Analytical ultracentrifugation: a powerful ‘new’ technology in drug discovery

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<ul><li><p>rire,</p><p>and should become a mainstay for target identification,</p><p>are more and more moving into focus. The method is additionally usefulto better describe the behavior of low-molecular weight drugs in</p><p>Introduction</p><p>The analytical ultracentrifuge is similar to the more familiar</p><p>not depend on a comparison to standards. Consequently,</p><p>sedimentation can be used to analyze the solution behavior</p><p>of nearly any type of molecule over a wide range of concen-</p><p>trations and in a wide variety of solvents. Furthermore, a</p><p>Drug Discovery Today: Technologies Vol. 1, No. 3 2004</p><p>ethpreparative centrifuge, except that the analytical ultracentri-</p><p>fuge is configured to determine the concentration distribu-</p><p>tions of molecules during sedimentation. Analytical</p><p>ultracentrifugation (AUC) provides first-principle hydrody-</p><p>namic and thermodynamic information about the size,</p><p>broad range of particle sizes might be analyzed by using</p><p>different rotor speeds. Sedimentation has the merits of being</p><p>rapid, non-destructive, and simple to use. Advances in data</p><p>analysis software have created new, significantly improved</p><p>tools for obtaining information about protein association</p><p>and the behavior of viruses. Finally, a new, highly sensitive</p><p>fluorescence detection optical system allows the analysis ofE-mail address: (T. Laue) tom.laue@unh.eduURL http://www.camis.unh.edu/target validation, lead optimization, formulation in drug</p><p>development and QA/QC. Recent studies have used</p><p>AUC to characterize the binding stoichiometry and</p><p>binding sites of an anti-tumor agent; of a hemoglobin-</p><p>stabilizing protein, and of a fibril growth inhibitor, and</p><p>to assess the causes of protein aggregation. The recent</p><p>addition of fluorescence to the existing absorbance and</p><p>interference detectors dramatically extends the flex-</p><p>ibility of analytical ultracentrifugation.</p><p>aqueous solution.Tom Laue from the Center to Advance Molecular Interaction Science,</p><p>an internationally acknowledged expert in the field, reviews latestadvances in technological as well as numerical methods. He summarizes</p><p>useful applications and compares it to other methods.</p><p>shape, molar mass, association energy, association stoichio-</p><p>metry and thermodynamic nonideality of molecules in solu-</p><p>tion. Because sedimentation relies on the principal property</p><p>of mass and the fundamental laws of gravitation, it is a</p><p>primary method for which the results are absolute and doAnalytical ultracenta powerful new tecdrug discoveryTom LaueCenter to Advance Molecular Interaction Science, University of New Hampsh</p><p>Analytical ultracentrifugation (AUC) is a powerful</p><p>means of characterizing the solution behavior of mole-</p><p>cules. Sedimentation velocity analysis, the preferred</p><p>AUC technique for characterizing complex systems,</p><p>has higher resolution, broader applicable range and</p><p>fewer solute/solvent limitations than gel-permeation</p><p>chromatography. The technique is simple to perform1740-6749/$ 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.11.012Rudman Hall 379, 46 College Road, Durham, NH 03824, USA</p><p>Section Editor:Oliver Zerbe Institute of Organic Chemistry, University ofZurich, Switzerland</p><p>Many drugs interfere with protein oligomerization or disruption of</p><p>proteinprotein interfaces. Analytical ultracentrifugation is a sensitivemethod to reveal the oligomeric state of proteins and thereby is able to</p><p>detect changes in such. This capability might become of even moreinterest in the context of diseases related to protein misfolding, whichTECHNOLOGIES</p><p>DRUG DISCOVERY</p><p>TODAY</p><p>Editors-in-Chief</p><p>Kelvin Lam Pfizer, Inc., USA</p><p>Henk Timmerman Vrije Universiteit, The N</p><p>Lead optimizationifugation:hnology in</p><p>erlandswww.drugdiscoverytoday.com 309</p></li><li><p>ability to detect trace quantities of components of interest in</p><p>the presence of high concentrations of background (e.g.