New PAINS, and their painful mechanisms

Pan-assay interference compounds – PAINS – are a topic that has come up repeatedly at Practical Fragments (see here, here, and here for starters). Indeed, they form the basis of our occasional (and controversial) “PAINS shaming” series (see here, here, here, and here). In a paper recently published online in J. Med. Chem., HTSPains-master Mike Walters (University of Minnesota) and collaborators at the Mayo Clinic College of Medicine and AstraZeneca characterize several new classes of PAINS (also covered at In the Pipeline). I was honored with the invitation to write a Viewpoint on the topic. Since both papers are open-access I’ll just briefly touch on a few key points here.

First, one of the only critics of the PAINS concept is concerned that PAINS were originally defined on the basis of their over-representation as screening hits in one set of assays. The new paper goes beyond empiricism to characterize mechanisms, which involve non-specific reactions with thiols. (The “non-specific” aspect is key to their undesirability, as covalent drugs can be quite attractive.)

Second, just as “not every clam will hurt you”, not every molecule with a PAINS substructure will show activity in every assay. These “structure-interference relationships” (SIR) can be mistaken for “structure-activity relationships” (SAR), making PAINS all the more insidious. The researchers explore some of the reasons for the observed SIR.

Third, one of the saddest parts of the new paper is a list of dozens of references in the supplemental information in which PAINS were reported as screening hits or probes. It’s a safe bet that most – if not all – of these should be disregarded.

Finally, publishing this list of new PAINS will allow people to steer clear of them. To borrow from Hippocrates: chemical space is big, life is short. Why waste time working with chemotypes known to be pathological? 

Heptares Get Bought

Yesterday brought news that Heptares was purchased by Sosei.  The deal is for 180$MM upfront and 220$MM in royalties.  Similar to the Astex deal, Sosei is not absorbing Heptares, but is keeping them as a wholly owned subsidiary.  In terms of clinical assets, Heptares didn't show up on the 2015 version of fragments in the clinic, but they do have a p1 asset, a M1 agonist.  As if we needed further validation, this deal shows the power of SBDD/FBDD and gives a good idea off how it is valued.  Congratulations to our friends at Heptares.

19F Target-based Screening Reduced to Practice

If you read this blog you know I love 19F NMR.  I am a big fan of it for ligand based screening or as a secondary screen in target-based mode.  Well, this paper is the first to use target-based NMR to screen small molecules.  Using 3-fluorotyrosine-labeled protein and using what they call Protein observed fluorine NMR (PrOF-NMR), the interrogate a PPI (CBP/p300-KIX) and determine this interaction's ligandability. 

Starting from the Maybridge Ro3 Library, they found 508 19F containing fragments.  These were put into 85 mixtures (5 or 6 compounds each) and screened at 833uM and 2.5 % DMSO (Figure 1, KG-501 is a control compound).  Each experiment was performed in 5 minutes (with a quick reference spectrum) which is faster than SOFAST HSQC (for 15N labeled proteins).  15 mixtures gave hits which upon deconvolution gave 4 actives (>2 SD in chemical shift), a 0.8% active rate.

Figure 1.  Typical 19F Data

 They then titrated the four confirmed fragments to determine the Kd, which for 3 of them was in the mM range.  They followed this up with Analog by Catalog and developed some SAR.  Lastly, they used H-N HSQC to verify that the compounds do bind and where they data shows.  They do. 

Some thoughts on this.  The use of 3FY is only one amino acid that can be used.  Fluorotryptophan can also be used, so this method can easily be applied to other systems.  Secondly, 19F can accomodate much larger pool sizes (within reason).  And there is no reason why both could not be used.  Of course, one of the things not noted here is that they produce single mutants in order to ID each individual residue.  I think you could live without residue specific assignments and still get tremendous value out of this method.  I would be curious what others think.  We bring up impractical tools all the time, so I really want to applaud this paper.  Here is a very practical new method for screening. 

MELK part 2: fragment deconstruction

Last week we highlighted a paper from Chris Johnson and collaborators at Astex and Janssen in which they used fragment-growing to develop a selective inhibitor of maternal embryonic leucine zipper kinase (MELK). The paper immediately following in ACS Med. Chem. Lett. also describes the team’s efforts to discover MELK inhibitors, but using a very different approach.

