soon.

20 July 2016

Fragments deliver a chemical probe for Family VIII bromodomains

Today’s post continues the theme of July as bromodomain month at Practical Fragments. The 61 human bromodomains (found in 46 proteins – some proteins have more than one) have been divided into eight families based on their sequences. Family VIII contains ten members, some of which are involved in keeping stem cells from differentiating. Two papers describe chemical probes that target some or most members of this family.

The first paper, which actually came out last year in Science Advances, is from a multinational group including Thomas Günther (Universität Freiburg), Stefan Knapp and Susanne Müller (both University of Oxford) and collaborators at Pfizer. The researchers started by screening libraries of acetyl lysine mimetics that had yielded inhibitors against other bromodomains. These came up empty; even promiscuous bromodomain inhibitors failed to hit Family VIII members. As is so often the case, when all else fails, the researchers turned to fragments. A thermal shift assay revealed that salicylic acid – the polypharmacological metabolite of aspirin – binds to the bromodomain PB1(5). Isothermal titration calorimetry (ITC) confirmed this result, providing a dissociation constant of 250 µM.

The researchers were also able to obtain a crystal structure of PB1(5) bound to salicylic acid in the acetyl lysine binding site common to all bromodomains, with the carbonyl making the usual hydrogen bond with a conserved asparagine. But whereas most other bromodomain binders make a water-mediated bridge to a conserved tyrosine, the phenol makes a direct hydrogen bond. The benzene ring also binds deeper in the pocket, displacing four highly conserved water molecules.

The subsequent medicinal chemistry optimization of this fragment is described in a paper published earlier this year in J. Med. Chem. by Dafydd Owen and colleagues at Pfizer, along with collaborators at the University of Oxford, DiscoveRx, Eurofins, the University of Massachusetts Worcester, and Johann Wolfgang Goethe University. Testing commercial and proprietary analogs of salicylic acid quickly revealed that uncharged enamides such as compound 2 were more effective at stabilizing PB1(5) against thermal denaturation than salicylic acid, and crystallography confirmed a similar binding mode.


Two rounds of library synthesis were conducted, first with 130 amines and then with 320 amines, with physicochemical properties of target compounds chosen in advance such that cLogP would range between 1 and 4. Seven family VIII bromodomains were screened in parallel, and compounds were identified with differing specificities. Some of the compounds were unstable in water, but introducing steric hindrance around the amine improved stability and led to compounds such as PFI-3. This is potent against the family VIII bromodomains PB1(5), SMARCA2A, and SMARCA4 and did not hit at least 40 other bromodomains tested. A related compound is active against more of the family VIII bromodomains while still maintaining good selectivity against other bromodomains.

Both of these probes are able to bind to family VIII bromodomains in cells and were used to explore the proteins’ biological roles. A variety of cellular phenotypic assays showed minimal changes, and the compounds do not appear to be toxic. They did attenuate myocyte or adipocyte differentiation, while PFI-3 caused embryonic stem cells to differentiate. One gets the impression that the researchers were hoping for more profound effects, but that’s why you make chemical probes in the first place. Whether or not these compounds will ultimately prove useful as drug leads, they should help to unravel some fiendishly complex biology.

15 July 2016

Fragments in the clinic: 2016 edition

There’s a new FBDD review out today in Nat. Rev. Drug Discovery. I know - there are lots of reviews each year - but this one is written by a who's who list of luminaries, including Steve Fesik (Vanderbilt), Rod Hubbard (Vernalis and University of York),  Wolfgang Jahnke (Novartis), and Harren Jhoti (Astex). I'm also an author so I'm undoubtedly biased, but I think it provides a nice overview of the field, especially for those who don't have time to read the recent book.

The review distills hard-won wisdom from two decades of work and covers practical decisions needed when using fragments: library design, screening methods, protein-ligand interactions, hit to lead strategies, and applications. Another useful feature is what I believe to be the most complete and up-to-date list of fragment-derived drugs that have entered clinical development. Where possible these include chemical structures, so definitely check it out.

The drugs themselves are listed below. Although it has not even been two years since the last compilation, it is exciting to see several promotions and new entrants. This table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). A few compounds from earlier lists have been removed because their fragment origins could not be confirmed. Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post.


Drug Company Target
Approved!

Vemurafenib Plexxikon B-Raf(V600E)
Venetoclax AbbVie/Genentech Selective Bcl-2
Phase 3

PLX3397 Plexxikon FMS, KIT, and FLT-3-ITD
Verubecestat Merck BACE1
AZD3293 AstraZeneca/Astex/Lilly BACE1
Phase 2

AT7519 Astex CDK1,2,4,5,9
AT9283  Astex Aurora, JAK2
AZD5363 AstraZeneca/Astex/CR-UK AKT
Erdafitinib J&J/Astex FGFR1-4
Indeglitazar Plexxikon pan-PPAR agonist
LY2886721 Lilly BACE1
LY517717 Lilly/Protherics FXa
Navitoclax (ABT-263) Abbott Bcl-2/Bcl-xL
NVP-AUY922 Vernalis/Novartis HSP90
Onalespib Astex HSP90
Phase 1

ABL001 Novartis BCR-ABL
ABT-518AbbottMMP-2 & 9
ABT-737AbbottBcl-2/Bcl-xL
ASTX660 Astex XIAP/cIAP1
AT13148AstexAKT, p70S6K, ROCK
AZD3839AstraZenecaBACE1
AZD5099AstraZenecaBacterial topoisomerase II
BCL201 Vernalis/Servier/Roche BCL-2
DG-051deCODELTA4H
IC-776Lilly/ICOSLFA-1
LP-261LocusTubulin
LY2811376LillyBACE1
PF06650833 Pfizer IRAK4
PLX5568Plexxikonkinase
SGX-393SGXBCR-ABL
SGX-523SGXMet
SNS-314SunesisAurora

The current list contains more than 30 clinical-stage drugs but is certainly incomplete, particularly in Phase I. If you know of any others (and can mention them) please leave a comment.

