29 October 2018

Capillary electrophoresis revisited

Among the various fragment-finding methods, capillary electrophoresis (CE) seems to be among the least-used, at least according to our polls. Indeed, we last wrote about CE in 2012, and since then the company that was popularizing the technique seems to have quietly dropped it from its website. A new paper by Marianne Fillet and collaborators at the University of Liege and the University of Namur in Analytica Chimica Acta presents a how-to guide for CE.

As we previously discussed, the most general CE assay involves filling a capillary with a protein solution as well as a “probe ligand” with affinity for the target protein. Interactions between the probe ligand and the protein will increase the migration time of the probe ligand compared to its progress through a capillary without protein (i.e., the probe ligand will move more slowly through the capillary in the presence of protein).

If a “test ligand” is introduced into the capillary and displaces the probe ligand, the migration time of the probe ligand will again decrease. By changing the concentration of test ligand and measuring the shift in migration time of the probe ligand, the affinity of the test ligand can be determined.

The researchers applied CE to thrombin, a drug target that is often used for validating fragment-finding methods. The low nanomolar inhibitor NAPAP was chosen as the probe ligand due to its strong chromophore (simplifying detection) and positive charge (allowing it to move in the electric field of the capillary). They tested three literature compounds with inhibition constants ranging from high nanomolar to high micromolar and found good agreement with published results.

Next, the researchers applied CE to a small library of fragments, generating several hits. They also describe a method for detecting irreversible binding: this involves screening the protein with an even higher concentration of probe ligand to see whether the test ligand itself can be displaced.

This is a nice study, but it perhaps also illustrates why the technique hasn’t caught on. First and most importantly is throughput; the runs shown are on the order of 14 minutes. Second, it does require a probe ligand. (Screening test ligands directly only works if they are positively charged.) On the other hand, CE can work with native protein, unlike immobilization-based techniques such as SPR and WAC.

Have you tried CE yourself – and if so how did it perform?

22 October 2018

Fragments vs Ras – part 3

Six years ago we highlighted papers from Genentech and Steve Fesik’s group reporting fragments that bind to Ras-family proteins, which are among the best validated but most difficult anti-cancer targets. The fragments bind some distance from the GTP-binding site, but can block Ras signaling by interfering with important protein-protein interactions. However, the most potent molecules reported bound at this site with just ~200 µM affinity, and we concluded by musing that “it still remains to be determined whether this is a ligandable site on the protein.” As reported recently in Nature Communications by Terence Rabbitts and collaborators at the University of Oxford, St. James University, Domainex, and the University of Aberystwyth, the answer appears to be yes.

The researchers screened HRas against 656 fragments, each at 200 µM, using SPR, resulting in 26 initial hits. These were tested again by SPR against active-form protein (bound to the GTP mimetic GTPγS) or inactive protein (bound to GDP). A single compound, Abd-1, was selective for the activated form of the protein, and did not bind when the protein was complexed to an antibody the researchers had previously generated that binds at the same PPI site.

Abd-1 had low affinity and was not particularly soluble, so the researchers looked for analogs with better properties, resulting in Abd-2, which binds to both HRas and KRas. Further growing in the direction taken by the Fesik group did not lead to significant improvements, but a breakthrough occurred when the researchers grew off a different region of the fragment, towards what looked to be the wall of the small pocket. As Trevor Perrior mentioned at the DOT meeting last month, this led to the opening up of a new channel and a substantial boost in affinity for Abd-5. Further growing allowed the researchers to trim off the right-hand portion entirely, leading to Abd-7, with mid-nanomolar activity and good ligand efficiency. Crystallography revealed that, despite the conformational changes, the core of Abd-7 still binds in the same location as Abd-2.

