Fragments vs membrane proteins with TINS

Fragment-based ligand discovery owes much of its success to the rise of biophysical techniques such as NMR, crystallography, and – more recently – surface plasmon resonance. These have allowed the discovery of fragments against a wide range of proteins, but one notable exception has been membrane proteins, the targets of more than half of marketed drugs. In a recent issue of Chemistry and Biology, Gregg Siegal and colleagues take a crack at this diverse group of proteins.

The researchers, from Leiden University, ZoBio, and elsewhere, use an NMR-based technique called target immobilized NMR screening, or TINS. In this method, a protein is immobilized onto a solid support. A reference protein is also immobilized; this reference is usually a well-characterized protein that does not bind to many small molecules. Each protein is then put into its own compartment of a two-compartment flow-cell, and this is inserted into an NMR spectrometer. Mixtures of fragments are then flowed through both chambers: those that interact with protein show a reduction in the amplitudes of their NMR spectra. By choosing fragments that show such a reduction for the target protein and not the reference protein, fragments that bind to the target can be differentiated from those that bind to proteins in general. After each NMR experiment, the fragments are washed away and replaced with a new set of fragments. TINS has been applied to a number of soluble proteins, as reviewed here. Remarkably, the immobilized protein samples often remain stable through hundreds of screening cycles.

Membrane proteins are notoriously difficult to crystallize or characterize by NMR. Moreover, it is often difficult to obtain enough protein to work with. However, since TINS relies on a decrease in signal from the fragment rather than a signal from the protein itself, Siegal and colleagues tested whether they could use the technology to discover fragments that bind to membrane proteins.

The researchers chose a protein called disulphide bond forming protein B (DsbB), which is found on the inner membrane of E. coli and other Gram-negative bacteria and may be important in virulence factor folding. One of the challenges of membrane proteins is keeping them properly folded, and the researchers used two different approaches to do this, either detergent micelles or “nanodiscs,” lipid bilyaers surrounded by a scaffold protein. Using less than 2 milligrams of DsbB, the researchers used TINS to screen a set of 1071 fragments in groups of about 5 each, with each fragment present at 500 micromolar concentration, a process that took 5 and a half days.

The TINS process led to 93 hits, a respectable hit rate of 8.7%. Each of these was then tested in a functional assay at 250 micromolar concentration, and more than half of the hits inhibited DsbB activity by at least 30%. Eight of these were subsequently characterized using full IC50 curves and kinetic analysis. The potencies were impressive, ranging from 7 micromolar to 193 micromolar, with ligand efficiencies as high as 0.45 kcal/mol/atom. DsbB has the advantage that it has been characterized structurally, and the researchers used chemical shift information from 2-dimensional NMR experiments to show that the fragments could be divided into two groups, with one set competing with a quinone cofactor and the other binding at a different site.

This paper demonstrates that it is possible to find fragments that bind to membrane proteins. Of course, the next question is, what can you do with the fragments? In this case there were structural data about the target, but this will not generally be true for membrane proteins, and in the absence of structure, advancing fragments to leads can be challenging. On the other hand, medicinal chemists have been developing drugs against membrane targets for decades without knowing their precise structures, so perhaps the challenge is as much psychological as scientific.

Allosteric FPPS inhibitors – not so negative

The protein farnesyl pyrophosphate synthase (FPPS) has long been a target of drugs for osteoporosis, and some data have suggested it could be a productive target for other diseases too. However, approved drugs that target FPPS contain two phosphonates, highly negatively charged moieties that, while nicely directing the drugs to bone, lead to low plasma and soft tissue levels. Researchers at Novartis have now identified fragments that bind to an allosteric site on FPPS and so lack the phosphonate moieties. An article in the September issue of Nature Chemical Biology describes the discovery of these fragments and how they were advanced to nanomolar inhibitors.

The researchers, led to Wolfgang Jahnke in Basel, Switzerland, started by screening a library of only 400 fragments using NMR. Several low affinity (millimolar) hits such as compound 1 were identified, but surprisingly these were not competitive with bisphoshonate drugs, and some even bound synergistically. Crystallography revealed that they were binding in a previously undiscovered allosteric site. (The same group also used fragment screening to explore an allosteric site in another protein.)


