February 4, 2013
In a recent LinkedIn Discussion post, Ben Davis posed the following question:
"Do any of the commercially available fragment libraries come with reference 1D NMR spectra acquired in aqueous solution?"
Most commercial vendors of fragments do not offer nuclear magnetic resonance (NMR) reference spectra with their compounds useful to fragment screeners; if anything, it is 100% organic solvent, at room temperature, at relatively low magnetic field strength. The NMR spectra of fragments and other small molecules are very solvent- and sample-dependent; different buffers, solvents, temperatures and magnet field strengths can generate large spectral differences for the exact same compound. As a result, NMR reference spectra acquired for fragments in organic solvent cannot be used to design fragment mixtures, one of the key advantages in NMR screening. Furthermore, solubility in organic solvent is no measure of solubility in the mostly aqueous buffer conditions typically used in NMR-based fragment screening.
At Emerald Bio, the leading protein resource, we routinely acquire NMR reference spectra for all our commercially-sourced fragment screening compounds as part of our quality control (QC) procedures. This is necessary to ensure the identity, the purity and the solubility of each fragment we use for screening campaigns. This data are further used to design cocktails of 9-10 fragments with minimal peak overlap for efficient STD-NMR screening in-house.
Recently, we selected a random set of commercial fragment compounds for analysis, and closely examined those that failed to better understand the reasons behind it. The most common reason for QC failure was insolubility (47%), followed by degradation or impurities (39%), and then spectral mismatch (17%) [Note: Compounds can acquire multiple QC designations, hence total incidences > 100% ]. Less than 4% of all compounds assayed failed due to solvent peak overlap or lack of non-exchangeable protons, both requirements for NMR screening. Failure rates were as high as 33% per individual vendor, with an overall average of 16% (see Figure 1). Although some vendor fragments yielded failures, ordering very few compounds from a single vendor did not change the outcome (8% from over 20 vendors ordering 10 or fewer fragments from each).
These results highlight the importance of implementing tight quality control measures for preliminary vetting of commercially-sourced materials, as well as maintaining and curating a fragment screening library. It also puts forth a statistical likelihood of around 10-15% failure, regardless of vendor. Most importantly, we have seen our methods reduce risks while accelerating drug discovery. Do these numbers make sense to you? How do they measure up with your fragment library?
Let us know what you think.
Figure 1. Percentage of NMR QC failure rates for a random set of commercially available fragment compounds. Fragments were first dissolved to 50 mM in deuterated dimethyl sulfoxide (d6-DMSO). Final NMR samples were 1.0 mM fragment in 50:100:350 µL d6-DMSO:D2O:low salt aqueous buffer. NMR data were acquired at 10°C at 500 MHz field strength.
October 10, 2012
Congratulations to Brian Kobilka and Jeff Lefkowitz for the 2012 Chemistry Nobel Prize !
Now the world will need to learn how to say 'G-protein coupled receptors'.
C O N G R A T U L A T I O N S ! ! ! !
April 11, 2012
The relatively new field of Fragment Based Drug Discovery received a much-needed boost late last year when the FDA approved the first drug whose genesis was screening of a fragment library (see reference below). The BRAF inhibitor Zelboraf (vemurafenib) grew out of biochemical screening of a panel of kinases at Plexxikon, followed by intensive X-ray crystallographic analysis of hits from the primary screen.
There are, however, many different methods that FBDD practitioners can employ in screening. If an amenable crystal system is available, soaking of crystals and XRD analysis can be performed. Ligand-observe NMR methods such as saturation transfer difference (STD-NMR) are also often used. SPR, thermal shift, and a variety of biochemical methods are likewise well-documented.
What methods should you use? The answer is usually complex, and depends on the nature and abundance of your target macromolecule, as well as the size of the fragment library you wish to screen. At Emerald, we currently rely heavily on X-ray, STD-NMR, SPR and thermal shift techniques, and we prefer to use more than one technique at a time to aid with prioritization and characterization of hits. There are a couple of upcoming opportunities to have this discussion in real time…
We are looking forward to meeting lots of our past and future collaborators at the Drug Discovery Chemistry (DDC) conference in San Diego April 16-19. Alex Burgin, CSO of Emerald BioStructures, will be giving a workshop as part of the FBDD session on April 16. He will be co-teaching a short course with Daniel Erlanson (author of the excellent blog practical fragments) entitled “Advanced Tools and Technologies for Fragment-Based Design." If you would like to speak personally with Alex, he will be available for the remainder of the week in San Diego. Additionally, Emerald is holding an informal MeetUp at the conference on April 17, from 6;30-8:30 PM. Interested parties should contact Diana Wetmore for location details.