11 July 2016

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.

05 July 2016

Fragments deliver a chemical probe for BRD9

Bromodomains have nothing to do with bromine. Rather, they are small (~110 amino acid) domains that recognize acetylated lysine residues, a common modification on histones, and are thus key epigenetic “readers”. Humans have more than 60 of them, and as you can imagine selectivity is not assured. However, fragments have proven very useful in targeting these proteins. Since the first mention of bromodomains on Practical Fragments back in 2011 the number of posts has been growing rapidly, so for the first time ever we’ve decided to devote an entire month to the topic.

In other words, July is bromodomain month! We’ll start with two papers against the bromodomain BRD9, part of the SWI/SNF chromatin remodeling complex that seems to be important for acute myeloid leukemia.

The first paper, in J. Med. Chem. (and open access), is published by Laetitia Martin and collaborators at Boehringer Ingelheim, University of Oxford, and Cold Spring Harbor. The researchers used three orthogonal biophysical screening methods: differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). A library of 1697 fragments was screened at 0.4 mM (DSF), 0.1 mM (SPR) or 0.5 mM (MST), and hits were then validated using 15N HSQC NMR. The 77 hits that confirmed were taken into crystallography, producing 55 structures.

Validation rates in the NMR secondary screen were excellent for DSF (94%) and SPR (84%) but less so for MST (31%). That said, of the 38 validated hits from MST, 29 were not found in either of the other techniques, and 14 of these produced crystal structures. This is a useful reminder that while screening cascades can whittle down many hits, they do run the risk of throwing out the proverbial babies along with the bathwater.

In parallel with the biophysical screens, a virtual screen of ~73,500 fragments was conducted using Glide to identify 208 fragments that were then tested using SPR and DSF. This led to 23 hits, 11 of which produced crystal structures.

Two of the more potent fragments were the structurally related compound 3 (from the biophysical screen) and compound 4 (from the virtual screen). Optimization started with compound 4 by adding electron donating groups to the phenyl ring to try to improve a stacking interaction observed in the crystal structure. This led to compound 10, and building out the other ring to make it more similar to fragment 3 led to BI-9564.


BI-9564 has low nanomolar activity in both a biochemical assay as well as isothermal titration calorimetry (ITC). It is also quite selective: among 48 other bromodomains, it only hits the closely related BRD7 and CECR, and it is >10-fold more potent on BRD9. None of a panel of 321 kinases were inhibited with IC50 < 5 µM, and only 2 of 55 GPCRs were inhibited. The compound is also cell active, reasonably soluble, has good pharmacokinetics in mice, and orally bioavailable. In short, BI-9564 is an excellent chemical probe – and is in fact being offered as such.

While we’re on the subject of BRD7 and BRD9, it’s worth noting another recent paper, this one in ChemBioChem from Ke Ruan and colleagues at the University of Science and Technology of China. The researchers screened their library of 890 fragments against BRD7 using three different ligand-detected NMR techniques: STD, WaterLOGSY, and CPMG. Fragments were screened in pools of 10 with each fragment present at 400 µM. This yielded just 10 hits, of which 5 confirmed when tested individually. Protein-observed NMR was then performed on these, suggesting that they all bind in the acetyl-lysine recognition sites; they have similar affinities for both BRD7 and BRD9, with dissociation constants between 22 and 600 µM. Crystallography confirmed the binding mode for one of the fragments bound to BRD9. Interestingly, this showed quite a bit of plasticity in the protein compared to the un-liganded structure. Indeed, the BI researchers suggest that different degrees of protein flexibility between BRD7 and BRD9 could account for the selectivity differences observed for BI-9564.

Stay tuned next week for more fragment-screening against a different class of bromodomains!

27 June 2016

Fragments vs Lp-PLA2 – less greasily

Human lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme involved in lipid metabolism that is implicated in multiple diseases, from atherosclerosis to Alzheimer’s. Because the natural substrates are lipophilic phospholipids, it is no surprise that reported inhibitors are also large and hydrophobic. A case in point is darapladib: with a molecular weight of 667 Da and a clogP of 8.3 this is a poster child for molecular obesity – and it also failed in phase 3 clinical trials. A new paper in J. Med. Chem. by Alison Woolford (Astex), Joseph Pero (GlaxoSmithKline) and colleagues describes an effort to discover less lipophilic inhibitors.

The researchers performed a screen of 1360 fragments using thermal shift and ligand-detected NMR and a smaller screen of 150 fragments using crystallography. This yielded 34 fragments that were ultimately characterized crystallographically; screening commercial and in-house collections for related fragments yielded another 16. Interestingly, rather than clustering at a single hot spot, these fragments bound to different regions of the extended active site, with some – such as fragment 6 – a full 13 Å from the catalytic center. This is reminiscent of a fragment campaign against soluble epoxide hydrolase, another enzyme with a long, hydrophobic active site.

In addition to binding in an interesting site, fragment 6 also has good affinity and ligand efficiency. Moreover, its binding site overlaps partly with that of fragment 5. Thus, the researchers merged the two fragments together, resulting in compound 7, with submicromolar activity.

Further structure-guided optimization, which included growing into a polar region of the protein, ultimately led to compound 16, with low nanomolar potency.