</p><p>excipient) molecules.</p><p>These two advances make AUC a first-choice technology</p><p>for the characterization of solutions.</p><p>The first advance: Lamm equation analysis</p><p>Sedimentation is described exactly by the second order,</p><p>differential Lamm equation [3]. In the past, extraction of s,</p><p>D, f and Mb from sedimentation velocity data was difficult,</p><p>particularly from complicated mixtures, because simple solu-</p><p>tions to the Lamm equation analysis did not exist. Seminal</p><p>work by Haschemeyer [10], along with clever computer pro-</p><p>gramming by Schuck [3] and Stafford [4], use computer-</p><p>generated solutions of the Lamm equation to fit sedimenta-</p><p>Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004small samples in complex mixtures, enabling the study of</p><p>valuable molecules under near in vivo conditions.</p><p>Background</p><p>Extensive reviews are available that cover the mathematics of</p><p>the sedimentation process [1,2] and data analysis [36].</p><p>Although knowledge of the theory occasionally is useful</p><p>for interpreting some phenomena, it is not needed to appreci-</p><p>ate the quantity and quality of data available from AUC. This</p><p>review will focus on what AUC can do, and will specifically</p><p>address the information available from sedimentation velo-</p><p>city (as opposed to sedimentation equilibrium) analysis.</p><p>The primary quantity of interest in SEDIMENTATION VELOCITY</p><p>(see Glossary) is the sedimentation coefficient, s, which has</p><p>both experimental and molecular definitions (Fig. 1). The</p><p>experimental definition is s v=a, where v is the rate (typi-cally a few microns per second) at which a molecule moves in</p><p>the gravitational field, a. Both v and a are readily measured</p><p>quantities. The molecular definition is s Mb/f, where Mb isthe buoyant mass (the molecules mass less the mass of</p><p>solvent it displaces), and f is the frictional coefficient, which</p><p>depends on the molecules size and shape. Both Mb and f are</p><p>useful parameters for characterizing a molecule.</p><p>It is also possible to determine the diffusion coefficient, D,</p><p>of a molecule from the shape of the sedimenting boundary</p><p>(Fig. 1). Because D = RT/f, where R is the gas constant, T the</p><p>temperature and f the same frictional coefficient as that in the</p><p>definition of s, it is possible to determine the buoyant mass,</p><p>Mb, from sedimentation velocity analysis as s/D = Mb/RT.</p><p>Conversion from the buoyant mass to the anhydrous mass</p><p>is straightforward [7], and has been automated for proteins</p><p>(Sednterp, download available at http://www.bbri.org/</p><p>Glossary</p><p>Lamm equation: a second order differential equation which has no</p><p>single analytical solution, but which can be solved using a mathematical</p><p>method called finite element analysis. Used in direct fitting of sedimenta-</p><p>tion velocity data.</p><p>Sedimentation velocity: an analytical ultracentrifuge method in which</p><p>the rate at which concentration boundaries move in a gravitational field is</p><p>monitored.RASMB/), so AUC provides a rigorous means of determining</p><p>solution mass.</p><p>Two recent advances in analytical ultracentrifugation</p><p>Analytical ultracentrifugation is an old method. However,</p><p>two recent advances have increased its utility profoundly.