Not only did Abd-7 bind tightly to KRas, it also inhibited the pathway in cell-based assays (albeit at 100-fold higher concentrations), presumably by blocking interactions with Ras-effector proteins. The compound also showed low micromolar activity against cancer lines with different Ras mutations in cell viability assays. The researchers note that “the observed discrepancy between affinity (in vitro Kd) and efficacy (IC50 in cells) is a known challenge that can be addressed through chemistry.” Other possible challenges include metabolic stability and oral bioavailability, neither of which is discussed. Nonetheless, the paper reveals that this site in Ras family proteins is ligandable. It is also a useful reminder that proteins can be remarkably plastic, and sometimes the best route forward really is by slamming into what appears to be a solid wall.

15 October 2018

FBLD 2018

Ten years ago, Vicki Nienaber (Zenobia) enlisted a small group of fellow enthusiasts to help her organize an independent fragment-based lead discovery conference in San Diego. That event was so successful that it was repeated in York in 2009, Philadelphia in 2010, San Francisco in 2012, Basel in 2014, and Cambridge (USA) in 2016. Last week, to celebrate its first decade, Derek Cole (Takeda), Rod Hubbard (University of York) and Chris Smith (COI) brought FBLD 2018 back to San Diego, along with some 200 fragment fans. With around 30 talks, more than 40 posters, and nearly 20 exhibitors, I won’t attempt to present a comprehensive overview, but just focus on broad themes.

Success Stories
I estimate that, in 2008, 14 fragment-based programs had entered the clinic, none of which had advanced beyond phase 2. That list has now grown to more than 40, so naturally success stories were a focus.

Andy Bell (Exscientia) discussed NMT inhibitors for malaria and the common cold (see here); the AI-driven approach took < 500 molecules to get to molecules with animal efficacy. Steve Woodhead (Takeda) revealed potent inhibitors of TBK1, a kinase involved in the innate immune response. It took just three months to go from a fragment hit to an animal-active lead, though unfortunately that molecule also showed apparent on-target toxicity. And Rosa María Rodríguez Sarmiento (Roche) described the discovery of COMT inhibitors (see here).

Mary Harner (BMS) described the discovery of sub-micromolar KAT II inhibitors in just a few months, enabled by parallel chemistry and the synthesis of 833 compounds. Several series turned out to be aggregators, and BMS has instituted a routine β-lactamase screen (an enzyme particularly sensitive to aggregators) to catch these early.

Keith McDaniel (AbbVie) described the discovery of the BET-family bromodomain inhibitor ABBV-075. This program also made rapid progress: just six months from the initial fragment hit, although the team did spend another year trying to find better molecules. This effort eventually paid off, as the same fragment has now led to a BD2-selective molecule, ABBV-744, that has recently entered the clinic.

And Paul Sprengeler (eFFECTOR) described the discovery of eFT508. This too was a rapid success: just 1 year and 170 compounds, enabled by 30 co-crystal structures, and in the end a dozen molecules competing for candidacy.

Notice that many of these projects moved quickly. Feel free to send this summary to anyone who worries that fragment programs move too slowly to be practical.

Technologies have always had a starring role in FBLD conferences, and this one was no exception. Ben Cravatt (Scripps) discussed his fragment-based target discovery methods (see here and here). As I speculated recently, he is now using these approaches to discover new protein degraders. And his "fully functionalized fragments" are being adopted by others, as described in a poster by Emma Grant and collaborators at GlaxoSmithKline and University of Strathclyde.

Surface plasmon resonance (SPR) was used routinely by many of the speakers, but there is plenty of room for innovation. John Quinn (Genentech) described how to extend kinetic measurements to the very fast and the very slow. John also noted that gathering kinetic data earlier to deprioritize series with slow on-rates may be wise. And for those who wonder about the limits of detection for SPR, John measured the affinity of imidazole for NTA: just 13.6 mM!

Miles Congreve (Sosei Heptares) described multiple methods applied to GPCR targets along with a number of success stories. He also noted that, in the PAR2 program we mentioned recently, fragments were able to identify a buried pocket that could not be found using DNA-encoded libraries of several billion members, presumably because the pocket would not be accessible to a DNA-bound ligand. Interestingly, this pocket could be detected computationally using FTMap, as shown in a poster presented by Amanda Wakefield (Boston University).