To follow up on these observations, the researchers tested 40 related compounds in the Novartis internal compound collection, again using NMR to detect binding. This led to the more potent compound 5, and two rounds of focused library assembly and screening led to low micromolar inhibitors such as compound 7. Structure-based design led to molecules such as compound 11, which has comparable potency to approved drugs that target FPPS. Compound 11 was further characterized using ITC and crystallography, and although its two carboxylic acids likely account for a relatively low cellular permeability, it does not show any affinity for bone.

Interestingly, a high-throughput screen conducted against FPPS did not yield any inhibitors with an IC50 better than 5 micromolar. So in this case not only did a fragment-based approach discover a new series of molecules against a new site on an old target, it succeeded where conventional HTS didn’t.

Protein-templated click chemistry – just add copper

We’ve written previously about protein-templated chemistry (here and here), in which a protein catalyzes the formation of a more potent inhibitor from two lower affinity fragments. Of course, proteins aren’t the only things that can catalyze reactions: copper is well-known to promote the cycloaddition between azides and alkynes. Like peanut butter and chocolate, it turns out that copper in the context of a protein can be even better than either alone, as reported in a recent issue of Angew. Chem. by an international team of researchers from Japan and the US.

The researchers were interested in using in situ click chemistry to discover inhibitors of histone deacetylases (HDACs), and they decided to see if they could use an activity assay to detect the formation of inhibitors formed in situ. They incubated two different hydroxamic-containing alkynes (known HDAC inhibibitors) with 15 different azides in the presence of HDAC8 and looked for enhanced inhibition of the enzyme. Of these 30 combinations, they found a single hit: the reaction of compound 1b with compound 2o (see figure).


However, there were several oddities. First, the linked compound (anti-3) is no more potent than the initial hydoxamic-containing fragment. Second, only the anti isomer was formed, despite the fact that the syn isomer is almost 10-fold more potent. Finally, the yields of anti-3 were much higher than typically observed in these sorts of experiments. This made the researchers suspicious, and after a series of experiments they determined that trace amounts of copper, most likely introduced in the synthesis of 1b, had incorporated into the active site of HDAC8 and were serving to accelerate the reaction. A small amount of copper in the absence of protein was unable to catalyze the reaction, nor was the protein alone when copper was carefully removed.

There are a number of interesting implications from this paper, but one in particular is rather sobering: in situ assembly screening does not necessarily yield the most potent inhibitor. I suspect this is a general feature of kinetically-guided methods of inhibitor discovery, but what do you think?

Fragments in the Clinic: 2010 Edition

It’s been a while since we last tried to tabulate all the drugs derived from fragment-based drug discovery that have entered the clinic. Below is an attempt, culled together from a variety of sources. Those that have been covered on Practical Fragments are hyperlinked to the relevant post.

I realize that some of these 18 drugs have been quietly discontinued, but I’m also sure I’m missing others that have entered the clinic. If you know of any, please add them in the comments.

Phase 3
PLX-4032 Plexxikon B-RafV600E inhibitor

Phase 2
ABT 263 Abbott Bcl-2/Bcl-xL inhibitor
ABT 869 Abbott VEGF & PDGFR inhibitor
AT9283 Astex Aurora inhibitor
LY-517717 Lilly/Protherics FXa inhibitor
Indeglitazar Plexxikon PPAR agonist
VER-52296/NVP-AUY-922 Hsp90 inhibitor

Phase 1
ABT-518 Abbott MMP-2 & 9 inhibitor
ABT-737 Abbott Bcl-2/Bcl-xL inhibitor
AT13387 Astex Hsp90 inhibitor
AT-7519 Astex CDK1,2,4,5 inhibitor
DG-051 deCODE LTA4H inhibitor
IC-776 Lilly/ICOS LFA-1 inhibitor
LP-261 Locus Tubulin inhibitor
PLX-5568 Plexxikon Kinase inhibitor
SGX-393 SGX Bcr-Abl inhibitor
SGX-523 SGX Met inhibitor
SNS-314 Sunesis Aurora inhibitor

Fragments in the Clinic: AT13387

We recently discussed BACE, a target that has been tackled by FBDD due to its intractability to other methods. The subject of this post is quite the opposite: the anticancer target Hsp90 has proven very amenable to a variety of approaches, including fragment methods (see here and here); close to a dozen compounds targeting Hsp90 are in the clinic. Now Astex has detailed their work in this area with two back-to-back papers in a recent issue of J. Med. Chem. describing the discovery of AT13387.

