Fragment linking leads to nanomolar leads for LDHA

The metabolic changes seen in cancer cells were first observed decades ago, but only recently have companies gotten serious about exploiting these changes for new therapies. One potential target is the enzyme lactate dehydrogenase, in particular the LDHA isoform. In a paper recently published in J. Med. Chem., Richard Ward and colleagues at AstraZeneca describe a fragment linking strategy to generate nanomolar leads for this enzyme.

An internal HTS campaign gave a hit rate of about 1%. Worse, none of these hits confirmed in ligand-observed NMR assays, and follow-up studies suggested that the activity was often caused by heavy metal impurities, particularly silver. (This is yet another form of false positive of which to beware.)

When HTS doesn’t succeed, fragments start looking more attractive, so the researchers used their NMR assay to screen about 1000 fragments in pools of 6, with each fragment present at 0.2-0.4 mM. This resulted in 44 hits; of the 27 of these chosen for follow-up 13 gave quantifiable binding, with affinities ranging between 0.3-4.2 mM. Happily, some of these (such as compound 12, below) could be soaked into crystals of LDHA, and structure determination showed these fragments bound in a pocket that normally binds the adenine portion of NADH. An SPR assay was developed and used to screen 350 analogs of some of these hits, and although some improvement in potency could be seen, ligand efficiencies remained more or less the same. (All Kd values in the figure below are taken from SPR data.)

The active site of LDHA is quite long, and so the researchers sought to span its length to obtain decent affinity. The adenine pocket where the identified fragments bind is located some distance away from the site where LDHA’s product lactate binds, and so linking fragments from both pockets would generate molecules spanning the desired region. Finding fragments that bind in the lactate pocket posed a challenge, however, as none of the first set of fragment appeared to bind there. Because lactate is negatively charged, the researchers assembled a specially-tailored 450 fragment library with a high proportion of acids and screened compounds at 2.5 mM using SPR. This screen resulted in 40 hits, and although many of them were nonspecific (they also bound to denatured LDHA!) some hits were specific, including compound 20.

A crystal structure revealed that fragment 20 bound, as expected, some distance from fragment 12, so the researchers generated libraries around both fragments to try to help bridge the gap. About 150 analogs were made around compound 12 and about 70 analogs were made around compound 20, resulting in compounds 24 and 25. Although not necessarily more potent than the initial fragments, crystallography revealed portions of these that were positioned more closely to one another, and linking them to form compound 26 gave a very satisfying boost in potency. This was actually the first linked compound made, and it was also the first compound to show activity in the enzymatic assay. Subsequent optimization was able to drive the potency down to low nanomolar (compound 34). Not surprisingly, the acidic nature of the compounds precluded cellular activity, but some diester derivatives showed sub-micromolar activity.

This is a thorough and engaging account of how fragment-based methods can tackle a difficult target. Although the compounds still need work, they represent good starting points for further optimization and for better exploring the validity of LDHA as a cancer target.

And a Reviewer Responds

A recent paper in J. Med. Chem was reviewed on this blog.  It was also noted by In the Pipeline as one of the worst papers ever published in that journal in a long time.  Today, both ITP and PF received an email from one of the reviewers of the paper, Dr. Elizabeth Howell. ITP has a post about this and a correspondence from the main author of the paper here.  I am reproducing her entire email to us below:

Dear Teddy Zartler and Derek Lowe,

I was made aware of your blogs on the Bastien et al. paper (April 18^th, How do these things get published? &April 17^th, Sometimes your compound sucks). I am an enzymologist and I was one of the reviewers of the paper. I would like to respond to your comments.

I have worked on DHFR since the 1980s. You may perhaps be unaware, but there are 2 different types of DHFRs. The chromosomal DHFR is the target of many drug design efforts and is the more well-known enzyme. A protein conferring resistance to trimethoprim, typified by R67 DHFR, was identified in the 1970s. This R-plasmid encoded DHFR (a type II DHFR) has an entirely different scaffold than the chromosomal DHFRs. I wonder if you thought the Bastien et al. paper was describing drug design efforts for the chromosomal DHFR and this misunderstanding is at the root of your concern? For example, Dr. Zettler [Ed: Zartler] used the term “mutant DHFR” in his post. Is an apple the same as a mutant orange? To me, R67 DHFR and chromosomal DHFR are like apples and oranges. While these 2 DHFRs catalyze the same reaction, they have different protein folds, different oligomerization states, different active sites (R67 has a single active site pore while chromosomal DHFR has a typical binding cleft) and the enzymes use different transition states (endo vs exo).

