FBLD 2012 Early Registration

40 superb speakers

The most beautiful city on the planet

And you

The biggest fragment conference of the year is happening in San Francisco this September.

We are still accepting posters, so if you have a fragment story to tell, this is the venue.

Early registration ends July 1, so sign up today!

To read about previous events in this series see here. And for a complete list of upcoming events, please see here.

Do You Need Structure To Prosecute Fragments?

As pointed out here, the majority of people think you need to have structure to successfully move fragments forward.  I, as has been noted previously, disagree and vehemently so.  However, if you fall in the category where you need that crutch you may be familiar with INPHARMA.  X-ray crystallography is the workhorse of structures, but in many cases (far more as we get into more and more complex targets), X-ray fails.  Old-fashioned solution NMR (with 15N, 13C, etc.) is just not fast enough to be a viable tool.  A few years back, INPHARMA was introduced to try to bridge the gap between the two methods.

  

INPHARMA (INter-ligand mapping for PHARmacophore MApping) is based upon robust ligand-based screening data.  Two weakly binding, and competitive, fragments are put into solution with the target.  If the compounds are weak enough (>10uM) it is possible to detect a NOE between protons on the two ligands.  This NOE is mediated by the active site, so the compounds must be binding in the active together (see below).  It is then possible to run very intense computational methods to determine the orientation of the ligands in the binding site (if the structure of the target is known).  

In this paper, Isabelle Krimm looks at INPHARMA's ability to discriminate different binding sites on a single target.  For this she uses, glycogen phosphorylase (a type 2 diabetes target) that has four distinct binding sites: active site, inhibitor site, allosteric site, and new allosteric site. 

Cpd 1 and 2 bind the inhibitor site, where Cpd 3 binds the new allosteric site.  Cpds 4 and 5 are proposed analogs of 2, Cpds 6 and 7 are analogs of 3, and Cpds 8 and 9 are "frequent-hitters".  NOESY experiments were recorded for the six fragments in the presence of 2 and 3.  All compounds exhibited intramolecular NOEs upon binding to the target.  Additionally, Cpds 2 and 4/5 showed intermolecular NOEs, as did Cpd 3 with 6, 7, 8, and 9.  Cpds 4 and 5 did not shows NOEs with 3, nor did 6, 7, 8, and 9 show NOEs to Cpd 2.  For fragments 4-7, competition data support that these are inter-ligand NOEs and that Cpds 8 and 9 are non-specific binders.  No intermolecular NOEs were seen to Cpd 1.  Cpd 1 has a IC50 of 1 uM, while Cpd 2 is 100uM.  This supports theoretical calculations that the two competitive binders must have binding constants no more than 8x different.  

In this paper, Lee et al. demonstrate the use of hyperpolarization to increase the sensitivity of INPHARMA. Dynamic Nuclear Polarization (DNP) uses electrons to transfer magnetization to nuclei leading to orders of magnitude increases in signal.  Lee et al. use DNP to increase the signal in INPHARMA significantly by hyperpolarizing one of the two competitive compounds, in a method they call HYPER-BIPO-NOE (Hyperpolarized binding pocket NOE)[which may be one of the worst not-even-acronyms ever]. 

1D HYPER-BIPO-NOE spectra, a) in full scale and
b) expanded to show transferred signals. Stacked spectra are, from top
to bottom: hyperpolarized ligand 1 with ligand 2 and protein (HYPERBIPO-
NOE); hyperpolarized ligand 1 with ligand 2, but without protein
(Control 1); hyperpolarized [D6]DMSO/D2O, with only ligand 2 and
protein (Control 2); Thermal spectrum of the HYPER-BIPO-NOE
sample (Thermal). The resonance from DMSO, which was suppressed
using presaturation, is designated by *.

This tremendous increase in signal can be extraordinarily useful for INPHARMA applications because they are able to obtain the inter-ligand NOE in a single scan.  The authors then go on to demonstrate that HYPER-BIPO-NOE-INPHARMA data is similar to STD-INPHARMA and can be used to generate binding poses. 

These two papers show that INPHARMA can be a useful tool to orient fragments similarly in a binding pocket.  INPHARMA requires that you have a weak binder that binds in a known site on the target.  For the current generation of targets this may exclude INPHARMA from being used.  It is also important to note that the choice of mixing time (which can range from 70-800ms) is a critical parameter.  The incorrect choice of the mixing time can lead to poor signal intensity and false negative results.  DNP, while turnkey if you have enough money, is not common in industry at all.  I would be surprised if HYPER-BIPO-NOE-INPHARMA actually gets any traction there at all.  There is also a poll with this post, please read and answer.

Fragments versus CDK4 and CDK6

The cyclin-dependent kinases (CDKs) were some of the earliest protein kinases targeted for drug discovery. They are important for cell-cycle progression, and thus cancer. However, selectivity among the multiple CDK family members has been challenging. In the June issue of ACS Med. Chem. Lett., researchers from Astex and Novartis describe the optimization of a fragment to a selective inhibitor of CDK4 and CDK6.

Astex has been working on CDKs for some time; one of Practical Fragments’ first posts described AT7519, an inhibitor of CDK1, 2, 4, 5, and 9 that is in multiple phase 2 clinical trials. In the new paper, the researchers start with a fairly potent CDK6 hit (fragment A). Crystallography suggested that replacing the pyrrole with a pyridine would provide better vectors from which to grow the fragment, leading to Compound B, which was still active. Growing in two directions then led to Compound 1, and extensive structure-based design led ultimately to Compound 6, which is selective for CDK4 and CDK6 over CDK1 and CDK2. In a panel of 35 additional off-target kinases, the compound displayed IC₅₀ values of 5 micromolar or worse. Compound 6 also showed target modulation in mice and tumor xenograft activity, albeit at fairly high doses.