The first paper, by Christopher Murray and colleagues, actually presents the discovery of two separate series of inhibitors. The researchers started with a library of about 1600 fragments and used NMR techniques (water LOGSY) to identify hits against Hsp90. Competition with ADP allowed them to identify molecules that bind to the nucleotide binding site. In all, 125 fragments were taken into crystallography, using both co-crystallography and soaking, resulting in 26 co-crystal structures. Four of these structures are described in some detail, with two leading to potent inhibitors. Throughout the process, isothermal titration calorimetry was used to measure dissociation constants.

In the first series, compound 1 was identified as a weak hit (see Figure 1). Virtual screening led to the purchase of a few variants, including compound 5, with roughly 100-fold improved affinity. Interestingly, the crystal structure of compound 1 bound to Hsp90 showed that the molecule was twisted around the bond connecting the two aromatic rings, despite this not being energetically optimal for the unbound molecule. By substituting the phenyl ring of compound 5 to stabilize this twisted conformation the researchers were able to improve the potency another 20-fold (compound 9), along with a boost in ligand efficiency. Further structural work suggested adding another chlorine to fill a lipophilic site as well as adding a solubilizing group, ultimately leading to compound 14, with low nanomolar binding affinity and low micromolar cell activity.

Figure 1

In the second series, compound 3 (which is actually itself a drug, ethamivan) had only modest ligand efficiency, but crystallography suggested that replacing the methoxy group with something slightly larger and more lipophilic would improve the interactions, a hypothesis borne out by the increased activity of compound 17 (see Figure 2). Increasing the lipophilicity of the amide side chain to take advantage of protein flexibility led to a further two orders of magnitude increase in potency (compound 28). Finally, the researchers were able to use the known binding mode of a natural product to add an additional hydroxyl group, leading to compound 31, with sub-nanomolar affinity (more than a million-fold more potent than the initial fragment!) and mid-nanomolar cell activity.

Figure 2

An impressive feature of both these examples is that, through the use of elegant medicinal chemistry, the researchers were able to improve ligand efficiency throughout the course of affinity improvement. Of course, it helps that they were working on a crystallographically friendly target for which several other groups had published extensive SAR, but these are nonetheless beautiful case studies. As the researchers point out, “in terms of the efficiency of the added groups, the two fragment to lead campaigns… are among the most efficient ever reported.”

But the story doesn’t end there. The second paper, by Andrew Woodhead and colleagues, describes the further optimization of compound 31 to the clinical candidate AT13387. Despite its impressive biochemical and cell potency, compound 31 had only modest activity in a mouse xenograft model, as well as a short plasma half-life. Not surprisingly the hydroxyl groups were found to be points of metabolism, but initial efforts at capping these or changing their electronics either proved detrimental to activity or did not improve the pharmacokinetics. This led to a medicinal chemistry focus on the isoindoline portion of the molecule: a number of positively charged moieties were added at various positions to try to change the overall properties of the molecule. Several substituents were tolerated, and seven related molecules were taken into preclinical candidate selection to look for optimal in vivo properties, solubility, and selectivity against P450 and hERG. AT13387 (see Figure 2) was chosen as the molecule having the best overall profile and entered human clinical trials for solid tumors.

This second paper is a valuable companion to the first: it is particularly notable that, on the simple measures of biochemical and cell potency, AT13387 is no better than compound 31. This emphasizes yet again that affinity is only the first step in drug discovery – it’s a long road from a good lead to the clinic, and an even longer road from there to a marketed drug.