New fragment screening methods are constantly evolving. One technology we find particularly intriguing is the “CEfrag” Capillary Electrophoresis technique of Selcia, Inc. This approach monitors the mobility shift of a probe ligand as both target and a cocktail of fragments move through a gel-filled capillary. The image below is a schematic of the technology from the Selcia Discovery website.
In addition to presentations at DDC, a scientist from Selcia will give a full description of their fragment screening method as a guest presenter in the Emerald BioStructures Webinar series. The webinar, titled "Complementing Biophysical and Structural Methods for Drug Discovery with Capillary Electrophoresis," can be watched live on April 17 at 11 am Pacific Time, or as an archived presentation any time after that. If you watch live there’ll be opportunities to send in questions for discussion at the end of the webinar.
Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S, Bremer R, Gillette S, Kong J, Haass NK, Sproesser K, Li L, Smalley KS, Fong D, Zhu YL, Marimuthu A, Nguyen H, Lam B, Liu J, Cheung I, Rice J, Suzuki Y, Luu C, Settachatgul C, Shellooe R, Cantwell J, Kim SH, Schlessinger J, Zhang KY, West BL, Powell B, Habets G, Zhang C, Ibrahim PN, Hirth P, Artis DR, Herlyn M, & Bollag G (2008). Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proceedings of the National Academy of Sciences of the United States of America, 105 (8), 3041-6 PMID: 18287029
April 2, 2012
Lipitor, Prevacid, and an estimated 20% of all drugs on the market contain one or more fluorine atoms. The relative lack of fluorine in the biological environment allows NMR spectroscopists to easily detect fluorine atoms on drugs, lead candidates and other small molecules when mixed with proteins. But does anybody actually use 19F- NMR?
Strong signal (high gyromagnetic ratio);
No isotopic enrichment (normal fluorine is NMR-active);
Wide chemical shift range (200 ppm versus 10 ppm for proton);
Zero background (proteins, DNA, RNA, etc. don't have fluorine).
The first use of 19F-NMR for biophysics dates back to the 1970s, using a Varian XL-100 (that's 100 MHz!) spectrometer. Bruce Dunlap and co-workers detected covalent AND non-covalent binding between the age-old cancer chemotherapeutic 5-fluoro uracil (5FU) and its enzymatic target, thymidylate synthase[1, 2]. 19F-NMR has also been successfully used as a protease inhibitor assay; tagging a target peptide with a single flurorine atom allows easy detection of cleaved peptide - and uncleaved peptide in the presence of a caspase inhibitor (see image). Now, fluorine NMR is being revisited for the purposes of fragment screening. Taking a lead from Claudio Dalvit, researchers at Amgen have retooled their systems and sorted their screening collection to conduct fluorine-based NMR fragment screening.
With such a range of applications, why is 19F not more widely used? Is it just instrumentation? Most X-tuneable probes "don't go to fluorine", requiring purchase of a dedicated 19F, 1H-decoupling probe and the amplifiers to power them. But are such capital expenditures worth the investment in discovery science?
Do you use 19F-NMR? If so, how often? And to what purpose?
Byrd RA, Dawson WH, Ellis PD, & Dunlap RB (1977). 19F nuclear magnetic resonance investigation of the ternary complex formed between native thymidylate synthetase, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate. Journal of the American Chemical Society, 99 (18), 6139-41 PMID: 893883
Byrd, R., Dawson, W., Ellis, P., & Dunlap, R. (1978). Elucidation of the detailed structures of the native and denatured ternary complexes of thymidylate synthetase via fluorine-19 NMR Journal of the American Chemical Society, 100 (24), 7478-7486 DOI: 10.1021/ja00492a007 Fattorusso, R., Jung, D., Crowell, K., Forino, M., & Pellecchia, M. (2005). Discovery of a Novel Class of Reversible Non-Peptide Caspase Inhibitors via a Structure-Based Approach Journal of Medicinal Chemistry, 48 (5), 1649-1656 DOI: 10.1021/jm0493212
Vulpetti A, Hommel U, Landrum G, Lewis R, & Dalvit C (2009). Design and NMR-based screening of LEF, a library of chemical fragments with different local environment of fluorine. Journal of the American Chemical Society, 131 (36), 12949-59 PMID: 19702332
Jordan JB, Poppe L, Xia X, Cheng AC, Sun Y, Michelsen K, Eastwood H, Schnier PD, Nixey T, & Zhong W (2012). Fragment based drug discovery: practical implementation based on ¹⁹F NMR spectroscopy. Journal of medicinal chemistry, 55 (2), 678-87 PMID: 22165820
April 2, 2012
As interest in membrane protein targets by both academic laboratories and large pharmacological companies grows, becoming versed in the topic of membrane proteins is more important than ever. After working with soluble proteins, the world of membrane protein structure can be intimidating to the novice structural biologist just entering the field. With over 80,000 structures present in the Protein Data Bank, searching through this vast number of entries for membrane protein structures can be akin to searching for a needle in a haystack. In this post I will highlight and discuss the features of three repositories of membrane protein structures: Membrane Proteins of Known 3D Structure, Membrane Protein Databank, and Protein Databank of Transmembrane Proteins.
