Fragment optimization without purification

Compound purification can be a major hassle: separating the desired product from starting materials, reagents, and byproducts often takes far longer than making the compound in the first place. As we’ve previously noted, this is especially true for small, polar fragments – which are particularly attractive for drugs. Two new papers address this challenge. (Shameless plug: my company Carmot Therapeutics also has a solution to this problem.)

In J. Med. Chem., Paul Brough and Vernalis colleagues describe their discovery of inhibitors of all four isoforms of pyruvate dehydrogenase kinase (PDHK), potential targets for diabetes and oncology. The ATP-binding site of these four enzymes is similar to that of oncology target HSP90, in which Vernalis has a long-standing interest.

A screen of 1063 fragments (each at 0.5 mM) against PDHK-2 yielded 78 hits that were positive in three different NMR-based assays and also ATP-competitive. These yielded a whopping 43 structures when soaked into crystals of the related isoform PDHK-3. Compound 6 was one, and the binding mode was very similar to that previously seen for the same fragment with HSP90. Fragment growing rapidly led to molecules such as compound 8, with low micromolar potency. This compound was almost equipotent against HSP90, but modeling suggested that it might be possible to further grow this molecule in a direction that would be accommodated in the PDHKs but not in HSP90.

The next step was to make a bunch of analogs, and here's where avoiding purification becomes advantageous. Specifically, the researchers turned to off-rate screening (ORS), which entails making compounds and then testing the impure mixtures using surface plasmon resonance (SPR) to look for those which dissociate more slowly. Since off-rate is not dependent on the concentration of ligand, a low yield shouldn’t change the results of the assay.

An initial library of 56 compounds led to the discovery of compound 18, and subsequent libraries and medicinal chemistry ultimately yielded VER-246608, which is a potent pan-PDHK inhibitor. As designed, it is also completely inactive against HSP90. The molecule is described more thoroughly in this Oncotarget paper, which reveals that despite activity against PDHKs in cells, VER-246608 is not particularly effective at slowing the proliferation of cancer cells. Still, it does appear to be a useful chemical probe for further exploring the biology of the PDHKs.

Shifting methods but staying with the theme of assaying impure compounds brings us to a paper in SLAS Discovery by Sten Ohlson, Brian Dymock, and colleagues at Nanyang Technological University and the National University of Singapore. The protein tested was HSP90, and the method used was weak affinity chromatography, or WAC (see here, here, and here).

Like SPR, WAC also uses an immobilized protein. However, whereas SPR provides the (kinetic) off-rate, WAC provides the (thermodynamic) dissociation constant, which is calculated from the change in retention time of the molecule as it passes through a column containing protein-bound resin. In this case the researchers synthesized a mixture of five different compounds which varied from 7-24% of the mixture. This crude sample was analyzed by WAC, and the resulting dissociation constants, ranging from 48-147 µM, were satisfactorily similar to the values obtained using pure compounds.

Both of these approaches should accelerate screening and facilitate the analysis of complicated mixtures, such as natural product extracts. It will be fun to watch for more examples.

Fragments vs PRC2 revisited: a chemical probe

Earlier this year we highlighted a paper from Novartis in which ligand deconstruction was used to deconstruct an HTS hit against the epigenetic target methyltransferase polychrome repressive complex 2 (PRC2). In a new J. Med. Chem. paper, Ying Huang and Novartis colleagues report a similar approach on a different HTS hit, ultimately yielding a promising chemical probe.

Compound 7 was identified as one of about 1400 hits from a high-throughput biochemical screen of 1.4 million molecules (described here at PLOS ONE). Crystallographic studies revealed that, like the previous molecule, this one also binds in the site on the EED subunit of PRC2 that normally recognizes trimethylated lysine 27 on histone H3.

Crystallography also suggested that the left half of the molecule didn’t seem to be making productive interactions with the protein. Lopping this off actually increased the activity and dramatically improved the ligand efficiency. Fragment-sized compound 8 was then subjected to extensive medicinal chemistry, ultimately resulting in EED226. Crystallography revealed that the binding modes of the initial hit and the final molecule are quite similar.

In addition to good biochemical activity, EED226 also shows good cell potency and impressive selectivity against other histone methyltransferases, kinases, and unrelated safety targets. It also shows excellent oral bioavailability in mice and acceptable pharmacokinetic properties. The compound caused complete tumor regression in a mouse xenograft model.

