Tenth Annual Fragment-based Drug Discovery Meeting...Teddy's Thoughts

Dan posted his thoughts here.  Like Dan, CHI put me to work: I chaired the first session in PPIs, co-taught (with Dan) our award winning FBDD course to 22 attendees (a new high which I think shows that interest in FBDD is still growing), moderated a breakfast roundtable on kinetics and thermodynamics,  judged posters.  All of this during weather which made the natives shiver, and me feel like spring is really here (64F and cloudy).  

First off, there was live tweeting of talks by me and a few others: @iceobar, @moleculesmith, and others.  Beware that the Dubai Diamond Conference was also going on that week.  

In the PPI track, just as last year, fragments were a key component to various projects.  Mark McCoy, Merck (and he taught me more about NMR than just about anyone, whether he will admit it or not) gave a great talk on HDM2-p53.  I took away a few things from his talk which I really liked.  Merck (legacy S-P) really relies on NMR structural information: HSQC-based screening, NMR-based Ki,  and NMR-driven docking.  I was particularly intrigued with the NMR-driven docking because they were able to generate 80+ models with a 75% success rate that was confirmed by X-ray (once that was enabled).  They were forced down this path because they went 2 years without a X-ray structure. 

Joe Patel of AZ talked on SOS-RAS.  What I liked was that AZ uses a modified Voldemort Rule (which Harren Jhoti incorrectly attributes to me; I am the Boswell to Rod Hubbard's Johnson): HAC less than/equal20, cLogP less than5, less than3 rings, less than5 rot bonds, less than 3 HBD, and less than 5 HBA.  Their initial X-ray screening ended up at a wall, so they went to a covalent approach. 

Troy Messick of the Wistar gave a nice talk on using fragments and SBDD to drug an "undruggable" target.  I think this is exactly this is exactly the kind of success that has led FBDD to be ubiquitous these days.  I have to admit I have and am working with the Wistar on the NMR component of their screening, so I may be biased. 

I won't go into the various talks from the FBDD track,  However, echoing Dan this is really a great conference.  My main take home themes is that FBDD is really mainstream.  It's no longer the red headed stepchild to other hit generation processes (apologies to my ginger friends).  Biophysics is also seeing a huge growth, having grown up with FBDD, but really finding a lot more uptake outside of that space.  Next week I will post summaries of roundtables and some useful information. 

Tenth Annual Fragment-based Drug Discovery Meeting

Last week marked the tenth anniversary of CHI’s three-day Drug Discovery Chemistry conference in San Diego. The conference consists of six tracks, with three happening simultaneously. The FBDD track is the only one which dates all the way back to the beginning in 2006. In fact, this is the oldest recurring fragment conference, predating both the Royal Society Fragments meetings as well as the independent FBLD meetings.

It’s worth reflecting on how far fragments have come since 2006. Back then, as Rod Hubbard (Vernalis and University of York) noted, most of the talks were prospective and methodological. Even as late as 2010 there were talks describing how dedicated fragment groups needed to be shielded from the larger organization. Now fragments are mainstream: a large fraction of the talks in the protein-protein interaction track involved fragments, as did both plenary keynote addresses to the entire conference.

Harren Jhoti’s keynote focused on lessons learned at Astex over the past 15 years. There has been some debate in the literature over ligand efficiency (LE), and one slide that struck me was a summary of 782 dissociation constants (measured by ITC) against 20 projects. The vast majority of these compounds had LE > 0.3 kcal/mol/atom. Given that Astex has put multiple fragment-derived drugs into the clinic and was acquired by Otsuka in one of the largest M&A events of 2013, the metric appears to have some utility.

Still, it’s important not to be dogmatic, particularly for difficult targets. Harren described a program for XIAP/cIAP where they started with an extremely weak fragment with LE < 0.2, but its binding mode was sufficiently interesting that they were willing to work on it. This program also revealed the importance of biophysical measurements, as functional activity was uninterpretable and even misleading until higher affinity compounds were discovered.

One theme throughout the conference was the observation that fragments bind at multiple sites on proteins. Harren noted that Astex researchers have found fragments bound (crystallographically) to 54 sites on 25 targets – an average of 2.2 sites per target. Some targets are even more site-rich: Joe Patel (AstraZeneca) performed a crystallographic screen on a complex of Ras and SOS and found four binding sites, including one previously discussed here. In this effort, 1200 fragments were screened in pools of 4, and in one case two fragments from the same pool each bound only when they were both present at the same time – each fragment alone showed no binding by NMR or crystallography.