The Membrane Proteins of Known 3D Structure database is curated by the lab of Dr. Stephen White at the University of California Irvine. The database is updated often, well-maintained, and contains structures of membrane proteins determined by both X-ray and electron diffraction methods (although you’ll find a few NMR structures in there as well). Membrane protein structures are divided into three classes on the site: (1) monotopic transmembrane proteins, (2) multi-pass beta-barrel transmembrane proteins, and (3) multi-pass alpha-helical transmembrane proteins. Each class of protein is the further sub-classified into functional groups via a drop-down menu. So if I wanted to look at available structures for autotransporters (my personal favorite), I would open the “Transmembrane Proteins: Beta-Barrel” drop-down menu and then select “Outer Membrane Autotransporters”. Proteins that have multiple entries (ie: different ligands bound) are then grouped together so you don’t have to go searching through a long list to find them all. The database is also accessible using text-based searches. At the time of writing of this post there were 956 entries in this database, 322 of those being unique.
The Membrane Protein Databank was started by the lab of Dr. Martin Caffrey, currently at the University of Limerick in Ireland. It is updated weekly and consists of membrane protein structures determined by X-ray diffraction, electron diffraction, NMR, and cryoelectron miscroscopy. The database is searchable by a number of different criteria including but not limited to: expression system, function, journal of publication, ligand, pH, resolution, alpha-helical vs. beta-sheet, crystallization method, and temperature. In addition, the statistics function makes doing searches like “How many structures have been solved using bicelles?” a breeze to answer. Simply select Crystallization Method for “Statistics on Membrane Protein Versus”, All Experimental Techniques for “As Appropriate, Limit Analysis by Experimental Technique”, and Bicelle for “As Approriate, Limit Analysis by Crystallization Method” and voila! At the time of the writing of this post the MPDB database held 1096 entries.
The Protein Data Bank of Transmembrane Proteins is maintained by the Institute of Enzymology in Hungary. The database is updated by an automated algorithm called TMDET that scans the entire PDB every week. This database is searchable by PDB code, PDB keyword, alpha-helical vs. beta sheet, and number of transmembrane segments. For example, if I wanted to ask the question “How many beta-barrel proteins structures exist that have 22 strands present?”, I would simply pick beta-barrels as the search type and then 22 as the number of transmembrane segments. This will return a list of 22-stranded beta-barrels with links to download the PDB text file for each entry. At the time of the writing of this post the TMDET algorithm has identified what it believes to be 1568 membrane proteins, 1348 of those alpha-helical and 219 of them beta-barrels.
Raman, P., Cherezov, V., & Caffrey, M. (2005). The Membrane Protein Data Bank Cellular and Molecular Life Sciences, 63 (1), 36-51 DOI: 10.1007/s00018-005-5350-6
Tusnády GE, Dosztányi Z, & Simon I (2004). Transmembrane proteins in the Protein Data Bank: identification and classification. Bioinformatics (Oxford, England), 20 (17), 2964-72 PMID: 15180935
March 22, 2012
We've almost gotten used to GPCR structure publications - but today I'm seeing two publications in Nature that ought to rock the world of GPCR structural biology: morphine receptors. Structures of antagonist bound µ-opioid receptor and κ-opioid receptor with bound antagonist. The impact of these structures can not be underestimated since these structures give insight into the most used (and abused) clinical drugs and their workings on an atom scale. In a nutshell, these structures connect atom scale molecular structure to human behavior.
Manglik, A., Kruse, A., Kobilka, T., Thian, F., Mathiesen, J., Sunahara, R., Pardo, L., Weis, W., Kobilka, B., & Granier, S. (2012). Crystal structure of the µ-opioid receptor bound to a morphinan antagonist Nature DOI: 10.1038/nature10954
Wu, H., Wacker, D., Mileni, M., Katritch, V., Han, G., Vardy, E., Liu, W., Thompson, A., Huang, X., Carroll, F., Mascarella, S., Westkaemper, R., Mosier, P., Roth, B., Cherezov, V., & Stevens, R. (2012). Structure of the human κ-opioid receptor in complex with JDTic Nature DOI: 10.1038/nature10939
Congratulations to the Kobilka and the Stevens teams!
February 18, 2012
What a staggering variety of access to GPCR binding pockets there is. The recently published GPCR structures shed new light on the exquisite architecture of GPCR binding pockets:
1. An internal hydrophobic binding pocket that is pretty much closed towards the aqueous phase was identified in the crystal structure of the sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist). Similar to rhodopsin, the ligand can access to the deep binding cavity from the hydrophobic section of the membrane.