Compound 16 has a molecular weight of 411 Da and a clogP of 3.4 and is correspondingly reasonably soluble (> 0.3 mM). Whereas darapladib showed a dramatic 700-fold potency decrease upon addition of human plasma – presumably due to nonspecific binding to other proteins – the decrease in potency for compound 16 is only 13-fold. Indeed, though darapladib is a picomolar binder, compound 16 is slightly more potent in plasma.

Unfortunately, compounds in this series turned out to have high clearance in rats, proving once again that lead optimization is often a frustrating game of whack a mole. Still, the fact that the researchers were able to develop smaller, more soluble inhibitors of an enzyme with such a lipophilic substrate gives hope that the game is perhaps winnable.

20 June 2016

19F-NMR-guided fragment linking on BACE1

Fragment growing has been the dominant strategy of most of the recent posts involving lead optimization, consistent with our poll results. However, fragment linking can be powerful too, as illustrated by the recent approval of venetoclax, which was derived from fragment linking. A recent paper in J. Med. Chem. by Brad Jordan and colleagues at Amgen provides another nice case study.

Amgen researchers had previously used fragment growing to discover inhibitors of BACE1, an Alzheimer’s target which has been heavily tackled by fragments. However, the most potent molecules in the series also inhibited the related aspartic protease cathepsin D (CatD), which could cause serious side effects. The researchers sought to gain selectivity by building inhibitors to occupy the so-called S3subpocket of BACE1. To do so, they used 19F-NMR to find fragments that would bind to BACE1 in the presence of a “blocking compound” that filled most of the active site but not the S3subpocket. This led to the discovery of seven fragments, the most potent being compound 3. Interestingly, this fragment only bound in the presence of the blocking compound as assessed both by NMR and SPR. Also, it could be competed by a compound that binds in the S3subocket.


Having thus identified a fragment that bound in the presence of one of their inhibitors, the researchers used interligand NOE (ILOE) to determine how the two compounds bind relative to one another. This supported the idea that compound 3 binds in the S3subpocket, and also suggested how the fragment could be linked onto the existing lead series, exemplified by compound 5. Just four compounds were designed and synthesized, and all of them were more potent than either of the starting points, with compound 9 being the best. More importantly, this compound also proved to be ~2000-fold selective for BACE1 over CatD in enzymatic and cell-based assays.

Despite the excellent (high picomolar) affinity of compound 9 for BACE1, this is actually about 25-fold worse than would be predicted by a simplistic additivity of binding energies – a not uncommon occurrence when linking molecules. Still, with its combined used of multiple NMR techniques and structure-based design to solve a specificity challenge, this paper is worth perusing.

15 June 2016

Covalent fragments writ large

We’ve written previously about irreversible covalent fragment-based lead discovery. The nice thing about irreversible inhibitors is that they have an infinite no off-rate: once they bind and react with a target, that protein is permanently out of action. A paper published today in Nature by Keriann Backus, Benjamin Cravatt, and colleagues at Scripps Research Institute takes this approach to a whole new level.

The researchers assembled a library of just over 50 fragments containing cysteine-reactive electrophiles, such as chloroacetamides and acrylamides; the average molecular weight was 284 Da. These were then screened against human cells or cell lysates using a proteomic approach called isotopic tandem orthogonal proteolysis-activity based protein profiling (isoTOP-ABPP). This technique, previously developed by the Cravatt laboratory, uses mass spectrometry to differentiate contents of treated and untreated cells and identify specific regions of proteins that are modified.

In all, 758 cysteine residues in 637 different proteins were found to be modified by at least one of the fragments. These included targets (such as BTK) with known covalent drugs as well as many proteins with no small molecule inhibitors. Even more exciting, this set included some particularly challenging classes of proteins, such as transcription factors and various adapter and scaffolding proteins. Most proteins only had a single modified cysteine, and these were not necessarily in the active site (see also here). Happily, computational docking did a good job of (retrospectively) predicting the modified cysteine residues.

The fragments themselves ranged significantly in how many cysteines they modified, from < 0.1% to > 15%, with a median of 3.8%. Interestingly, the correlation with intrinsic electrophilicity – as measured by reaction with the small molecule thiol glutathione – was fairly weak. This suggests that the fragments are modifying proteins based on other properties, such as specific interactions between fragment and protein.

The initial studies were done using cell lysates at high (500 µM) fragment concentrations. Follow-up studies in whole cells using 50-200 µM fragment gave similar results, with 64% of the cysteines from the lysate experiments reacting with the same fragments in cells, even at the lower concentrations. Interestingly though, four fragment-cysteine interactions were found only in cells and not in lysates.

One class of proteins you might expect reactive fragments to hit are cysteine proteases, such as the caspases, and indeed one chloroacetamide-containing fragment reacted with the active site cysteine of caspase-8 (CASP8). Surprisingly though, this fragment showed only marginal activity in an inhibition assay, and subsequent experiments revealed that it is selective for the inactive zymogen (or proenzyme) form of the protein, thereby preventing activation. This fragment does not react with the related caspases 2, 3, 6, or 9, though it does hit CASP10. Modest modifications led to a compound that was also selective for CASP8 over CASP10. These two molecules were used to show that both CASP8 and CASP10 appear to be essential for extrinsic apoptosis in primary human T cells, but not in the immortalized Jurkat T-cell line.

Of course, it will be essential to rigorously characterize any covalent molecules used as probes. Chloroacetamides are well-known electrophiles – so well known in fact that they are generally excluded from screening libraries, including those that helped define the original PAINS filters. A single digit percentage hit rate means that any given covalent fragment could easily hit hundreds of proteins. The researchers here do careful control experiments – such as using an inactive enantiomer and extensive proteomic analyses – but someone less careful could easily mislead themselves and others. Done rigorously, though, this is an exciting approach that may well increase the number of ligandable targets.