A separate paper in Nat. Chem. Biol. further characterizes EED226. A chemoproteomics study of a labeled version of EED226 revealed that it is remarkably selective for the PRC2 complex in human cell lysates. Also, EED226 is active against mutant cell lines that are resistant to other PRC2 inhibitors currently in the clinic, which are competitive with the cofactor rather than the trimethylated lysine residue. In fact, EED226 can bind to the PRC2 complex simultaneously with these other inhibitors, so dosing both together could give improved efficacy and slow the emergence of resistance.

As with the previous post on this target, the discovery of EED226 is a nice example of fragment-assisted drug discovery (FADD). Unlike that case, in which fragmentation led to an initial loss in potency, here trimming back the molecule paid immediate dividends.

Artists often talk about finding a sculpture within a stone by cutting away excess material. It is rewarding to see that chemists can use the same strategy.

Unexplored but promising fragments

Sir James Black famously said that the best way to find a new drug is to start with an existing one. A drug not only has to bind to a target with reasonable affinity, it also has to survive an onslaught of metabolic insults – and avoid doing too much collateral damage. Compound libraries are often populated with derivatives of known drugs, but as Richard Taylor and collaborators at UCB and Bohicket Pharma Consulting show in a recent J. Med. Chem. paper, there is plenty of untapped chemical real estate out there.

The researchers started by deconstructing all FDA-approved drugs into component rings. As they’ve previously shown (and presented), this gives a surprisingly small set: just 95 monocyclic rings (such as benzene and succinimide), 124 bicycles (purine and quinazoline), and 58 tricycles.

Next, they computationally combined these rings with one another in various ways, focusing on monocycles and bicycles to maintain low molecular weights. For example, one set contained all combinations of drug-derived monocycles connected either to another monocycle or to a bicycle by linkers containing up to four bonds. That provides about 14.4 million possibilities. Among commercially available molecules, about 1.6 million are monocycles connected to another monocycle or a bicycle by up to four bonds, but many of these monocycles and bicycles have never appeared in a drug. Remarkably, the overlap among the computed and commercial sets is less than 58,000 compounds: only about 3% of relevant commercial compounds contain two rings which have both appeared in a drug.

Of course, chemical space is large; how do things fare among fragments? The researchers examined a subset of theoretical molecules having two monocycles or a monocycle and bicycle connected by just two bonds and with molecular weights less than 280 Da. They also allowed “decoration” with a fluorine atom or a methyl, amino, or hydroxyl group. This provided 421,929 molecules – a sizable number but, as the researchers note, a small enough set to be tractable with computational docking approaches.

Even with this fragment set the commercial availability is less than 1%. In fact, less than half of the decorated monocycles and less than 40% of the decorated bicycles are for sale. This seems like a ripe business opportunity for enterprising vendors of fragments. Unfortunately the researchers do not provide a comprehensive list of structures, but the analysis would be relatively straightforward to repeat.

This paper draws similar conclusions to one we highlighted a couple years ago focused on kinase inhibitors. Some chemists enjoy the challenge of making entirely novel molecules, but it may be worth taking another look at more conventional pharmacophores, particularly when they are connected in new ways.

Ligand efficiency invalidated!

Practical Fragments has had quite a few posts on ligand efficiency (see here, here, and here, for starters). Ligand efficiency (LE) is defined simply as the free energy of binding for a ligand divided by the number of heavy atoms in the ligand. One of the criticisms of LE is that the definition of free energy depends on the definition of standard state, which may be different on different planets. With the discovery of silicon-based life on Venus, this is no longer just an academic argument. Indeed, a recent paper in Venusian Analytical, Physical, & Inorganic Discoveries describes an excellent case study.

Professor Perelandra and colleagues at East Eistla University performed a crystallograhic fragment screen on the enzyme silica hydratase, which is essential for the life cycle of the viciously parasitic Crystalline Horde. Fragment 1 binds in the active site, and although it has low affinity, structure-guided medicinal chemistry rapidly led to compound 42, with low nM activity in vitro and good efficacy in a silicon resorption model.

Things get even more interesting when you calculate the ligand efficiency values. The Venusians define standard temperature and pressure very differently from us. More importantly, they don't believe that standard state concentration should be 1 M. Given the extreme conditions on their home world, they choose a standard state concentration of 10 M.

LE = - ΔG/HA

(where HA = number of non-hydrogen atoms)

Thus, LE_Venus = -RTln(K_D/[A]⁰)/HA

(where T = 737 K and [A]⁰ = 10 M)

Using our terracentric definitions, the (impressive) LE of the fragment hit stays roughly the same during optimization, suggesting that the medicinal chemists have done a good job. However, by Venusian standards, the LE decreases!

This rock-solid example shows that Dr. Saysno was right: ligand efficiency is arbitrary and should never be used – on Venus.