Crystal Structure of a Lipid G Protein–Coupled Receptor
Michael A. Hanson, Christopher B. Roth, Euijung Jo,Mark T. Griffith, Fiona L. Scott, Greg Reinhart, Hans Desale, Bryan Clemons, Stuart M. Cahalan, Stephan C. Schuerer, M. Germana Sanna, Gye Won Han, Peter Kuhn, Hugh Rosen, Raymond C. Stevens
Science Vol. 335 no. 6070 pp. 851-855; 2012
2. In addition to this conventional ligand binding location, there seems to be a site close to the intracellular surface of GPCRs that serves as an alternate way to modulate GPCR activity in A2A adrenergic receptor and unrelated GPCRs. The structure of the A2A adenosine receptor with a Fab fragment (Fab2839) reveals how it penetrates the receptor with its CDR-H3 domain. The crux is that the interaction is similar to that of the activated b2-adrenergic receptor and that of opsin with a bound peptide; interestingly, the binding of the Fab fragment inactivates the A2A adenosine receptor. This discovery could have huge ramifications, since it offers an alternate way to modulate GPCR activity, without occupying the standard central ligand binding pocket.
Hino, T., Arakawa, T., Iwanari, H., Yurugi-Kobayashi, T., Ikeda-Suno, C., Nakada-Nakura, Y., Kusano-Arai, O., Weyand, S., Shimamura, T., Nomura, N., Cameron, A., Kobayashi, T., Hamakubo, T., Iwata, S., & Murata, T. (2012). G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody Nature DOI:10.1038/nature10750
All this news is phantastic! The more we look the more we see.
January 31, 2012
Congratulations to the publication of the first GPCR structure in 2012! These congrats go to the Kobilka, Haga and Kobayashi teams that reported the X-ray crystallographic structure of the M2 muscarinic acetylcholine receptor this weeks' issue of Nature. The bound antagonist (3-quinuclidinyl-benzilate) is bound in the center of a long aqueous tubular structure that has not seen before in any GPCR. Apart from the common arrangement of the 7 TMs, the comparison of the shape of the M2 binding pockets with those of other receptors (b2, A2A, CXCR4, D3 and H1 receptor) shows that the binding modes are very different from each other (as compared to b2, A2A, CXCR4, D3 and H1 receptors, see Fig.4 here). The ligand binding pocket is really a channel that goes two thirds through the membrane, and opening up the remainder would likely convert this GPCR into a water pore. Turns out that the M2 binding pocket is very similar amongst the family of muscarinic acetylcholine receptors, explaining the difficulty of developing specific ligands.
As seen with many other GPCR structures, T4 lysozyme inserting into the third intracellular loop provides for strong packing interactions between layers in the M2-T4L crystal. No surprise here. As of writing this, the coordinates are not yet available (access code 3UON) but should be released soon.
PS: More GPCR structures to come: e.g. the yet unpublished S1P1 and k-opioid receptor structure are advertised here
November 7, 2011
Identifying individual amino acid residues within a GPCR and comparing these across different receptors is a routine task that’s helped by a widely accepted nomenclature system: that of Ballesteros and Weinstein.
Juan A. Ballesteros, Harel Weinstein (1995). Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors Methods in Neurosciences, 25, 366-428 DOI: 10.1016/S1043-9471(05)80049-7
You could look up the nomenclature rules in the original paper or find the rules online and apply them to the particular amino acid sequence you’re working with. This is a bit cumbersome, isn’t it?
Good news: here’s a simple way to check the Ballesteros&Weinstein nomenclature with the Sequence Tool provided by http://www.gpcr.org. Just call up the target sequence and hover over a particular amino acid to extract the Ballesteros&Weinstein(B&W) code:
A simple way to call up the Ballesteros&Weinstein(B&W) code for a particular amino acid in a GPCR target
I like simple.
November 7, 2011
When it comes to expression systems, insect cells have been the primary supplier of GPCR protein for crystallographic studies. Notable exceptions are:
Rhodopsins can be obtained from the retina of eyes from Squid or cows and alternatively be expressed in COS cells.
The Histamine H1 receptor expressed in Pichia pastoris was used to determine its crystal structure
To attendees and listeners of the "GPCR expression for biophysical and structural studies" webinar: There was a question at the very end of the webinar and I'd like to correct the answer that I gave. While it is true that insect cells are an important source for heterologous expression of GPCRs, they're not the sole source of GPCR material for crystallization.
Histamine 1 receptor, expressed in Pichia pastoris as a fusion protein with Lysozyme as described in
Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R, Cherezov V, Liu W, Han GW, Kobayashi T, Stevens RC, & Iwata S (2011). Structure of the human histamine H1 receptor complex with doxepin. Nature, 475 (7354), 65-70 PMID: 21697825
Thanks to the listeners for making me aware of this!