13 June 2016

Fragments vs MetAP2: reversible inhibitors

Methionine aminopeptidases, or MetAPs, cleave the N-terminal methionine residue from newly translated proteins. The human enzyme MetAP2 is a potential target for obesity, as demonstrated by the impressive clinical results of beloranib. But this drug hasn't been approved, and patients have died while taking it. Beloranib is an irreversible inhibitor that may also hit other targets, so researchers at Takeda California have been seeking non-covalent inhibitors. They report their results in two recent papers in Bioorg. Med. Chem. Lett.

In the first paper, Zacharia Cheruvallath and colleagues describe a biochemical fragment screen of ~5000 fragments (11-19 non-hydrogen atoms) conducted at 0.1 mM. This produced an impressive number of hits (110 compounds with > 20 % inhibition), which were triaged based on both ligand efficiency and LLE, ultimately yielding 16 interesting fragments. In particular, fragment 6 is remarkably potent.
Crystallography was not successful for any of the fragments. Undeterred, the researchers performed classic “SAR by catalog” (and corporate collection) to develop a binding model. This quickly revealed that the hydroxyl group was unnecessary. It also suggested that one of the indazole nitrogen atoms might be interacting with an active site metal ion, and that the bromine might be pointing towards a hydrophobic pocket where the side chain of the methionine substrate normally binds. Growing led to compound 16, and a closely related compound was characterized crystallographically bound to the protein, confirming the model. Further optimization led to compound 38, with low nanomolar potency in both biochemical and cell-based assays, excellent selectivity against a panel of >100 other targets, good oral bioavailability, and reasonable pharmacokinetics. This compound caused dose-dependent weight loss in a mouse model of obesity.

The second paper, by Christopher McBride and colleagues, involved more dramatic changes to the fragment. The indazole 4 is very potent, but indazoles are quite common in the literature, so the researchers sought to scaffold-hop to a novel core. This led them to design compound 6’, and using some of the SAR from the previous series ultimately led to compound 10. As with compound 38 above, this compound showed good cell-based activity, acceptable pharmacokinetics, oral bioavailability, and a clean profile against > 100 off-targets at 10 µM. It also showed measurable weight loss in a rodent model of obesity.

The question sometimes arises as to how many fragment hits are necessary for a program to move forward. These two papers show that a single fragment can be elaborated to two very different lead series with animal efficacy. In contrast to some of our recent posts, these efforts did not initially require crystallography. There are many ways to advance fragments, and no single technique is essential.

06 June 2016

Fragments vs Dengue virus polymerase

Dengue fever, evocatively called “breakbone fever” for the severe pain it can inflict, is caused by a mosquito-borne virus that infects hundreds of millions of people each year. There are no approved antiviral treatments. Two papers from researchers at the Novartis Institute for Tropical Diseases and the University of Texas Galveston provide some promising early leads.

The first, in J. Biol. Chem., by Christian Noble, Pei-Yong Shi, and collaborators, describes a crystallographic screen of 1408 fragments against Dengue virus RNA-dependent RNA polymerase (DENV RdRp), which is highly conserved among the four serotypes of Dengue virus. Crystals were soaked in pools of eight fragments, with each present at only 0.625 mM, ten to one hundred times lower than other recent crystallographic screens. Perhaps because of this low concentration, only a single hit was identified – compound JF-31-MG46. The crystal structure revealed that the molecule binds in the “palm subdomain” of the protein, which is analogous to a druggable site on the hepatitis C virus protein.

Surface plasmon resonance (SPR) showed that this fragment had a dissociation constant of 0.21 mM against RdRp from serotype 3 and 0.61 mM against RdRp from serotype 4, suggesting weak but real binding. Isothermal titration calorimetry (ITC) was not successful, perhaps because of compound solubility, but replacing the terminal phenyl group with a thiophene led to more potent compounds which could be characterized both by SPR and ITC. The compounds were also active in an enzymatic assay, with IC50 values comparable to their affinities.


The second paper, by Fumiaki Yokokawa and collaborators and published in J. Med. Chem., describes the optimization of these fragments. Fragment growing was performed to try to displace a bound water molecule, resulting in the low micromolar compound 17. Compounds that contain carboxylic acids often have low cell permeability, so several bioiosteres were tested to try to replace this moiety, and compound 23 showed increased affinity. However, this compound was still quite polar, showed poor permeability, and no cell activity. Adding a lipophilic substituent and decreasing the acidity led to compound 27, with nanomolar affinity and enzymatic inhibition of all four Dengue virus serotypes. Importantly, this compound also showed low micromolar activity against all four serotypes in cell assays.

The J. Med. Chem. paper notes that a high-throughput screen against RdRp had been plagued with false positives. One validated low micromolar hit was optimized to nanomolar potency, but this was very lipophilic and displayed no cell activity. It is interesting that the fragment-derived leads initially displayed no cell activity for the opposite reason: they were too polar. This is a useful reminder that physicochemical properties matter. The successful optimization of the fragment-derived series suggests that it can be easier to make leads more lipophilic than less.

01 June 2016

Fragment library vendors - 2016 version

It's been two years since we last updated our list of commercial fragment libraries, and there have been several changes. The prompt for updating the list is a new Perspective published in J. Med. Chem. by György M. Keserű & György G. Ferenczy (Hungarian Academy of Sciences), Mike Hann & Stephen Pickett (GlaxoSmithKline), Chris Murray (Astex), and me. This covers all aspects of fragment library design, so definitely check it out.

One table in the Perspective compares various libraries, both commercial and proprietary. One of the manuscript reviewers asked if we could evaluate the various vendors, particularly given some negative experiences with commercial compounds. Such direct criticism (and praise!) can be awkward in the peer-reviewed literature, but is more acceptable in an online forum - think of Yelp for library suppliers. Please comment (anonymously if desired) if you've had experiences, positive or negative, with these vendors, and please feel free to add any we omitted.