Troy Messick (Wistar) described his work against the EBNA1 protein from Epstein-Barr virus. An HTS screen of 600,000 compounds came up with at best marginal hits, but soaking 100 different Maybridge fragments into protein crystals led to 20 structures, with fragments bound to four different sites. Some of these fragments were then merged to give cell-active compounds with good oral bioavailability.

Rather than exploring different ligands binding at different sites, Ravi Kurumbail (Pfizer) described an interesting case of the same ligand binding at different sites. A screen against the kinase ITK identified a (large) fragment that could bind both in the adenine binding pocket as well as a nearby pocket, as determined crystallographically. Determining the affinities of the same fragment for the two sites necessitated some clever SPR and enzymology, but did lead to a highly selective series.

In terms of targets, BCL-family proteins were certainly well-represented, featuring heavily in talks by Chudi Ndubaku (Genentech, selective Bcl-x_L inhibitors), Mike Serrano-Wu (Broad Institute, MCL-1 inhibitors), Zaneta Nikolovska-Coleska (University of Michigan, MCL-1), Roman Manetsch (Northeastern, Bcl-x_L and MCL-1), and Andrew Petros (AbbVie, BCL-2 and MCL-1). Of course, it was AbbVie (neé Abbott) that pioneered BCL inhibitors as well as FBLD in general, and I was happy to hear that there is a renaissance occurring there, with fragment approaches being applied to all targets, even those undergoing HTS.

Finally, there were some interesting practical lessons on library design. Peter Kutchukian described how the Merck fragment library was rebuilt to incorporate more attractive molecules that chemists would be excited to pursue. There is an ongoing debate as to whether a fragment library should be maximally diverse or contain related compounds to provide some SAR directly out of the screen, and in the case of the Merck library the decision was to target roughly five analogs in the primary library, with a secondary set of available fragments for follow-up studies.

The utility of having related fragments in a library was illustrated in a talk by Mark Hixon (Takeda) about their COMT program. A HTS screen had failed, and even a screen of 11,000 fragments came up with only 3 hits (with an additional close analog found by catalog screening). Remarkably, all of these are extremely closely related, but other analogs in the library didn’t show up; had they not had multiple representatives of this chemotype in their library they would have come up empty-handed.

In the interest of space I’ll close here. Teddy will post his thoughts later this week, and please share your own. CHI has announced that next year’s meeting will be held in San Diego the week of April 19. And there are still several great events on the calendar for this year!

Tethering versus RNA

Last week we highlighted one of the less common fragment-finding methods, and today we turn to another. Tethering uses reversible disulfide exchange chemistry to trap thiol-containing fragments near binding sites. Back when we developed the technology at Sunesis we used cysteine residues in proteins. We occasionally discussed applying it to nucleic acids, but at the time it was hard to make a good business case. Now that microRNAs (miRNAs) have become hot, there is more interest in going after nucleic acid targets, and in a recent paper in Molecules Kiet Tran and Peter Beal (UC Davis) and Michelle Arkin (UC San Francisco) have done just that.

The researchers were interested in an RNA sequence that is cleaved in cells to generate miR-21, a potential cancer target. The idea is to find small molecules that bind to pre-miR-21 and prevent its processing to the mature miRNA. To perform Tethering, the researchers first introduced a thiol group into adenosine and incorporated this into RNA. They made two separate versions of pre-miR-21, with the modified adenosine at a different site in each, and also made a control RNA with a completely different sequence.

Next, they incubated the modified RNAs with 30 different disulfide-containing small molecules under partially reducing conditions and used mass spectrometry to identify those that covalently bound. As expected most showed minimal binding, but there were a couple hits. One of these, a 2-phenylquinoline, bound to both modified versions of the pre-mR-21 as well the control RNA, suggesting non-specific binding. In fact, 2-phenylquinoline is a known intercalator, so while its identification is not surprising, it does validate the ability of Tethering to identify binders. The other hit, however, appeared to be specific for one of the two pre-mR-21 sequences.

Of course, there is still a long way to go; it is unclear how much affinity the hit has for the RNA, or how specific it would prove if tested against a large panel of decoy RNAs. A key challenge for Tethering – as with many fragment-finding methods – is figuring out what to do with a hit. This is all the more true with RNA, about which we’ve written several times over the years. Still, one nice feature of Tethering is that it allows one to target a specific site of interest. Also, the covalent (disulfide) bond helps with both crystallography and modeling. It will be fun to watch this story develop.