The authors note that selectivity was a key goal, and that in the course of optimization they were willing to sacrifice potency against their desired targets in order to avoid hitting CDK1 and CDK2. The success of this strategy illustrates again the importance of maximizing ligand efficiency at the outset, as drops in LE can then be used to “pay” for other desirable properties. (Note also that the drop in LLE_AT is not quite as severe as the drop in LE.)

Some enthusiasts have argued that fragments provide a more efficient path to the clinic, and this can certainly be the case, as illustrated by the rapid progress of vemurafenib from fragment to drug. However, the current paper illustrates that advancing fragments can still require considerable resources: with 36 authors on two continents, it is clear that this project was not a walk in the park. It is, however, another illustration of starting with a fragment to develop a useful molecule.

Capillary Electrophoresis

One of the fun aspects of fragment-based lead discovery is the number of ingenious biophysical methods for finding low-affinity fragments. In a recent issue of J. Biomol. Screen., Carol Austin and colleagues at Selcia describe their approach, capillary electrophoresis, which they term CEfrag.

Capillary electrophoresis itself has been around for quite a while. It involves applying a high voltage across a thin capillary filled with liquid; a solution to be analyzed is injected, and the voltage causes migration of analytes (for example, proteins or small molecules). Analyte movement through the capillary depends on charge and “hydrodynamic radius,” which is a function of molecular size and shape. In the case of CEfrag, the idea is to start with a reporter molecule that can be readily detected, for example via UV absorbance. Under a standard set of conditions, this “probe ligand” will have a characteristic mobility. If an excess of protein that binds to the probe ligand is present in the running buffer, the migration time will shift. If an inhibitor is also present in the running buffer, this will prevent the probe ligand from binding to the protein, also causing the migration time to change. By running different concentrations of inhibitor and measuring the changes in mobility, the inhibition constant can be determined.

The researchers demonstrated their approach using that old work-horse of FBLD, the cancer target Hsp90. The known Hsp90 inhibitor radicicol was used as the probe ligand. A total of 609 fragments were screened individually at an initial concentration of 0.5 mM, yielding 42 fragments that reproducibly inhibited radicicol mobility by 20% or more. This ~7% hit rate is similar to that found by others for this target.

Only 12 of the 42 hits identified by CEfrag were also detected in a confirmatory fluorescence polarization (FP) assay, of which only 5 gave measurable IC₅₀ values. However, FP is not ideal for evaluating fragments. In fact, one of the CE hits that didn’t reproduce by FP was ethamivan, the starting fragment for the program that ultimately led to Astex’s AT13387, now in a phase 2 clinical trial for GIST.

To get a better sense of the quality of the CE hits, the researchers put 6 fragments into crystallography trials: 3 hits from both CE and FP, two from CE alone, and one that hit neither. The negative control didn’t produce a structure, whereas two of the FP-confirmed hits produced co-crystal structures (the one that did not had solubility issues). One of the two CE-only hits (ethamivan) also did.

With a throughput of 100 compounds per day per instrument, this is not a high-throughput method, but it is comparable to many other biophysical approaches. Also, the low protein consumption and ability to use unmodified protein are selling points. Have you tried CEfrag? If so, what do you think?

Fragments versus Ras – Part 2

Practical Fragments recently highlighted a paper from Genentech in which researchers there discovered fragments that block the activity of the prominent oncology target Ras. Illustrating just how much interest there is in this protein, Stephen Fesik and colleagues at Vanderbilt University have just reported results of their own work in Angew. Chem. Int. Ed.

Fesik is famous for SAR by NMR, the first truly practical approach to fragment-based lead discovery. In the current work, the researchers also used NMR (HSQC with ¹⁵N-labeled protein) to screen 11,000 fragments, yielding about 140 binders to the GDP-bound form of K-Ras. A number of these were then further characterized crystallographically: of 20 cocrystal structures obtained, all of them were found to bind in the same hydrophobic pocket identified by the Genentech researchers. Fesik and colleagues also noticed a nearby, electronegative cleft, and grew one of their fragments (compound 1) to take advantage of this. This led to compound 12, the most potent compound reported. In addition to binding to the GDP-bound form of K-Ras as assessed by NMR, this compound also inhibited Sos-mediated nucleotide exchange in a functional assay.

Overlaying one of these compounds (blue – similar to compound 12) with the Genentech compound DCAI (red) reveals that while both compounds bind in the same hydrophobic pocket, they make very different contacts.

Of course, it still remains to be determined whether this is a ligandable site on the protein (ie, whether these – or any – molecules can be advanced to high potency). Given the importance of Ras, it’s certain that lots of people are doing their best to find out.

Poll results: how big are your fragments?

The poll results are in, and it looks like most of the 46 respondents look askance at fragments that are larger than about 20 heavy atoms:

Since each non-hydrogen atom adds about 13 Da to a molecule, this means fragment sizes are limited to about 260 Da, well below the Rule of 3. And some folks are even more stringent – 30% of respondents set an upper limit of 16 non-hydrogen atoms, or about 210 Da.

By way of comparison, I looked at the size of fragments in a fairly large (albeit somewhat dated) review. Of the 42 fragments reported, 79% consist of 20 or fewer heavy atoms, so clearly this is a fruitful area.

Of course, as the graph above shows, larger fragments have been discovered and advanced, but perhaps it is generally better to avoid the more obese fragments.