Pedro Serrano (Takeda) described a variety of biophysical methods applied to GPCRs, the most stunning of which is an SPR microscope capable of performing kinetic binding assays on whole cells. He has tested this Biosensing Instrument on four different GPCRs, and although there are technical challenges, the data seem usable.

But the light shone most brightly on crystallography, illuminated by Stephen Burley (Protein Data Bank) among others. In order to justify continued public funding and free access (yes, there were suggestions to put the PDB behind a paywall), the PDB was asked to demonstrate its usefulness to society. Their analysis found that of the 210 new molecular entities (NMEs) approved by the FDA from 2010 through 2016, 184 had PDB entries for the target and/or the NME – for a total of 5914 structures, 95% of which were crystallographic. Most of these structures had been deposited at least 10 years before the drug was approved, so in many cases they probably played an important role.

John Barker described how Evotec has jumped into high-throughput screening by crystallography in a collaboration with the Diamond Light Source, which is now capable of doing 700 soaks per day. They have run 10 screens over the past 18 months with a small library of 320 fragments, with hit rates typically around 8%.

We have written about how high concentrations can improve success in crystal soaking experiments, and both Chris Murray and Dominic Tisi of Astex described how they’ve taken this to an extreme: 1 M soaks, with the fragment dissolved directly in the soaking solutions. Obviously this requires highly soluble fragments, so they’ve built a library of 81 “MiniFrags” having on average just 6.4 non-hydrogen atoms. They have tested these against five targets that diffract to high resolution and have found impressively high hit rates of 20-60%, compared to the 2-20% in the original 100 mM soaks for the same targets. Some of the sites are exploited by previously reported inhibitors or substrates, while others are new. And while the “universal fragment” 4-bromopyrazole did well, 1,2,3-triazole did even better – binding to all five targets in a total of 22 sites.

Crystallographers should not become complacent. Gabe Lander (Scripps) gave an update on cryo-EM, which we’ve written about here. The number of cryo-EM structures deposited in the PDB eclipsed those from NMR in 2016, and resolution continues to improve, with the current (as of late September) record at 1.56 Å. Still, the technique is not nearly as fast as crystallography: best case is 8 hours from data collection to refinement, although Gabe did think that 10 structures per day would be possible within the next few years. And Chris Murray noted that, if present trends continue, “we’ll all be doing cryo-EM in five years’ time.” Backing this up, he showed what I suspect may be the first clear density map of a fragment bound to a test protein.

This was the last major fragment event of the year, but next year’s calendar is already shaping up nicely. And mark your calendar for September 2020, when FBLD 2020 will move to the original Cambridge (UK).

06 October 2018

Fragments in the clinic: 2018 edition

To celebrate FBLD 2018, we're updating the list of FBLD-derived drugs. The current list contains 40 molecules - 25% more than the last compilation two years ago. As always, this table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). 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.


VenetoclaxAbbVie/GenentechSelective Bcl-2
Phase 3

PLX3397PlexxikonCSF1R, KIT
Phase 2

AT9283 AstexAurora, JAK2
IndeglitazarPlexxikonpan-PPAR agonist
Navitoclax (ABT-263)AbbottBcl-2/Bcl-xL
Phase 1

ABT-518AbbottMMP-2 & 9
AT13148AstexAKT, p70S6K, ROCK
AZD5099AstraZenecaBacterial topoisomerase II
BI 691751Boehringer IngelheimLTA4H
MAK683NovartisPRC2 EED

I have no doubt that this list is incomplete, particularly in Phase 1. If you know of any others (and can mention them) please leave a comment.

01 October 2018

Sixteenth Annual Discovery on Target

CHI’s Discovery on Target took place in Boston last week. With >1300 attendees from over two dozen countries, this is the older, larger cousin of the San Diego DDC meeting; at some points ten tracks were running simultaneously. Although more heavily focused on biology, there were still plenty of talks of interest to fragment folks.