FYI, I am attaching a pdf file [Ed:shown below as two jpgs] showing the 2 different structures and a Table comparing the binding constants and active site volumes of the 2 enzymes (emailed to Drs. Lowe & Zartler).

A few observations about R67 DHFR (I am happy to provide citations supporting these data if requested):

●R67 DHFR is a homotetramer and each monomer has a fold that is related to an SH3 domain.

●The R67 tetramer has exact 222 symmetry and a SINGLE active site pore.

●For each cofactor (or substrate) binding site in R67 DHFR, there must be four related sites from the symmetry. However a total of 2 ligands bind per pore, either 2 substrates, 2 cofactors or 1 molecule of each. Only the latter is productive and results in catalysis.

●The classical chromosomal DHFR inhibitors (trimethoprim & methotrexate) do not inhibit R67 DHFR very well (K_i values of 150mM and &500mM respectively).

The above observations (& others) lead to the hypothesis that R67 DHFR has a promiscuous binding surface that can accommodate both ligands. The 222 symmetry imposed on the single active site pore of R67 DHFR is utilized by Bastien et al. in their drug design process as they construct "dimeric" ligands. Again, I wonder if the bloggers did not realize a different DHFR was targeted, as for example, Dr. Lowe says “The authors string these things together into huge dimeric molecules, apparently because they think that this is a good idea, but they get no data to support this hypothesis at all.” The hypothesis of Bastien et al regarding symmetry related binding sites comes from the previous crystal structures of the R plasmid enzyme, by stoichiometries derived from time resolved fluorescence and ITC binding studies and by the ability of R67 to catalyze a transhydrogenase reaction (albeit weakly).

With regard to the bloggers comment on electon density interpretation, It is the 222 symmetry that makes it difficult to deconvolute electron density of crystal structures describing bound complexes in R67 DHFR , please see the attached figure of the symmetry problem in this enzyme.

In regards to my review of the paper, I read the paper carefully and wrote 2 pages of comments in my initial review. I also read the revised manuscript. In my opinion, the paper was a first step in the design of an inhibitor that would target this unusual binding site and have some specificity for the R67 DHFR vs. the chromosomal DHFR. To my knowledge, this is the first drug design effort to target R67 DHFR activity. I stand by my decision, the paper is worthy of publication.

Sincerely, Liz Howell, Professor, Biochemistry, Cellular & Molecular Biology Dept., University of TN, Knoxville, TN

Comments

Dan and I have been talking about the blog here at the CHI Drug Discovery conference we are both attending. The blog was born of a night having beers here five years ago. We were wondering why we barely ever get comments (and if you exclude Peter Kenny the total asymptotically approaching zero). If you just look at the blog we look like two cranks writing a blog no one reads (and Dan would be the crazier one because he writes more of the posts). However, our web traffic is good and constantly increasing. We have wondered if it is because people don't want to comment from work (where Big Brother is always watching). Or if people have nothing to say (I find that hard to believe). So, what can we do to get you all to comment more?
Dan's latest post on  the FBDD conference is a good example.  I KNOW many of your were there, but no one has anything to add?

Seventh Annual Fragment-Based Drug Discovery Meeting

CHI’s annual FBDD meeting took place in San Diego this week, and since this was the first time in a while both Teddy and I have been in the same place we’ve decided to make this a joint post. As with last year this does not aim to be comprehensive.

One of the highlights of the conference was a set of three talks on BACE1 inhibitors from Amgen (Ted Judd), Lilly (David Timm), and Pfizer (Ivan Efremov), the first two of which have been discussed here and here. It’s nice to see fragments playing a pivotal role in delivering advanced leads and – at least in the case of Lilly and Merck – clinical candidates against what has been one of the most difficult drug targets in industry.

Speaking of difficult targets, Till Maurer of Genentech gave a lovely presentation on using NMR-based fragment screening to discover inhibitors of the holy grail of oncology, Ras. They’ve recently published some of this story, which we’ll highlight in an upcoming post.

A common question is ‘how often do you find the same fragment using different methods?’ (see here for an ongoing discussion on LinkedIn). Cynthia Shuman from GE gave a nice case study in which she screened the protein PARP15 against 987 fragments using a Biacore T200. Of the 15 fragments with shapely, well-behaved sensorgrams, 14 were confirmed by NMR. On the other hand, only one of these hits was detected in a differential scanning fluorimetry (thermal melt) assay.

Marcel Verdonk at Astex described general trends from mining in-house and published data. After looking at 43 in-house targets, he found that 8000 compounds had been tested against 2 or more proteins, and after plotting by molecular weight found that, consistent with the original Hannian model, larger compounds are more selective. In a separate analysis of 53 fragments that had been advanced to leads, he found that in most cases the initial fragment maintained roughly the same position and orientation from start to finish.