</p><p>The first advance is the use of direct fitting to the LAMM EQUA-</p><p>TION (see Glossary) for the analysis of sedimentation velocity</p><p>data [3]. Programs which use this analysis method are able to</p><p>detect, quantify and characterize tiny quantities of solution</p><p>contaminants. The second advance is the addition of fluor-</p><p>310 www.drugdiscoverytoday.comescence detection [8,9], which extends the useable concen-</p><p>trations into the picomolar range, as well as providing the</p><p>Figure 1. Scans of the absorbance (A) at 230 nm as a function of radial</p><p>position (r) taken at 8 min intervals during a sedimentation velocity</p><p>experiment. Initially, there was a uniform concentration from the air</p><p>liquid meniscus (spike at 6.1 cm) to the bottom of the cell (7.1 cm). The</p><p>left-to-right progress of the boundary is marked by the arrows, and the</p><p>spreading of the boundary as it moves down the cell is highlighted by the</p><p>dots. The shape of the curves is described exactly by the Lamm equation,</p><p>so that analysis of the curves eliminates nearly all of the noise [3].tion velocity data quickly and rigorously. Because neither</p><p>stochastic nor systematic noise are solutions to the Lamm</p><p>equation, they are filtered out, thus allowing very tiny sedi-</p><p>menting boundaries to be detected (Fig. 2) and analyzed</p><p>accurately (Fig. 3). The key point is that very weak signals</p><p>can be detected and provide important molecular informa-</p><p>tion. The sedimentation coefficient of trace quantities (&gt;1%)</p><p>of aggregates or cleavage products might be determined, and</p><p>even complex mixtures of aggregates might be resolved and</p><p>quantified. Although trace quantities less than 1% might be</p><p>detected, reliable estimates of the amount and size of the</p></li><li><p>the analytical ultracentrifuge is useable from 1000 to</p><p>60,000 rpm, a very wide range of gravitational fields might</p><p>be used in AUC. Earlier analysis methods, however, had</p><p>difficulty working with very small molecules (M &lt; 3000)</p><p>and very large molecules (M &gt; 10,000,000). The advent of</p><p>Lamm equation analysis removes both of these limitations.</p><p>Consequently, sedimentation velocity analysis recently has</p><p>provided unique and useful information on the association of</p><p>small peptides [11], as well as on the heterogeneity and</p><p>aggregation of viruses [12].</p><p>In addition to providing s, D, f and Mb for molecules, Lamm</p><p>equation analysis might be used to determine the association</p><p>constants and stoichiometries, even for complex assembly</p><p>schemes (Fig. 4) [3,4]. Recently, these analysis programs have</p><p>been extended to analyze the combined data from several</p><p>experiments, including data acquired using different first-</p><p>principle methods [6]. In addition to Lamm equation analy-</p><p>sis, programs based on thermodynamic first principles are</p><p>available for the analysis of sedimentation equilibrium data</p><p>[13]. Discussion of these programs is beyond the scope of this</p><p>review. It is recommended that interested readers sign up for</p><p>the RASMB e-mail forum (http://www.bbri.org/rasmb/),</p><p>explore web sites (http://www.analyticalultracentrifugation.</p><p>com/, http://www.ap-lab.com/) and participate in workshops</p><p>devoted to the sedimentation analysis of interacting systems</p><p>Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead optimization</p><p>Figure 2. The incredible ability of Lamm equation analysis to detect</p><p>trace quantities of sedimenting material is demonstrated here. Shown</p><p>are fluorescence intensity data for a 1.4 pM monoclonal antibody (goat</p><p>anti-mouse IgG) that had been labeled with Alexa-488 dye (5 dye/protein), with the intensities acquired at a 50% gain setting. The totalmaterial can require replicate measurements (see Outstand-</p><p>ing issues).</p><p>signal is12 intensity units (on a scale from 0 to 4095). Data (dots) foronly five scans are shown. The fitted data also are shown (solid lines),</p><p>slightly offset from the raw data for clarity.Lamm equation analysis also broadens the applications for</p><p>sedimentation velocity. For example, because the gravita-</p><p>tional field depends on the square of the rotor speed, and</p><p>Figure 3. The continuous size-distribution (c(s)) analysis for the data in</p><p>Fig. 2 reveals a peak at 6.8 s, which is the correct value for IgG. Theanalysis included all 200 of the scans acquired during the experiment</p><p>(105,000 data points), which were analyzed using Sedfit version 8.9</p><p>(http://www.analyticalultracentrifugation.com/) and fit with an rms of</p><p>0.29 