In fact, the second paper doesn’t really start from fragments. The researchers were interested in designing compounds that bind MELK in a particular fashion: type II inhibitors fit into the hinge region but also insert themselves deep into a back pocket which is accessible when the so-called DFG-loop swings open (DFG-out). Since Type II inhibitors make more interactions with the kinase, they have the potential to be more selective.

The problem was that all previous crystal structures of MELK were “DFG-in”, so the researchers couldn’t use crystal soaking. Instead, they turned to structure-based design for molecules that would be able to span the hinge region and the back pocket. Happily, they succeeded with compound 2, and further optimization led to the low nanomolar compound 7. Co-crystallization experiments using a related molecule revealed that the compound binds as expected with MELK in the DFG-out conformation.

Compound 7 was tested in a panel of 243 kinases and inhibited 31 of them >50% at 1 µM; besides MELK, six other kinases were inhibited with IC₅₀ values < 100 nM. This isn’t terrible, but it is far from the selectivity seen with MELK-T1, the Type I inhibitor discussed last week. Thus, one can’t assume that Type II binders will necessarily be more selective than Type I binders.

Fragments enter the picture at the very end of the paper, when the researchers “deconstructed” their molecules. Simply removing the aminomethyl group from compound 2 to give compound 8 reduced affinity by more than ten-fold. This was expected because crystallography had already revealed that this moiety makes electrostatic interactions with aspartate and glutamate residues in the protein.

More surprisingly, removing the phenyl group from compound 8 produced a molecule with greater affinity and ligand efficiency than the initial compound 2! The researchers determined the crystal structure of this (compound 9) bound to MELK and found that, in contrast to the other molecules, it binds in the DFG-in conformation. The isoquinoline hinge binder actually binds in a similar manner as it does for its DFG-out binding cousins, it’s just the back pocket that is cut off. The researchers speculate that the DFG-in conformation of the protein may be lower energy, giving the edge to compounds that bind to this state. Whether or not this is the case, it is certainly another reminder of the remarkable plasticity of proteins.

Fragments vs MELK part 1: a chemical probe

As we recently noted, there are hundreds of human kinases in the human genome, and figuring out what they do is not easy. Selective small molecule inhibitors can probe an uncharacterized kinase’s function, but these don’t exist for the majority of proteins. Such was the case for maternal embryonic leucine zipper kinase (MELK), a potential anti-cancer target. In a recent paper in ACS Med. Chem. Lett., Chris Johnson and collaborators at Astex and Janssen describe how they created one.

The researchers started with a screen of the ~1500 Astex fragment library using both ligand-observed NMR and protein thermal shift assays. Hits were soaked into crystals of MELK, resulting in “a large number” of structures of fragments bound to the hinge-binding region. It is certainly possible to develop selective inhibitors that bind to the hinge region, but doing so is seldom straightforward, and thus the researchers were particularly interested in unusual fragments. Compound 1 caught their attention because it makes just a single hydrogen bond with the hinge region via the carbonyl oxygen in the fragment; most other hinge binders form two or three hydrogen bonds.

Compound 1 was well-positioned for fragment growing, and the addition of a phenol moiety (compound 2) led to a nice boost in potency and maintenance of ligand efficiency. Crystallography revealed that the phenolic oxygen was both a hydrogen bond donor as well as an acceptor, and replacing this moiety with a pyrazole led to compound 4, with slightly better potency. Pyrazoles themselves are often hinge binders (two of Astex’s clinical compounds, AT9283 and AT7519, contain them), but crystallography revealed that the binding orientation remained the same as in the original hit.

Expanding the aliphatic ring of compound 1 by one methylene gave compound 5, with a higher affinity and a slightly better fit to the protein, and adding bits from compound 4 gave mid-nanomolar compound 7, or MELK-T1. Impressively, the researchers improved the ligand efficiency of their molecules even as they became larger.

Finally, the moment of truth: would optimizing a fragment with an unusual hinge-binder lead to a selective inhibitor? The researchers tested MELK-T1 in a panel of 235 kinases, and happily only 6 were inhibited >50% at 1 µM [compound]. Notably, these did not include AMPK, which has 60% identity to MELK in the kinase domain. MELK-T1 was also cell-permeable.