Note that this list only includes companies that sell their libraries (as opposed to just using them internally).

ACB Blocks: 1280 compounds, 19F NMR-oriented, RO3 compliant, predicted to be soluble, purity >96%

Analyticon: 213 compounds, fragments from nature, RO3 compliant, high solubility, purity >95%

Asinex: >22,000 compounds

ChemBridge: >7000 compounds, RO3 compliant with predicted solubility; minimum purity 90% by 1H NMR

ChemDiv: >4000 3D fragments

Enamine: Multiple subsets including >18,000 RO3 compliant, ~1800 "Golden", and >126,000 with < 20 heavy atoms. Also separate fluorinated, brominated, sp3-rich, and covalent subsets.

InFarmatik: 1700 member consolidated library with different subsets (3D, GPCR, kinase)

IOTA: 1500 diverse, mainly RO3 compliant fragments

Integrex: 1500 compounds with diversity in shape and chemical structure, RO3 allowing one violation

Key Organics: ~26,000 compounds total with multiple subsets including 1166 with assured solubility and RO3 compliant as well as brominated, fluorinated, and CNS-directed fragments

Life Chemicals: 31,000 fragments of which 14,000 are RO3 compliant; also fluorinated, brominated, covalent, Fsp3-enriched, and covalent subsets

Maybridge: >30,000 fragments in total. The 2500 Diversity collection is guranteed soluble at 200 mM in DMSO and 1 mM in PBS.  NMR spectra are available (in organic solvent). It is available in many formats, from powder to DMSO-d6 solution. A smaller 1000-fragment subset is also available.

Otava: >12,000 fragments with various subsets including fluorinated, brominated, and metal-chelating

Prestwick: 910 mainly derived from drugs, RO3 compliant

Timtec: 3200 compounds, structurally diverse with predicted high solubility

Vitas-M: ~19,000 fragments, RO3 compliant

Zenobia:  968 fragments from different design paradigms, cores from drugs, higher Fsp3, flexible cores

30 May 2016

Fragments in Texas

The meeting Development of Novel Therapies through Fragment Based Drug Discovery was held last week in Houston, Texas, organized by the Gulf Coast Consortium for Quantitative Biomedical Sciences. Although it was only a single day, it was packed, with thirteen speakers, a couple vendor lunch talks, and some two dozen posters. Below is just a flavor – please add your own impressions if you were there.

I kicked off the first session by giving an overview of FBDD, highlighting both pitfalls and successes. Beth Knapp-Reed (GlaxoSmithKline) then discussed efforts against LDHA, a target previously tackled successfully using fragment linking (see here and here). In this case, an HTS screen of 1.9 million compounds produced only a single hit that resulted in a crystal structure, while fragment screens yielded 16 structures at three binding sites. An NMR-based functional screen (using 13C-labeled substrate) was key to obtaining robust SAR, and using information from both the HTS hit and the fragments ultimately led to nanomolar inhibitors. Next, Tom Davies provided an overview of Astex’s discovery platform, focusing on a success with KEAP1. We recently highlighted research suggesting that crystallography should be used as a primary screen, which Astex does for some targets. Tom noted that doing so currently takes about a month, though only after spending somewhere between 3 and 12 months establishing a robust protein construct as well as crystallization and soaking conditions.

Jane Withka opened the next session by discussing the continuing evolution and use of the Pfizer fragment library. Out of 32 targets screened, only one produced no hits, and this was a particularly flexible protein. Interestingly, despite being relatively balanced among basic, acidic, and neutral members, hits were strong enriched in neutral compounds and strongly depleted in basic fragments. Neutral fragments were exactly what was sought by Daniel Cheney (Bristol-Meyers Squibb), who discussed successful efforts to replace a basic amidine moiety in Factor VIIa inhibitors. And Brad Jordan (Amgen) discussed the successful application of 19F NMR screening to find fragments that could be linked to previously discovered inhibitors to obtain selective picomolar inhibitors of BACE1.

Alex Waterson (Vanderbilt) started the first afternoon session by discussing how fragments had been successfully applied to RPA, RAS, and MCL-1. In the last case, the best compounds now have dissociation constants in the picomolar range, are active in cells, and show activity in xenograft models. IND-enabling studies are slated to begin as early as this year, with the hope of developing a cousin of venetoclax. Inna Krieger (Texas A&M) described how fragments could be used to understand the mechanism of M. tuberculosis malate synthase, while Dawn George (AbbVie) described selective (but inexplicably toxic) PKCθ inhibitors, which are now being made available to researchers to probe the biology. Finally, Damian Young (Baylor) gave an update on his sp3-carbon enriched fragments. Jane had mentioned that following up on hits with multiple stereocenters was not always easy, but Damian’s DOS approach efficiently and systematically yields each possibility. Whether these will meet the Safran-Zunft challenge remains to be seen.

The last session was focused on success stories. Michael Mesleh (Broad Institute) discussed Cubist’s bacterial DNA gyrase inhibitors. Marion Lanier (Takeda) described how fragment screening and careful medicinal chemistry led to a low nanomolar, selective inhibitor of BTK. With a molecular weight of just 318, the molecule is scarcely larger than a Texas fragment, and has good pharmacokinetics and activity in a rat arthritis model. And Yi Liu (Kura) discussed the optimization of covalent KRAS inhibitors originally discovered using Tethering.

