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Weak Affinity Chromatography
Molecules are forced through a column containing molecules known to bind to the specified weak affinity molecules (In red).
AcronymWAC
ClassificationChromatography
Analytesorganic molecules
biomolecules
ions
polymers

This article is still under construction. I am working on this for a class. Please do not move until April 21st.

This article is closely related to Chromatography.

Weak affinity chromatography[1] (WAC) is a robust and reproducible technology used for affinity screening of small molecules. The process is based on the principles of affinity chromatography in which molecules are separated based on their attraction to an immobilized ligand within the chromatography column. Weak affinity chromatography is used specifically for those molecules that do not bind tightly enough to normal ligands within the column and would therefore otherwise pass through the column before they were able to be analyzed. This technique has been proven useful most commonly in drug development.[2][3] WAC is an affinity-based liquid chromatographic technique that separates chemical compounds based on their different weak affinities to an immobilized target in a chromatography column.

Overview edit

The separation of fragments through affinity chromatography is based on the retention time of those fragments. As molecules are pushed through a loaded chromatography column, those fragments with the strongest affinity towards the stationary phase in the column will remain in the column longer and be eluted later than those molecules with the weakest affinity. The differences in elution times can then be used to distinguish the fragments that have been separated. By computing the obtained retention times of analyzed compounds, the affinity measure and ranking of affinity in the solution can be determined. Weak affinity chromatography is capable of capturing the elution of molecules that pass too quickly through normal chromatography columns. Separation of these weak affinity molecules is being used specifically in fragment-based drug design.

Process edit

 
Example of an interchangeable weak affinity chromatography column

Weak affinity chromatography closely resembles those processes used in high-performance liquid chromatography (HPLC). WAC experiments have been conducted using an adapted HPLC machine with interchangeable columns[4]. A column is prepared using microparticulate silica beads approximately 8-12µm in diameter. These beads are porous, with variable pore size depending on the target fragment size. The protein of interest is then immobilized onto this chromatography column. Each fragment to be analyzed is injected in buffer solution into the column using a pressurized pump to push the molecules through the column. The affinity of the fragments will then affect the elution time and the individually separated fragments can be detected with either UV spectroscopy or mass spectrometry[5]. The affinity separation is based on the binding properties of the stationary phase within the column. Fragments in the range of 1mM-10µM in dissociation constant (KD) can be separated using this method. Weak affinity chromatography is capable to recording elution times not typically seen in the range of HPLC machines. The speed of the weak affinity binding in the column leads to the possibility for high-resolution chromatography of multiple analytes in the selected matrix.[6]

Data Reporting edit

 
Example of a chromatogram. The x-axis represents the elution times, with peaks on the left denoting the fastest moving molecules, those with the weakest affinity. The y-axis represents the signal strength based on the measurements from a secondary detection system

Data gathered using weak affinity chromatography is recorded on a chromatogram. This is a visual representation of the separation of the small molecules in the mixture. The chromatography system separates the molecules based on elution time from the column. This is represented on the x-axis. A second identification technique such as mass spectrometry or ultraviolet–visible spectroscopy is then used to establish the strength of the signal for each eluted group of molecules.[7] This data is represented on the y-axis.

Common Affinity Targets edit

The weak affinity chromatography technology is demonstrated against a number of different protein targets that exhibit transient binding. The most common of these targets are very small molecule types such as proteases, kinases, chaperones and protein–protein interaction (PPI) targets. These molecules make up the structure of larger compounds, specifically those involved in therapeutic activity. WAC has been used mainly for analysis and characterization of carbohydrates[8] and for chiral separations[9]


Applications edit

Fragment Based Drug Discovery edit

Fragment-based drug discovery (FBDD), also known as fragment-based lead discovery, is a process by which lead compounds are identified. These compounds are predicted to be therapeutically useful, but still require further modification and development. Lead compounds tend to exhibit weak affinity and can therefore not be identified using conventional chromatography methods.[10] In combination with other methods of detection such as mass spectroscopy, weak affinity chromatography has been shown to be effective in the detection of kinase targets in the fragment screening process.[11]

See Also edit


References edit

  1. ^ Zopf, D.; S. Ohlson (1990). "Weak-affinity chromatography". Nature. 346 (6279): 87–88. doi:10.1038/346087a0. ISSN 0028-0836.
  2. ^ Duong-Thi, M. D.; Meiby, E.; Bergström, M.; Fex, T.; Isaksson, R.; Ohlson, S. (2011). "Weak affinity chromatography as a new approach for fragment screening in drug discovery". Analytical Biochemistry. 414 (1): 138–146. doi:10.1016/j.ab.2011.02.022. PMID 21352794.
  3. ^ Meiby, E.; Simmonite, H.; Le Strat, L.; Davis, B.; Matassova, N.; Moore, J. D.; Mrosek, M.; Murray, J.; Hubbard, R. E.; Ohlson, S. (2013). "Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90". Analytical Chemistry. 85 (14): 6756–6766. doi:10.1021/ac400715t. PMID 23806099.
  4. ^ Magnus Strandh, Håkan S. Andersson, and Sten Ohlson. Weak Affinity Chromatography.
  5. ^ http://practicalfragments.blogspot.com/2011/03/weak-affinity-chromatography-wac.html
  6. ^ Weak Affinity Chromatography. Molecular Interactions in Bioseparations 1993, pp 15-25. Sten Ohlson, David Zopf.
  7. ^ Feng, JJ; Lv, T; Liu, L; Xie, L; Xing, YW; Min, CY; Xie, HP. Mass spectrum selectivity-based chromatogram baseline-shift elimination and its application in metabonomics of liver toxicity. CHEMOMETRICS AND INTELLIGENT LABORATORY SYSTEMS. Volume: 127, Pages: 112-120, Published: AUG 15 2013
  8. ^ M. Bergström, S. Liu, K.L. Kiick, S. Ohlson. Cholera toxin inhibitors studied with high-performance liquid affinity chromatography: a robust method to evaluate receptor–ligand interactions.Chem. Biol. Drug Des., 73 (2009), pp. 132–141
  9. ^ J. Haginaka.Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations.J. Chromatogr. A, 906 (2001), pp. 253–273
  10. ^ FRAGMENT-BASED DRUG DISCOVERY AND X-RAY CRYSTALLOGRAPHY. Erlanson, DA (Erlanson, Daniel A.). Book Series: Topics in Current Chemistry. Volume: 317, Pages: 1-32, Published: 2012
  11. ^ Meiby, E; Knapp, S; Elkins, JM; Ohlson, S. Fragment screening of cyclin G-associated kinase by weak affinity chromatography. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. Volume: 404, Issue: 8, Pages: 2417-2425, Published: NOV 2012.

Category:Chromatography Category:Drug discovery


 

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