Substrate activity screening for irreversible PAD3 inhibitors

Of all the ways to find fragments, one of the more unusual is substrate activity screening, or SAS, which we first discussed here. The idea is to make and screen libraries of potential enzyme substrates and transform the best ones into inhibitors. In a new paper in J. Am. Chem. Soc., Jon Ellman and coworkers at Yale University describe how they used SAS to discover irreversible inhibitors of protein arginine deiminase 3 (PAD3), a potential target for neurodegenerative diseases.

The four human PADs (conveniently named PAD1-4) transform arginine residues in proteins to citrulline residues, with subtypes distributed differently across different tissues. The researchers started by making a library of more than 200 fragment-sized guanidines (the unique side-chain moiety in arginine) as potential substrates. These were then screened in a colorimetric assay. Several compounds were found to be processed by the enzyme, though all were very weak substrates (K_m > 10 mM).

Next, the best substrates from three different chemical series were optimized for activity. For example, substrate 4a was grown to substrate 15a.

Finally, the substrates were converted to irreversible inhibitors by replacing the guanidine with a known chloroacetamide warhead. This coopts the natural mechanism of the enzyme, which relies on covalent bond formation between an active-site cysteine residue and the substrate. Within a given series, the better the substrate, the better the resulting inhibitor (for example, inhibitor 15b is more potent than inhibitor 4b). However, these correlations did not hold across series.

The best compounds were also tested for selectivity, and some of them were at least 10-fold selective for PAD3 over the other three PADs.

Last year we highlighted a paper that described several difficulties encountered (and overcome) using SAS to find inhibitors of the protease urokinase. (The comments to that post are well worth reading as they include contributions from the corresponding author of the paper as well as a former Ellman postdoc who is using SAS.) However, according to the current paper, SAS was relatively straightforward for PAD3 – another confirmation that different targets require different approaches.

Fragment-based methods in drug discovery

FBLD generates a plethora of reviews, as evidenced by Practical Fragments’ annual round-ups (see for example 2014, 2013, and 2012). However, for the past three years there have been no new books. The drought has now ended, starting with the publication of Methods in Molecular Biology Volume 1289, edited by Anthony E. Klon of Pennsylvania Drug Discovery Institute. Computational chemistry is probably one of the most rapidly changing disciplines within FBLD, and thus it is appropriate that this is the primary focus.

The book is part of the Springer Protocols series, which offers highly specific step-by-step instructions. Many of the chapters have a common organization: Introduction, Materials, Methods, and Notes. While this can work well for established molecular biology techniques such as cloning, it can be trickier to apply to computational approaches. Some of the chapters are quite brief and assume extensive specialized knowledge, while others are extremely detailed. Of course, it is impossible to satisfy everyone; hopefully the following summary will help you find what is most useful for you.

Part I (Preparation) consists of five short chapters. The first is by Rachelle Bienstock, editor of the most recent (and also computationally intensive) book. As we’ve noted, water plays a pivotal role in protein-ligand interactions, and Rachelle concisely but thoroughly summarizes available computational methods. Chapter 2, by Yu Zhou and Niu Huang at the National Institute of Biological Sciences in Beijing, outlines how to use DOCK to assess binding site druggability. In chapter 3, Raed Khashan (King Faisal University, Saudi Arabia) describes a free software tool called FragVLib for generating virtual fragment libraries to compare different ligand binding pockets. Chapter 4, by Jennifer Ludington (formerly of Locus Pharmaceuticals), discusses practical issues in preparing a virtual fragment library, such as conformer and partial charge assignment. Finally, in chapter 5 Peter Kutchukian discusses how he and his Merck colleagues enlisted medicinal chemists to help fill the gaps in their fragment collection.

The second section is titled Simulation. In chapter 6, Kevin Teuscher and Haitao Ji (University of Utah) summarize “fragment hopping,” including an extensive table of available software tools. Chapter 7, by Olgun Guvench (University of New England), Alexander MacKerrel (University of Maryland), and coworkers describes SILCS: site identification by ligand competitive saturation. This program, developed by SilcsBio LLC, soaks proteins in virtual solutions containing very tiny fragments (think propane and methanol) to look for binding sites. Molecular dynamics simulations include methods to prevent aggregation of the ligands or denaturation of the protein.