This was my first visit to Houston, and I was struck by the number of researchers who had relocated from around the world, particularly from (previously) large(r) pharma companies. Whenever scientists meet the talk often turns to funding shortages, but not here: everyone seemed to have plenty of money and resources, and one of the organizers announced that he was trying to fill several positions. This was the first major fragment meeting in Texas but likely not the last – there is talk of turning it into a recurring event. And there are still several good upcoming events this year; early registration for FBLD 2016 closes in just a few weeks.

23 May 2016

Calculating hotspots in detail

In the eight years since Practical Fragments first started, Moore’s law has held strong and computational power has increased accordingly. Last year we described how tools such as FTMap can be used to identify hot spots – regions on proteins where fragments are most likely to bind. Although FTMap is quite successful at identifying these, it is less able to point to specific interactions (such as hydrogen bond donors or acceptors) that are likely to drive binding. In other words, computational chemists have become adept at identifying where fragments might bind but lag in predicting how. A new paper in J. Med. Chem. by Chris Radoux at the Cambridge Crystallographic Data Centre and collaborators at UCB and the University of Cambridge addresses this challenge.

The approach starts with a set of three simple molecular probes: toluene, to look for hydrophobic interactions; aniline, to look for hydrogen bond acceptors; and cyclohexa-2,5-dien-1-one, to look for hydrogen bond donors. These probes are larger than those (such as ethanol) used in many other programs, the idea being that too-small molecules might find hot spots so small as to be useless. Indeed, with 7 non-hydrogen atoms, these probes are near the low end of the consensus size for fragments.

Calculations are performed on protein structures – either with no ligand bound or with a bound ligand computationally removed – to determine whether each surface atom of the protein is a hydrogen bond donor, acceptor, or hydrophobic, as well as how exposed the particular atom is. The three probes are then mapped onto the proteins to look for favorable interactions. Regions where multiple probes can bind are scored higher, with hotspots defined as those regions of the protein having the highest scores. The type of probe with the highest score also describes what type of interactions are likely to be favorable at various regions within a given hot spot. Although the researchers note that multiple software packages could be used for these calculations, they used a program called SuperStar, and calculations took just a few minutes on an ordinary laptop.

To validate the approach, the researchers used a previously published data set (discussed here) of 21 fragment-to-lead pairs against a variety of proteins for which crystal structures and binding affinities were available. In general, the method was able to identify the fragment binding site quite effectively; the one outright failure was on the fragment with the lowest affinity, which also had poorly resolved electron density in the crystal structure. Importantly, the fragments tended to have the highest scores, with added portions of the leads scoring lower. This data set was used to calibrate the scoring system for identifying hot spots, as well as specific molecular interactions within each hot spot.

Having thus validated the approach, the researchers took a more detailed look at two published fragment-to-lead programs for protein kinase B and pantothenate synthetase. In both these cases, group efficiency analyses had previously been performed to establish which portions of the ligands contributed most significantly to binding. Gratifyingly, the computations correctly predicted these.

Overall this approach appears promising. At a minimum, it is another tool for assessing the ligandability of potential targets. More significantly, by highlighting the hottest bits of hot spots, it could be useful for medicinal chemists trying to optimize and grow fragments and leads. Unfortunately, as currently described, the process will require a skilled modeler. It would be nice if the authors built a simple web-based interface for people to upload pdb files for analysis, as is the case for FTMap. Also, all the data presented are retrospective – a prospective example would be the true test. Does anyone have experience to share?

16 May 2016

Fragments vs JAK – but phototoxicity

The four members of the JAK family of kinases have received plenty of attention due to their role in inflammation. Two drugs that inhibit these targets, tofacitinib and ruxolitinib, have been approved for rheumatoid arthritis and myelofibrosis, respectively. Psoriasis is another possible indication, but for this disease a topical drug might be useful, particularly one with low systemic bioavailability. The search for such molecules is the subject of a paper recently published in ACS Med. Chem. Lett. by Andrea Ritźen and colleagues at LEO Pharma.

The researchers screened 500 fragments at 100 µM each using surface plasmon resonance (SPR) against JAK2. Hits were then tested in a biochemical assay against JAK1; in general, there was good correlation, suggesting a (desirable) lack of selectivity between the two family members. One of the more attractive hits was compound 1, which was characterized crystallographically bound to JAK2.


Compound 1 is an indazole, which is often seen in kinase inhibitors binding to the so-called hinge binding site where the adenine of ATP binds. Other indazoles have previously been reported as JAK2 inhibitors. Nonetheless, the sulfonamide moiety of compound 1 provides an interesting new vector. Moreover, a search of published structures suggested that adding a phenol moiety could make additional contacts to the proteins. This led to the design and synthesis of compound 2, which showed a dramatic boost in potency as well as measurable cell activity. Further optimization led ultimately to compound 34, with low nanomolar biochemical activity and mid-nanomolar cell activity. Compound 34 was also reasonably selective in a panel of 20 kinases.

A topical drug needs to be stable in sunlight, but unfortunately compound 34 showed phototoxicity. This led to the testing of a few other compounds, revealing that even the initial fragment 1 is unstable over a period of a few hours in simulated outdoor light. Indazole itself seems reasonably stable, suggesting that perhaps adding different substituents could fix the problem.

This is a brief but satisfying example of using published information as well as medicinal chemistry to advance a fragment hit. Although the program does not appear to have led to a drug lead, it is laudable that the researchers describe the photoinstability of the indazoles. With these moieties appearing so frequently in campaigns against kinases, this could be a valuable cautionary tale to others pursuing similar scaffolds.

09 May 2016

Fragments vs KEAP1 – crystallographically

Last week we discussed how soaking crystals in high concentrations of fragments could identify useful molecules. Indeed, last month’s Drug Discovery Chemistry conference featured a talk by Tom Davies (Astex) illustrating the power of this approach. In a recent paper in J. Med. Chem., Tom, Jeffrey Kerns (GlaxoSmithKline), and their collaborators provide the full story.