Michael Shultz (Novartis) provocatively asked “do we need to change the definition of drug-like properties?” Long-time readers will recall that his earlier papers on ligand efficiency led to considerable debate, which seems to have been settled to everyone’s satisfaction with the exception of Dr. Saysno.

His new study, which has just published in J. Med. Chem., analyzes the molecular properties of all 750 oral drugs approved in the US between 1900 and 2017. Contrary to what strict rule of five advocates might expect, the molecular weight has increased over the past couple decades, as has the number of hydrogen bond acceptors. In contrast, the number of hydrogen bond donors (#HBD) has remained constant, suggesting that this may be more important for oral bioavailability. (Indeed, #HBD is the only Lipinski rule not broken by venetoclax.) Although Shultz did not examine “three dimensionality,” he laudably includes all the raw data – including SMILES – in the supporting information. This will be a useful resource for data-driven debates.

Molecular properties are carefully considered by Ashley Adams, who discussed the four fragment libraries used at AbbVie. The first is a 4000-member “rule of three” compliant library. For tougher targets, a 9000-member Ro3.5 library is available, as is a specialized fluorine library for 19F NMR (2000 members) and a 1000-member “biophysics” library, in which all compounds are less than 200 Da. Fragment optimization is often challenging, and since the C-H bond is most common but perhaps least explored, the AbbVie database is annotated with references on C-H bond activation relevant to each fragment.

Anil Padyana spoke about the metabolic enzymes being targeted at Agios. As we mentioned recently, these are very difficult targets, so the researchers often use parallel (as opposed to nested) screening using different techniques to minimize false negatives. Anil also described an interesting SPR assay in which fragments were introduced to the protein after the addition of an activating substrate.

High-quality protein constructs are essential for any fragment screen, and Jan Schultz described ZoBio’s technology for generating these. The company’s “protein domain trapping” approach entails high-throughput generation and screening of tens or hundreds of thousands of truncations of a given protein and rapidly selecting stable, high-expressing, and active variants.

Trevor Perrior mentioned that Domainex has a similar technology, which has been able to produce soluble protein domains in 90% of its attempts. Trevor also described a separate project in which a 656-fragment compound library was screened using SPR against the enzyme RAS. They found fragments that bind in a previously discovered site but, unlike the earlier work, the Domainex researchers were able to optimize these to nanomolar inhibitors.

Another success story was presented by Dean Brown (AstraZeneca), who described a collaboration with Heptares to discover inhibitors of protease-activated receptor 2 (PAR2). As the name suggests, this GPCR is activated when a protease cleaves the N-terminus, allowing the remaining N-terminal residues to fold back and activate the GPCR. The researchers used a stabilized form of PAR2 in an SPR screen of 4000 fragments and obtained >100 binders in multiple series. This led to AZ8838, which blocks signaling by binding in an allosteric pocket. It also has a slow off-rate, which is often an attractive feature – particularly in the context of intramolecular activation.

A number of talks were focused on protein degraders such as PROTACs (PROteolysis-TArgeting Chimeras). These are generally two-part molecules connected by a linker: one part binds to a target of interest, while the other engages the cellular degradation machinery to destroy the target. As Shanique Alabi, a graduate student in Craig Crews's lab at Yale demonstrated, the molecules are catalytic – a single PROTAC molecule can cause the destruction of multiple copies of a target protein. This “event-driven” pharmacology is thus different from most historical drugs, which are “occupancy-driven.” Is there a role for fragments?

One of the strengths of FBLD is that if a ligandable site exists, it can be found. As Astex demonstrated, the majority of proteins seem to have secondary sites, away from the active site. Although some of these may be allosteric, others probably have no functional activity, particularly in the case of protein-protein interactions where secondary sites may be located some distance from the interface. The power of degraders is that non-functional sites can be made functional. The power of FBLD is that it can find small-molecule binding sites, which could then be used as anchoring sites for one side of a degrader. Watch this space!