Chapter 8, by Álvaro Cortés-Cabrera, Federico Gago (Universidad de Alcalá, Madrid) and Antonio Morreale (Repsol Technology Center, Madrid), describes how ligand efficiency indices can be used to guide fragment growing. Of course, metric skeptics will still ask, “sure it works in practice, but does it work in theory?” And in chapter 9, Jui-Chih Wang and Jung-Hsin Lin (Academia Sinica, Taipei) introduce a new scoring function for fragment-docking, including several pages of detailed instructions for implementing it in AutoDock. As we’ve noted, calculating binding affinities for fragments can be difficult, and the new function seems to be accurate to about ±2.1 kcal/mol for a series of compounds tested

Part III, Design, begins with another chapter by Rachelle Bienstock in which she outlines the process of fragment-based ligand design, highlighting various software tools available at each stage. This includes library design, growing, linking, and downstream considerations such as ADME. Chapter 11, by Zenon Konteatis of Agios, is a brief primer of the process, including an example for the kinase TGF-beta. The last chapter in this section, by Jennifer Ludington, focuses on binding site analysis to assess whether a protein site is druggable (or at least ligandable). She focuses on the procedure used at Locus Pharmaceuticals, which involved soaking a virtual protein in a solution containing fragments and then lowering the chemical potential of the system until only the tightest fragments remain bound. Clusters of probe fragments indicate possible hot spots.

Finally, Part IV consists of Case Studies, starting with a chapter on kinase inhibitors by Jon Erickson (Lilly). More than a third of FBLD-derived clinical candidates target kinases, so it is always good to have an updated overview, though there is at least one structural error.

The last two chapters are both by Frank Guarnieri, founder of Locus Pharmaceuticals and currently at Virginia Commonwealth University School of Medicine. These are highly opinionated (with lots of first-person singular pronouns) and fun to read. They both describe the simulated annealing of chemical potential (SACP) approach that formed the basis of Locus (and is also discussed by Jennifer Ludington above). Chapter 14 describes a small molecule erythropoietin (EPO) mimetic program. The protein EPO binds to and activates a dimeric receptor, and a small molecule functional mimetic would indeed be an exciting breakthrough. Unfortunately, the primary data presented are not compelling, and I remain unpersuaded, though perhaps readers are aware of more convincing evidence.

Chapter 15 describes the Locus program to develop a highly selective orally available p38 inhibitor. The discussion offers a rare window into life at a small biotech, including disagreements over strategies and interpretation of data. It now appears that p38 is probably not a good target for inflammation, which had unfortunate repercussions:

The business decision at Locus to put so many resources into this program along with other questionable business decisions resulted in the company going bankrupt after about 10 years in existence.

Some of the most important lessons are negative, and it’s nice to see these appear in print. Success stories are inspirational, but this chapter is a healthy reminder of the very many things that must succeed for fragment-based approaches to yield new drugs.

When a Lead is a Lead

As we keep on saying, epigenetics is big.  So, today we present another paper on an old friend, BRD4.  This paper is a follow up from previous work where they used docking and X-ray to find the thiazolidinone fragment hit that was elaborated as shown below (Figure 1), but with potency in the single digit micromolar in vitro and double digit in cellulo. 

Figure 1.  Previous work from these authors.

In this work, they continue developing this scaffold investigating the reversed sulfonamide(Figure 2)

Figure 2.  Reversed Sulfonamide

which had significantly improved activity.  Cyclo-aliphatic rings showed increases in potency, but with larger rings also decreasing ligand efficiency.  Aromatic rings decreased potency and larger groups (rings with linkers) were not tolerated at all.  

The crystal structure of the cyclopentyl derivative was solved and was seen to have a different binding mode from the original fragment.  In this case, the WPF shelf is NOT the major binding site for the compound.  In the end, they ended up with

Figure 3.  End Result of this study.  

This is compound is potent (albeit not super potent), ligand efficient, with cell-based activity,  selectivity, and good PK properties.   What I really like is that final sentence of the conclusion:

a promising BRD4inhibitor and a useful lead for further anticancer drug development.

Shapely fragments

Tired of all those planar aromatics in your compound collection? Three-dimensional fragments are all the rage these days, and chemical suppliers are happy to oblige. After the stunning success of their FUNK library, SerpentesOleum has come out with a new offering, Tesseract Products (TP). All of the TP fragments are guaranteed to be nice, plump, and squeezably soft. For example:

Even more exciting, the company has hired a crack team of physicists to produce a line of 4-dimensional fragments with principal moments of inertia greater than 1. Don't delay, order your TP today, and wipe away the 2-D blues!