The researchers were interested in the protein KEAP1, which binds to and blocks the activity of the transcription factor NRF2. Small electrophilic molecules covalently react with KEAP1, causing NRF2 to dissociate and upregulate various cytoprotective genes, which could be useful for a variety of diseases. Indeed, this is at least partly how the approved drug dimethyl fumarate seems to work. However, dimethyl fumarate is quite reactive; a more specific molecule could have a better therapeutic profile, and would certainly be useful for probing the complex biology. With this in mind, the researchers sought a non-covalent inhibitor.

NRF2 interacts with a bowl-shaped “Kelch domain” of KEAP1 largely through electrostatic interactions. Thus, not only is this a challenging protein-protein interaction, coming up with a cell-permeable molecule is all the more difficult. The researchers soaked crystals of the Kelch domain against 330 fragments overnight at concentrations of 5-50 mM. Fragments were observed binding in three adjacent hot-spots, and although no functional activity could be detected, the binding modes suggested a path forward.

Growing from fragment 1 toward a hot-spot occupied by an aromatic fragment (magenta below) led to compound 4, with detectable activity in a fluorescence-polarization assay as well as clear binding in an isothermal titration calorimetry (ITC) experiment. Growing compound 4 toward another hot-spot occupied by a sulfonamide-containing fragment (green below) led to the sub-micromolar compound 6, and further optimization resulted in the low nanomolar compound 7. As is often (but not always) the case, the fragment portion of the final molecule binds in virtually the same position as the initial fragment 1 (blue).


Comparison with the structure of the NRF2 peptide reveals that the carboxylic acid in compound 7 binds in a very similar fashion to a glutamate residue in the peptide, and some of the other peptide contacts are also mirrored, but with very different moieties. Importantly, compound 7 has only a single negative charge, balanced lipophilicity, and fills the binding pocket more effectively than the peptide. These properties translate to good biological activity in multiple different cell-based assays, where the compound causes NRF2 translocation to the nucleus, upregulates appropriate gene expression, and prevents glutathione depletion when cells are treated with an organic peroxide. Although the pharmacokinetics have yet to be optimized, it also shows encouraging activity in a rat model of ozone exposure. Finally, it is reasonably selective in a panel of 49 undesirable off-target proteins. All these properties make this at least an excellent chemical probe.

There are several important lessons in this paper. First, as we’ve noted previously, it is possible to replace a highly polar peptide with a much more drug-like molecule. Second, fragments don’t need measurable affinity to be useful starting points. Crystallography is ideally suited for finding such fragments, though Astex researchers reported a similar success story with NMR last year. Finally, progressing these fragments won’t necessarily be easy, as reflected in the 25 authors on the paper. That said, such intensive efforts can pay off, as illustrated last month by the approval of venetoclax against another difficult protein-protein interaction.

02 May 2016

A strong case for crystallography first

We noted last week that one theme of the recent CHI FBDD meeting was the increasing throughput of crystallography. Crystal structures can provide the clearest information on binding modes, and a key function of standard screening cascades is to whittle the number of fragments down to manageably small numbers for crystal soaking. Only a few groups have used crystallography as a primary screen. A team led by Gerhard Klebe at Philipps-Universität Marburg argues in ACS Chem. Biol. that crystallography should be brought to the forefront.

The researchers were interested in the model protein endothiapepsin. As discussed last year, they had previously screened this protein against a library of 361 compounds using six different methods, and the agreement among methods was – to put it charitably – poor. Nonetheless, many hits that did not confirm in orthogonal assays produced crystal structures when soaked into the protein. Thus emboldened, the researchers decided to soak all 361 fragments individually into crystals of endothiapepsin. This resulted in 71 structures, a hit rate of 20%, higher than any of the other methods (which ranged from 2-17%). Even more shocking, 31 of the fragments were not identified by any of the other methods, and another 21 were only identified by one other method. Thus, a cascade of any two assays would have found, at best, only a quarter of the crystallographically validated hits.

In agreement with other recent work, the fragments bound in multiple locations, including eight subsites within the binding cleft as well as three potentially allosteric sites. Not all of these sites were found using other methods.

But are these fragments so weak as to be uninteresting? To find out, the researchers performed isothermal titration calorimetry (ITC) to determine dissociation constants for 59 of the crystallographic hits. Three of the 21 most potent (submillimolar) binders were not detected by any of the other methods, and another seven were only found by one.

What factors led to this crystallographic bonanza? First, the researchers used the very high concentration of 90 mM for each fragment (in practice sometimes <90 mM because of precipitation). Not surprisingly, solubility was important: 97% of the hits had solubilities of at least 1 mM in aqueous buffer, and the soaking solution contained 10% DMSO as well as plenty of glycerol and PEG. Achieving such high concentrations is harder when multiple fragments are present, and the researchers argue from some of their historical data that the common use of cocktails lowers success rates.

How did different methods compare? Interestingly, functional assays such as high-concentration screening or reporter-displacement assays fared best, while electrospray ionization mass-spectrometry (ESI-MS) and microscale thermophoresis (MST) were close to random. This is in marked contrast to other reports for ESI-MS and MST, and the researchers are careful to note that “the choice and success of the individual biophysical screens likely depend on the target and expertise of the involved research groups.”

Primary crystallographic screening was an early strategy at Astex, and although this may not have been fully feasible 15 years ago, it seems they were on the right track. Of course, not all targets are amenable to crystallography, and not everyone has ready access to a synchrotron beam with lots of automation. But for those that are, it might be time to drop the pre-screens and step directly into the light.