This is a classic example of FBLD enabled by a robust protein construct suitable for crystal soaking. Getting to MELK-T1 required the synthesis of only ~35 compounds, and should lead to some interesting biology. At the same time, the researchers took a different approach to come up with another series of leads, which will be the subject of my next post.

Structure based Design on Membrane Proteins

GPCRs are a big target class, which have historically be unamenable to FBDD/SBDD.  However, recent work has changed this thinking.   Membrane proteins are being viewed as increasingly ligandable and amenable to FBDD.  In this paper, Vass and colleagues show their computational approach to indentifying multiple fragment binding sites amenable to linking.  

Recent clinical evidence supports the effectiveness of dual dopamine D2 and D3 antagonists or partial agonists in schizophrenia, depression, and bipolar mania. D2 antagonism is required for the antipsychotic effect, and D3 antagonism contributes to cognitive enhancement and reduced catalepsy.  Dual acting compounds should show higher activity to D3 than D2 (due to differential expression levels).  To this end, they apply their sequential docking protocol to identify potential points for fragment linking on the D3 crystal structure and D2 homology model.  These two targets have almost identical primary binding sites, but selectivity can be modulated through the secondary site.

In short, their in house fragment library consisted of 196 amine containing fragments for the primary site.  Second library of 266 fragments of cyclohexyl or piperidines.  Then, the first library was
docked to the apo receptor structures,then the docking poses were merged with the receptor, new grids were constructed including the merged ligands, and the second fragment library was docked to the partially occupied binding sites.  

Table 1.

As shown in Table 1, they synthesized three of their compounds and did generate potent and selective D3/D2 antagonists.  Linking is hard.   It still comes down to the right linker and all that entails.  Finding that right linker is made much easier by having structural data, as shown here.  This is a nice example of experimentally verifying in silico predictions. 

Fragments vs DsbA: targeting bacterial virulence

The quest for new antibacterial agents can seem quixotic: no sooner have you found a killer molecule than the bugs have developed resistance to it. Evolution is hard to beat, particularly when it comes down to life or death. But what if you could lower the stakes? Many bacteria express virulence factors that are not essential for survival but are important for colonizing their host. Perhaps targeting these would be less prone to generating resistance.

Virulence factors often contain disulfide bonds, and the bacterial protein DsbA is essential for catalyzing their formation. In a paper published recently in Angew. Chem. Int. Ed., Begoña Heras (formerly University of Queensland), Jamie Simpson and Martin Scanlon (Monash University) and collaborators describe a fragment-based approach against the E. coli. version of this target. (See also here for Derek Lowe’s thoughts.)

The researchers started with an STD NMR screen of an 1132 fragment library from Maybridge, with compounds in pools of 3 to 5 (each at 0.3 mM). This yielded 171 hits, 37 of which showed appreciable chemical shift perturbations (CSPs) in a two-dimensional HSQC ¹⁵N-¹H NMR assay. All of these were relatively weak, with none showing saturation at 1 mM fragment concentration, but they all appeared to be binding in a hydrophobic groove adjacent to the active site.

The 37 hits clustered into eight different structural subclasses, one of which – the phenylthiazoles – is described in detail. The Monash fragment library was designed with SAR-by-catalog in mind, and 22 commercial analogs were purchased and tested in the HSQC assay to assess the SAR. Several of the compounds were soaked into crystals of DsbA, in one case leading to a structure in which two fragments were bound stacked on top of each other in the hydrophobic groove. However, this binding mode was inconsistent with the NMR data, and indeed co-crystallography of the same fragment revealed a 1:1 complex with the protein, also in the hydrophobic groove. (As an aside, this is an interesting case of crystal soaking and co-crystallization giving different results; are readers aware of others?)

The crystal structure was used to inform fragment-growing, ultimately leading to molecules with dissociation constants around 0.2 mM as assessed by surface plasmon resonance and with similar IC₅₀ values in a functional assay. One of these compounds was also tested against E. coli. DsbA is not needed for bacterial growth in rich media but is necessary for motility, and happily the assays showed just this – the compound did not affect growth but did inhibit cell motility.

Although the molecules are still too weak to answer the question of whether targeting DsbA will be a viable antibacterial strategy in vivo, this paper presents promising starting points, along with a wealth of data (including 78 pages of supporting information!) And if you want to learn more, Martin Scanlon is one of the organizers of the FBLD symposium at Pacifichem this December – so you can ask him questions in person in Honolulu!