Rod Hubbard, Teddy and I all ran round-table discussions, but only Teddy kept notes, which are summarized here.

The topic started as a discussion of 2D vs. 3D fragment libraries. In the recent Pfizer fragment build, a group of diverse chemists eyed every compound, and at least five had to agree to each molecule before it went into the library. 

The discussion went briefly to the old fight: Are nitros masked amines or noxious moieties?  The table ended up agreeing that if you would remove the nitro group or change it anyways, why put it in the first place?

We then dove right back into the 2D vs. 3D debate. Kinases seem to love 2D fragments, while other classes of targets seem to NEED 3D fragments. One idea discussed was 3D fragments as complements to 2D fragments. It was mentioned that 3D fragment libraries would need to be MUCH larger to cover equivalent chemical space. I thought the idea that 3D fragments would be exploring “vector” space rather than “chemistry” space would mean that you could go with a much smaller library, if you want to use it for vector space searching. It was also proposed that 2D fragments tend to be much smaller (~150-180Da-ish) and 3D fragments would be, by necessity, bigger (~250 Da).

The topic then changed to SBDD (structure-based drug discovery) as part of FBDD. Most people at the table were of the opinion that they wouldn’t use FBDD (on a normal priority target) without SBDD. And with SBDD, you don’t need 3D fragments to explore vector space since you will have the X-ray to guide you. 

The point was made that 3D fragments also HAVE to be scaffolds which would end up in the final compound. If you are simply using a scaffold to explore vector space without any hope of it ending up in the final product, why bother. [TZ Note: I think this is very shortsighted and people do not understand that targets will most likely NOT have SBDD to guide them, at least in the fragment-based lead generation stage.] 

The question was asked to the table: is FBDD a valid approach in the absence of structure? Three people said yes, the rest (~8) said no. All three who said yes were small company/ CRO people. Everyone agrees if you do go with FBDD without structure you need to have a HEAVY investment in biophysics to characterize the protein and the hits. 

One person asked about low confirmation hits following a fragment screen: the table agreed that this is most likely the result of the library being “wrong”. We finished the discussion by asking if 2D fragments are more pan-class (not targeted to a specific class of targets) and 3D fragments may be more target-focused. The resounding answer was “Who knows, but why would they be?”

As this post is already getting long we’ll stop here, but for those of you who were also at the conference please add your comments. And if you missed this one, there are still several exciting upcoming events this year!

Sometimes Your Compound Sucks

Folks, let me wax grouchy. But, first, let me explain. My kids (10 and 7) play sports. I try to inculcate them with a sense of fairplay and sportsmanship. From their first game, I have always said to them: two rules to playing sports. 1. Never blame the refs (umps). 2. Never blame the equipment. It has gotten me through a lot of losses, hurt feelings, and it generally makes me feel like a good dad. I then take the money I am putting away for when they need therapy later in life for all the bad things I will/have done and buy beer. Trust me, this will come into play in at the end.
I have been trying to figure out how to review this paper from Bastien et al. It hasn't been easy. The kinder gentler version of Teddy is just not a hit. Go figure. This paper is aimed at delivering J. Med Chem. quality data derived from fragments on a mutant DHFR. DHFR is a long time drug target and is currently used in many approved therapies.

The paper all started out so well, with description of the fragment library, you know, fragments, "generally comprise[d} cycles -- often aromatic-- and were generally of an elongated structure." Seriously, in J. Med. Chem., they described their fragment library that way. There is no supplementary information.
With this excellently characterized fragment library, the screened all 100 compounds (what a herculean effort) and found 7 compounds in the high micromolar to millimolar range.
This is what they found...oh, and no inhibition in the biochemical assay for follow up compounds 1a, 4a,b,c,5a, and 6a. The depth of the SAR startled me. They did extensive SAR which led to this:
Now, starting from fragments which had pretty decent ligand efficiency, there were able to generate these horrendous monsters, with absolutely revolting LE. They then point out that these are "poorly optimized compounds." DUH.
They then did docking of the molecules and it shows that these symmetrical molecules for a U shaped molecule and suggest that intermolecular stacking may occur in solution. They then point out that this may be due to limitation in the docking software. Then tried to soak (9) into crystals, but there was a change in the active site pore electron density. And, again another excuse, "The pore lies on the crystallographic symmetry axes, combined with the fact that the ligand did not appear to be present at full occupancy, it prove impossible to interpret the electron density." YET, in the very next sentence, seriously the very next one, "From what could be seen, the density was not consistent with the U-shaped conformation of compound 9 as suggested by the docking results, but the poor quality of the electron density PRECLUDES DRAWING CONCLUSIONS [emphasis mine] on the actual bound conformation of the inhibitor."
So, now at this point, my brain literally explodes like this:
One more highly quotable line is: "Notwithstanding the precise mode of binding, symmetry appears to play a key role in binding in selectivity. " I assure you all I am NOT making this up.