intensity units.Figure 4. Titration of Alexa-488 labeled, goat anti-mouse IgG into a</p><p>fixed concentration of mouse IgG, in 100 mM KCl, 20 mM Tris pH 8,</p><p>0.1 mg/ml ovalbumin. The data have been normalized so that the</p><p>distributions might be presented on a single graph. For each concentra-</p><p>tion of labeled IgG, distributions are shown for samples with and without</p><p>a fixed concentration of mouse IgG. In the absence of mouse IgG, the</p><p>labeled IgG has a peak at 6.8 s, consistent with it being a monomer. The</p><p>shift of the labeled IgG to higher s when unlabeled mouse IgG is added is</p><p>the result of complex formation. Interestingly, at the highest concentra-</p><p>tion of label, the distribution shifts back to lower values of s, consistent</p><p>with the solution being in antibody excess. More detailed analysis of the</p><p>curves would be necessary to determine the size of the complexes. All ofthese data were acquired in a single experiment.</p><p>www.drugdiscoverytoday.com 311</p></li><li><p>Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004</p><p>diffe</p><p>er,</p><p>er th</p><p>erea(e.g. that held at the University of Connecticut, http://</p><p>www.ucc.uconn.edu/wwwbiotc/UAF.html).</p><p>The second advance: fluorescence detection</p><p>The fundamental measurement needed for the analysis of</p><p>sedimentation velocity is the radial concentration distribu-</p><p>tion (Fig. 1). The three optical detectors available for the</p><p>analytical ultracentrifuge are compared in Table 1. The three</p><p>Table 1. Available optical detection systems for AUC</p><p>Property Absorbance</p><p>Sensitivitya 0.1 ODd (190800 nm)</p><p>Radial resolutionb 20 mm</p><p>Data acquisition timec 120 s/sample</p><p>Sensitivity ++</p><p>Selectivity ++</p><p>Precision of analysis ++</p><p>Resolution of analysis ++</p><p>Notes Easiest to use</p><p>When to use Need selectivity</p><p>Added sensitivity</p><p>Non-dialyzable</p><p>buffer components</p><p>a Typical minimum useful concentration for analysis. Because the optical systems rely onb The radial distance between independent concentration readings. The smaller this numbc How long it takes to acquire a concentration profile at the radial resolution listed. The short</p><p>The absorbance and interference optical systems acquire data from one sample at a time, wh</p><p>speeds below 6000 rpm, the time to acquire data is determined by the rotor period.d Absorbance unit.detectors have complementary applications. Absorbance and</p><p>interference detectors have been available for some time, and</p><p>an analysis of their strengths and weaknesses is available</p><p>(Technical Bulletin 1821a, available from http://www.beck-</p><p>man.com/resourcecenter/literature/BioLit). The recent addi-</p><p>tion of the fluorescence detector (Aviv Biomedical, Inc,</p><p>http://www.avivbiomedical.com/) for the Beckman Coulter</p><p>XLA/I analytical ultracentrifuge (http://www.beckman.com/</p><p>) significantly broadens AUC analysis two ways. First, fluor-</p><p>escence detection allows AUC analysis of very dilute solutions</p><p>(Fig. 2), which is required for the analysis of very tight</p><p>interactions or characterizing trace quantities of a solute.</p><p>Second, the selectivity of fluorescence detection means that</p><p>it is possible to perform rigorous AUC analysis of a particular</p><p>molecule of interest in a very complex mixture (e.g. cytosol,</p><p>serum). This means that the rigor of AUC analysis can be</p><p>brought to bear on questions that heretofore were out-of-</p><p>bounds to physical analysis. In particular, it is possible to</p><p>determine the size of complexes under conditions that are far</p><p>closer to in vivo than is possible with most other first-principle</p><p>physical methods. Simple pull-down experiments can be</p><p>performed using fluorescently labeled and unlabeled antibo-</p><p>dies to probe for protein-protein complex formation (Fig. 4).</p><p>312 www.drugdiscoverytoday.comFluorescently labeled ligands, such as a phospholipid, might</p><p>be used as a probe for binding an...</p></li></ul>