30 January 2010

Commercial fragments – 2010 edition

A particularly active discussion on LinkedIn has prompted us to update the list of commercially available fragments - looks like a number of new additions since our last post. Note that we are restricting this list to suppliers specifically offering fragments, as opposed to general compound libraries (which of course will likely contain many fragments, but will make this list too unwieldy, and are probably less of a go-to source for people just entering the field).

4500 Rule of 3 (RO3) compounds
1800 compounds MW < 250, solubility > 0.1 mM (PBS) and > 10 mM (DMSO)

ChemBridge Corporation
~5000 RO3 fragments

~1900 fragments, expanding to "3-D" fragments

1190 fragments w. strict Rule of 3
11,717 fragment extension set

140 “3-D” fragments
198 diverse fragments

Iota Pharmaceuticals
Focused on fragment-based discovery
1500 fragments available for purchase
4000 additional fragments in collaboration with Vitas-M

Key Organics
6335 RO3 fragments

Life Chemicals
22,000 fragments w. MW < 300, clogP < 3
9000 fragments with rotatable bond, PSA, HBA limits

Maybridge (Thermo Fisher Scientific)
30,000 fragment library (MW < 350)
1000 RO3 fragments w. aqueous solubility > 1mM
1500 Br- and 5300 F- containing fragments

~3800 fragments, mostly RO3 (increased HB acceptors for kinases)
All have at least one ring; filtered to remove certain functionalities

Prestwick Chemical
720 fragments including known drugs, RO3 compliant

Pyxis (with Merachem)
317 RO3 fragments from drugs and natural products
Filtered to remove certain functionalities

Zenobia Therapeutics
352 very small fragments (Avg. MW 155)
Verified solubility at 200 mM in DMSO

I’ve also been told that BioFocus sells fragments, but can’t find this on their web-site.

Finally, Cambridge MedChem Consulting has a nice list with more detailed descriptions of many of these suppliers.

Are there companies we’re missing? Does anyone have any experience with any of these that you would like to share?

24 January 2010

Pinning fragments on Pin1

Pin1, a potential anti-cancer target, catalyzes isomerization around phosphoserine-proline and phosphothreonine-proline bonds. Its binding site is relatively shallow, complicating efforts to discover small, non-peptidic inhibitors. In a recent paper in Bioorg. Med. Chem. Lett., Jonathan Moore and colleagues at Vernalis describe their fragment-based approach to tackling this problem.

The researchers used NMR-screening of roughly 1200 fragments to identify five that competed known inhibitors; Compound 4 (see figure) was the most potent, with an IC50 of 16 micromolar in a functional assay. NMR experiments showed a weaker binding interaction, on the order of 200 micromolar, and surface-plasmon resonance (SPR) experiments were even less conclusive: the compound showed super-stoichiometric binding, indicating that multiple molecules were interacting with the enzyme rather than sitting specifically in the binding site. However, the researchers were able to obtain a crystal structure showing that the molecule binds in a hydrophobic pocket at the active site. Although they don’t mention it in this paper, in public presentations the researchers have reported seeing additional molecules of Compound 4 pile on top of each other, essentially forming a stack on top of the protein. Perhaps, as the authors suggest in the supplementary material, this is an example of a particularly insidious aggregation phenomenon: a legitimate hit that can also form aggregates.

In order to access other parts of the protein, and reduce the propensity for aggregation, the researchers relied on analog screening and modeling to generate Compound 18a, which is a more “three-dimensional” molecule. Further elaboration led to a series of compounds such as 19e, with low nanomolar potency, as well as one-to-one binding in the SPR assay.

Despite their potency, these molecules were inactive in cell-based assays, likely due to high polar surface areas and their resulting low cell permeabilities. To fix this, the researchers replaced the benzimidazole fragment with a naphthyl group, which led to a decrease in biochemical potency but did lead to cell-active molecules such as Compound 23b. Moreover, a crystal structure revealed that this molecule binds in a similar manner to the original fragment 4.

This paper exemplifies another example of fragment-assisted lead discovery: the original fragment morphed from an indole to a benzimidazole to a napthyl group, yet the final molecule still owes a debt to the initial fragment.

11 January 2010

There and back again: fragments and BACE-1

One of the many interesting talks at FBLD 2009 was by Daniel Wyss of Schering-Plough on how fragment-based screening was used to discover potent and selective inhibitors of the Alzheimer’s disease target BACE-1. Two papers published online in J. Med. Chem. now begin to tell the full story.

BACE-1 is an aspartyl protease, a class of enzymes that has proven to be druggable, as illustrated by the number of HIV-1 protease inhibitors on the market. However, BACE-1 has an unusually shallow, flexible, and hydrophilic active site, and its location in the brain means that candidate drugs need to be particularly small with a limited number of hydrogen bond donors.