There may be some redeeming social value in the paper, but I give up in finding it. Bastien et al., stop blaming the refs and the equipment!!!

Library design, search methods, and applications of fragment-based drug design

This is the title of a new book, edited by Rachelle Bienstock, which comes out of two symposia she organized at recent ACS Meetings (one of which is summarized here).

The book starts with an overview of fragment-based drug design by Bienstock. This is a thorough summary of the talks in both symposia, including those that did not end up as full chapters. This chapter also includes a useful table of available software relevant for FBLD.

The longest section of the book is devoted to designing and searching fragment libraries. In chapter 2, Dimitar Hristozov and colleagues at Eli Lilly and the University of Sheffield describe an algorithm for the de novo design of new molecules based on known reactions. Chapter 3, by John Badger of DeltaG Technologies, addresses the question of how to design libraries for crystallographic screening, with some emphasis on software. Chapter 4, by Ammar Abdo and Naomie Salim at the University Teknologi Malaysia, describes a “Bayesian interference network” for virtual screening as an alternative to conventional similarity searching. And chapter 5, by François Moriaud and colleagues at MEDIT, describes the researchers’ mining of the protein data bank (PDB) to understand the relationship between ligands and their protein pockets. This information is used for generating bioiosteric replacements and to generate new compound libraries for specific targets.

The next section of the book is focused on docking. In chapter 6, Zsolt Zsoldos of SimBioSys describes their high-speed eHiTS (electronic High Throughput Screening) engine for docking fragments. This chapter delves deeply into a statistical scoring function, and should be of particular interest for the mathematically-inclined. Chapter 7, by Peter Kolb at UCSF, discusses DAIM (Decomposition and Identification of Molecules), a program designed to break larger molecules into fragments, as well as computational methods for docking these derived fragments. He describes the use of this software to discover high affinity ligands for the kinase EphB4 and other targets.

The last section is devoted to fragment growing and linking. Chapter 8, by Eugene Shakhnovich and colleagues at Harvard, discusses FOG (Fragment Optimized Growth), which grows molecules by adding fragments such that the resulting molecules resemble those in a training set. This allows one to focus on regions of chemical space that are believed to be particularly productive or drug-like. And finally, in chapter 9 Zenobia founder Vicki Nienaber describes how fragment-based approaches are ideally suited for discovering drugs targeting the central nervous system.

By my count this marks the fifth book completely dedicated to fragment-based lead discovery, but its focus on computational methods still sets it apart from the others. That’s the fun thing about fragments: there’s something for everyone.

FBLD 2012 - registration now open

We are delighted to announce that registration is now open for the FBLD 2012 meeting to be held in San Francisco from 23rd to 26th September this year. Please visit the conference site and click through to registration.

This promises to be THE major fragment-based discovery conference of the past two years (you can read impressions of FBLD 2010 and FBLD 2009). A great list of speakers has already confirmed, and we still have plenty of room for more talks and posters. We plan to have some themed sessions around particular targets and approaches - these will be announced on the website as they are arranged.

In addition to great science, we have worked hard to keep the costs down, both for registration and hotel.

So - visit the site http://www.fbldconference.org.

Early bird rates expire on the 1st July 2012.

For a complete list of fragment-based events please see here.

Looking forward to seeing you in San Francisco!

Universal fragments

Third world diseases are popular academic drug targets, perhaps none more so than malaria. In a recent paper in Bio-Medicinal Chemical Letters, researchers from the University of Durak report their efforts to discover inhibitors of falcipain-1, a cysteine protease important for the parasite that causes malaria.

The researchers performed a functional screen of a small fragment library. Among many hits, the most potent were compounds 1 and 2, both with high ligand efficiencies. When these were linked together the resulting compound 3 had an improved potency, and adding a small substituent to yield compound 4 gave a sub-micromolar inhibitor.

Remarkably, not only was compound 4 potent against falcipain-1, it was also potent against several other important disease targets. In recognition of this pan-potency, the researchers have named compound 4 “hitinane.” Whether or not this discovery will lead to a drug, it undoubtedly will fuel many papers, patents, and grant proposals.