Enthalpy Arrays

As regular readers know, there are lots of ways to find fragments, each with its own strengths and weaknesses. Isothermal titration calorimetry (ITC) is useful for being able to extract thermodynamic values from an experiment, but it tends to be low-throughput and is thus used more as a secondary rather than a primary assay. To make calorimetry more convenient, Michael Recht and colleagues at Palo Alto Research Center have constructed nanocalorimeters. They describe using these “enthalpy arrays” for fragment screening in a paper just published online in the Journal of Biomolecular Screening.

In a typical ITC experiment, a protein is mixed with a ligand and the tiny temperature change that occurs upon binding is detected. In the case of nanocalorimeters, up to 96 detectors are arranged on a plate, and sample volumes are typically a few hundred nanoliters. At this scale, the enthalpy of binding becomes very challenging to measure, but it is possible to measure the heat generated during the course of a reaction as an enzyme processes its substrate. This allows one to follow the reaction in real time without any dyes, labels, or artificial substrates. Also, since an inhibitor will change the reaction profile in a predictable manner, one can determine its mechanism of action.

Each detector in the enthalpy array is set up such that droplets are rapidly mixed together in sets of two. In the experimental set, one droplet contains the protein of interest while the other contains a substrate and a fragment. (Each detector also incorporates a control pair – one droplet containing the substrate/fragment mixture and another containing bovine serum albumin.) In the current paper, the researchers were looking for competitive inhibitors of PDE4A10, a phosphodiesterase implicated in inflammatory disorders. The protein was present at a final concentration of 5 micromolar, substrate was at 2 mM, and each fragment was present at up to 2 mM. 160 very small fragments (average molecular weight only 154 Da) were screened individually, resulting in 11 competitive hits with Ki < 2 mM; 2 other hits displayed more complex kinetics.

The 11 competitive hits were characterized in more detail; the most potent had a Ki of 0.32 mM and the most ligand efficient had LE = 0.43 kcal/mol-atom. In collaboration with Vicki Nienaber and colleagues at Zenobia, all 11 of these were taken into crystallography experiments. This proved challenging: unliganded PDE4A10 crystals suitable for fragment soaking could not be grown, necessitating more labor-intensive co-crystallography. Unfortunately, although some crystals were obtained, they did not diffract at high enough resolution to unambiguously fit the electron density of the fragments, though there was evidence for binding in the active site. The researchers were able to crystallize PDE4A10 with pentoxifylline, a known phosphodiesterase inhibitor. Since many of the fragments have structural features reminiscent of other phosphodiesterase inhibitors, this suggests starting points for modeling.

As described in this paper, enthalpy arrays could be used as a primary screen for fragment hits with defined modes of action before follow-up by slower methods. Although in this particular case crystallography was not successful determining co-crystal structures of the novel fragments, in a recent talk Michael described a related system which did yield good crystal structures. I look forward to seeing additional applications of this approach.

ACS Spring Meeting 2010

The spring national meeting of the American Chemical Society has just concluded in (uncharacteristically sunny) San Francisco. The main fragment event was a full day session organized by Rachelle Bienstock of the NIH. The theme was “Fragment based drug discovery: success stories due to novel computational methods applications.” Rachelle is planning on getting some of the speakers to write chapters for a book, so I won’t do more than give a very brief overview here.

The session was very multinational, with speakers from France, Germany, Russia, and the UK, in addition to the US, and a good mix of companies and academics. On the computational corporate side John MacCuish from Mesa Analytics described the molecular shape fingerprints approach, Carsten Detering of BioSolveIT provided several examples of applying his company’s methods for fragment linking and scaffold hopping, and Francois Delfaud of MEDIT described mining the pdb for protein-fragment interactions and applying this to Eg5 inhibitors. On the computational academic side, Tobias Lippert of the Center for Bioinformatics in Hamburg discussed the Qsearch program, Vladimer Poroikov of the Institute of Biomedical Chemistry in Moscow discussed PASS, which relies on a large training set to predict actives and inactives, and Dima Kozakov of Boston University presented the FTMap approach for predicting fragment-binding pockets in protein-protein interactions.

Moving away from the purely computational, Yongjin Xu of Novartis described the application of virtual fragment screening to identify p38 and BRaf inhibitors, Vicki Nienaber of Zenobia described iterative fragment screening to identify potent and selective LRRK2 inhibitors, and I presented Carmot’s Chemotype Evolution approach. Finally, GPCRs appear to be increasingly amenable to FBLD; Richard Law of Evotec presented a number of applications of computational methods to various programs including histamine receptors, while Miles Congreve of Heptares presented their StaR Technology for generating stabilized GPCRs suitable for SPR, NMR, and crystallography and discussed applications to the adenosine A2A receptor and the beta-1 adrenoreceptor. In the later case, the researchers were able to obtain 9 co-crystal structures and found that agonists and antagonists bound somewhat differently.

There were also a few other relevant posters and talks throughout the conference. For example, I learned that Locus Pharmaceuticals has transformed itself into Ansaris; Fouzia Machrouhi presented a poster on developing nanomolar inhibitors of the protein kinase AMPK.

Finally, Andrew Woodhead presented an update on Astex’s CDK2 program. One of the earliest posts on Practical Fragments described Astex’s fragment-based discovery of AT7519, which is in clinical trials for cancer. However, with an oral bioavailability of less than 1%, this compound is administered intravenously. Extensive medicinal chemistry ultimately revealed that a relatively minor change – capping the secondary amine with a methyl sulfonamide – led to a molecule with dramatically improved oral bioavailabilty. This molecule, AT9311, also retains good activity in mouse xenograft models. This is a useful reminder that fragment-based methods are not a replacement for solid (and inevitably subsequent) medicinal chemistry.

HIV protease vs fragments

HIV protease (HIV PR) is a well-known and successfully exploited Achilles heel (OK, maybe more of an Alexandrian sword) of the virus HIV. Although there is no shortage of successful drugs on the market that target this enzyme, resistance is an issue, and new approaches are always welcome. To this end, researchers led by C. David Stout at Scripps Research Institute have performed a fragment screen against HIV protease, the results of which are reported in the March issue of Chemical Biology and Drug Design.

This is really a crystallography paper, and gives a thorough, nuts-and-bolts description of doing a crystallographic fragment screen. The authors screened a library of 384 commercially available fragments with an average molecular weight of only 142 Da. Some of this work was done at Active Sight, and although I believe Active Sight has closed, some of the folks have moved to Zenobia, so I suspect their fragment library incorporates some of the same features.

The researchers used five different crystal forms of HIV PR and examined a total of 808 crystals, 507 of which were co-crystallization experiments and 301 of which had the fragments soaked into crystals that had previously been grown. In all, 378 data sets were collected. Most of these were done in the presence of an active site inhibitor, thus specifically targeting the search for fragments that bind outside of the active site. Three fragments were identified binding to two different sites, and these data have been deposited in the protein data bank. The authors argue that these fragments could be binding to allosteric sites that might keep the protease in its “closed,” inhibited conformation.

Like the recent p53 example, there is still a long way to go: it is not even clear that these fragments have functional activity. Still, the discovery of these small-molecule binding sites illustrates that fragment methods can reveal something new even about an enzyme as well-characterized as HIV protease.