The first paper discusses how Wang and colleagues used 15N-HSQC NMR screening of a library of ~10,000 compounds, about half of them fragments, against the BACE-1 catalytic domain. This resulted in 9 distinct classes of hits, some of which were as potent as 30 micromolar as judged by NMR-based dissociation constants. The compound most extensively pursued was compound 2 (see figure), an isothiourea. Some 200 analogs of this were present in the corporate collection (an advantage of working in big pharma!), and 15 of these showed activity in an enzymatic assay, of which compound 3 was the most potent. Extensive NMR analysis and an X-ray crystal structure revealed that the isothiourea makes hydrogen-bond contacts to both catalytic aspartates and extends towards the S1 pocket and S3 subpocket (S3sp).

Isothioureas are potentially toxic and unstable, so structure-based design was applied to replace this moiety. One outcome was a series of 2-aminopyridines such as compound 4. Unfortunately, although they showed measurable binding by NMR and some could even be characterized crystallographically, most had little or no activity in a BACE-1 functional assay.

That’s where the second paper by Zhu and colleagues comes in. Careful analysis of the crystal structure of compound 3 combined with parallel synthesis led to a series of iminohydantoins (or cyclic acylguanidines) such as compound 23. Interestingly, a close analog of this compound made similar contacts but flipped to another orientation. These different binding modes complicated medicinal chemistry efforts, requiring parallel chemistry and NMR-based screening (as most early compounds were at best only weakly active). Ultimately this effort yielded potent inhibitors such as compound 39, with nanomolar biochemical activity. However, the compound also has a clogP of 7.5, a molecular weight of more than 500 Da, and only modest bioavailability, so turning this into a brain-active drug could be problematic.

Strikingly, truncating a large portion of the molecule (to generate compound 40) yielded a much smaller compound that, despite its reduced biochemical potency, had an improved ligand efficiency, as well as measurable brain penetration. Further simplification led to compound 41, which, being rule-of-three compliant, can be considered a fragment. In other words, these two papers report the optimization of a low-affinity fragment to a high-affinity ligand and on to a medium affinity fragment. Given that the second paper is subtitled “Part 1”, we can look forward to reading further chapters.

Many FBLD publications report the rapid discovery of new leads against established targets such as kinases or Hsp90. BACE-1 is just the opposite: references suggest that the program had been in place since before 2004. These papers, along with previous papers (for example here and here) on BACE-1 from AstraZeneca and Astex, illustrate that FBLD is also powerful for discovering leads against difficult targets.

06 January 2010

Fragments in the Clinic: DG-051

Last August we highlighted work from deCODE on their leukotriene A4 hydrolase (LTA4H) program. That paper described the construction of a fragment library based on naturally occurring compounds, crystallographic screening against LTA4H, and optimization of inhibitors for this cardiovascular disease target. In a new paper published in J. Med. Chem., the researchers provide a fuller description of the discovery of the resulting clinical compound, DG-051.

As noted in the previous paper, crystallographic screening of deCODE’s fragment library identified several hydrophobic hits such as Compound 6 (see figure). At the same time, the researchers were aware of research from Searle that had produced inhibitors such as Compound 5. Appending the pyrrolidine of this compound onto deCODE’s fragment led to a modest increase in potency (Compound 9), though the resultant compound was still orders of magnitude weaker than Compound 5. Crystallography suggested a couple bad interactions in Compound 9 compared to Compound 5, so the researchers modified Compound 5 to generate Compound 14, which was active in a whole blood assay but suffered from rapid metabolism. Replacing the central methylene with an oxygen and adding a chlorine (Compound 17) improved biochemical potency slightly while dramatically improving pharmacokinetics.

Several crystal structures of LTA4H showed an acetate ion bound to the catalytic zinc, and the researchers sought to combine this “fragment” with their existing series, generating clinical compound DG-051. This did not lead to an improvement in biochemical potency (and actually decreased ligand efficiency), but it did lead to a roughly ten-fold improvement in potency in the whole-blood assay, as well as improvements in solubility and DMPK parameters. Interestingly, both enantiomers were equipotent, and the S-enantiomer was ultimately chosen due to ease of synthesis.

This story could be seen as an example of what has been called “fragment-assisted drug discovery:” unlike AT9283 or Indeglitazar, the fragments identified (acetate aside) didn’t end up in the clinical compound, and it could be argued that the initial lead was taken from the literature. But information gleaned studying the fragments fed into the design of a molecule that was sufficiently active, stable, selective, and novel for development.

The article states that DG-051 entered phase 2 clinical trials “for the prevention of myocardial infarction and stroke”, although no reports of development appear in clinicaltrials.gov. Also, in what has been an all-too-common event over the past year, deCODE filed for Chapter 11 bankruptcy and announced that it planned to sell “substantially all of its assets.” Practical Fragments wishes the best of luck to all the folks there. Happily the structural biology and fragment-screening group has (re)gained independence as Emerald BioStructures.