25 April 2016

Eleventh Annual Fragment-based Drug Discovery Meeting

The first major fragment event of 2016, CHI’s Drug Discovery Chemistry, was held last week in San Diego. FBDD was the main focus of one track, and fragments played starring roles in several of the others as well, including inflammation, protein-protein interactions, and epigenetics. Also, for the first time this year the event included a one-day symposium on biophysical approaches, which also included plenty of fragments.

In agreement with our polls, surface plasmon resonance (SPR) received at least a mention in most of the talks. John Quinn (Genentech) gave an excellent overview of the technique, packed with lots of practical advice. At Genentech fragments are screened at 0.5 mM in 1% DMSO at 10°C using gradient injection, which permits calculation of affinities and ligand efficiencies directly from the primary screen. Confirmation of SPR hits in NMR is an impressive 80%. A key source of potential error in calculating affinities is rebinding, in which a fragment dissociates from one receptor and rebinds to another. That problem can be reduced by increasing the flow rate and minimizing the amount of protein immobilized to the surface. Doing so also lowers the signal and necessitates greater sensitivity, but happily the baseline noise has decreased by 10-fold in the past decade.

Some talks focused on using SPR for less conventional applications. Paul Belcher (GE) described using the Biacore S200 to measure fragments binding to wild-type GPCRs. In some cases this provided different hits than those detected against thermally stabilized GPCRs. And Phillip Schwartz (Takeda) described using SPR to characterize extremely potent covalent inhibitors for which standard enzymatic assays can produce misleading results. These screens require exotic conditions to regenerate the chip, so it helps that the SensiQ instrument has particularly durable plumbing.

In theory, SPR can be used to measure the thermodynamics of binding by running samples at different temperatures, but John Quinn pointed out that enthalpic interactions dominate for most fragments, so the extra effort may not be worthwhile. Several years ago many researchers felt that enthalpically driven binders might be more selective or generally superior. Today more people are realizing that thermodynamics is not quite so simple, and Ben Davis (Vernalis) may have put the nail in the coffin by showing that, for a set of 22 compounds, enthalpy and entropy of binding could vary wildly simply by changing the buffer from HEPES to PBS! (Free energy of binding remained the same with either buffer.)

Thermal shift assays (TSA or DSF) continued to be controversial, with Ben finding lack of agreement between the magnitude of the shift and affinity, though there was a correlation with success in crystal trials. In contrast, Mary Harner (BMS) reported good agreement between thermal shift and affinity. She also found that it seemed to work better when the fragments bound in deep pockets than when they bound closer to the surface. However, Rumin Zhang (Merck), who has tested more than 200 proteins using TSA, mentioned that some HCV protease inhibitors could be detected despite the shallow active site. Rumin also pointed out that a low response could indicate poor quality protein – if most of the protein is unfolded it might be fine for biochemical assays but not for TSA. Negative thermal shifts are common and, according to Rumin, sometimes lead to structures, though others found this to be the case less often.

What to do when assays don’t agree was the subject of lively discussion. Mary Harner noted that out of 19 targets screened in the past two years at BMS using NMR, SPR, and TSA, 45% of the BMS library hit in at least 1 assay. However, 68% of hits showed up in only a single assay. Retesting these did lead to more agreement, but even many of the hits that didn’t confirm in other assays ultimately led to leads. All techniques are subject to false negatives and false positives, so lack of agreement shouldn’t necessarily be cause for alarm. Indeed, Ben noted that multiple different soaking conditions often need to be attempted to obtain crystal structures of bound fragments.

Crystallography in general is benefiting from dramatic advances in automation. Jose Marquez described the fully automated system at the EMBL Grenoble Outstation, which is open to academic collaborators. And Radek Nowak (Structural Genomics Consortium, Oxford) discussed the automated crystal harvesting at the Diamond Light Source, which is capable of handling 200 crystals per hour. He also revealed a program called PANDAA (to be released soon) that speeds up the analysis of crystallographic data.

Crystallography was used as a primary screen against KEAP1, as discussed by Tom Davies (Astex). A subset of 330 of the most soluble fragments was tested in pools of four, which revealed several hot spots on the protein. Interestingly, an in-house computational screen had not identified all of these hot spots, though Adrian Whitty (Boston University) noted that they could be detected with FTMap. The fragments themselves bound exceptionally weakly, but intensive optimization led to a low nanomolar inhibitor.

Another case in which extremely weak fragments turned out to be useful was described by Matthias Frech (EMD Serono). A full HTS failed to find any confirmed hits against cyclophilin D, but screening by SPR produced 168 fragments, of which six were characterized crystallographically. Although these were all mM, with unimpressive ligand efficiencies, they could be linked or merged with known ligands to produce multiple leads – a process which took roughly one year from the beginning of the screen. Matthias noted that sometimes fragment efforts are started too late to make a difference, and that it is essential to not be dogmatic.

Huifen Chen discussed Genentech's MAP4K4 program. Of 2361 fragments screened by SPR, 225 had affinities better than 2 mM. Crystallography was tough, so docking was used instead, with 17 fragments pursued intensively for six months, ultimately leading to two lead series (see here and here), though one required bold changes to the core. This program is a nice reminder of why having multiple fragment hits can be useful, as the other 15 fragments didn’t pan out.

Finally, George Doherty (AbbVie) gave a good overview of the program behind recently approved venetoclax, which involved hundreds of scientists over two decades. He also described intensive medicinal chemistry which led to a second generation compound, ABT-731, with improved solubility and oral bioavailability.

We missed Teddy at this meeting, and there is plenty more to discuss, so please add your comments. If you did not attend, several excellent events are still coming up this year. And mark your calendar for 2017, when CHI returns to San Diego April 24-26.