Although mass spectrometry (MS) dates back to 1897, and high-performance liquid chromatography (LC) to the 1970s, scientists are still developing methods to enhance their capabilities. Rapid advancements in the pharmaceutical and biotechnology fields are the main drivers behind new developments. Many efforts are stemming from academic labs, which are opening new avenues for these technologies.
LC/MS is now a multibillion dollar business with tandem LC/MS comprising the majority sector. In this article, a handful of researchers provide a preview of some of the topics that will be covered at “Pittcon 2009” next month.
A researcher in the University of Michigan’s chemistry department is developing analytical methods to study changes in metabolites within pancreatic islets of Langerhans cells. Charles Evans, Ph.D., says that this involves developing new liquid-chromatography methods, miniaturizing LC separations using capillary columns and multidimensional separation.
“By developing higher-resolution separation methods, we can more effectively take a complicated sample and break it up into all its components,” he says.
Islet cells contain beta cells, which are responsible for secreting insulin and maintaining constant blood glucose levels. “We are studying the metabolites present in these beta cells, which may be involved in pathways associated with diabetes development when they fail and are unable to secrete adequate insulin quantities.”
The multidimensional, 2-D separation involves two different liquid chromatography columns. A standard separation is performed first on one column and the fractions collected. These fractions are further analyzed in the second column; the columns must be different.
“Select two different columns that have as little correlation as possible between the separation on the first dimension as on the second dimension,” Dr. Evans suggests. For example, perform reverse-phase separation as one dimension and ion-exchange separation as the second dimension.
He says that the metabolites they are primarily interested in detecting are those in central carbon metabolism, glycolysis, and the TCA cycle. Additional energy transfer metabolites like NAD, NADH, ATP, and ADP can also be measured.
“One change we detect is that there are more central carbon metabolites present in the cells as a response to stimulation with glucose. We also detect changes in the energy metabolites. We’re still sorting out what all these changes mean,” states Dr. Evans.
The overall goal of this research is to develop methods to help improve the understanding of the biochemical mechanisms that underlie Type 2 diabetes. “We’re hoping that being able to measure these compounds will detect changes associated with diabetes along the course of the disease.” Dr. Evans adds that this method could have potential use for many other components—whether in disease research or understanding biochemical mechanisms that change based on environmental factors.
Mobile-Phase Modifiers
Researchers at Purdue University are working on an HPLC-MS/MS method for metabolite identification.
Ionic liquids have an advantage over organic solvents like methanol in that they don’t denature proteins as much and enable protein separation in their native confirmation. This is the driving force behind the research of Neil Danielson, Ph.D., professor of chemistry at Miami University, Ohio.
“We have been separating small molecules like caffeine and aromatic carboxylic acids by reversed-phase liquid chromatography (RPLC) using ionic liquid modifiers like ethylammonium formate (EAF) and methylammonium formate (MAF) as replacements for methanol in the mobile phase.”
Furthermore, Dr. Danielson has discovered that MAF has an advantage over EAF in that it has a lower viscosity, which makes it more compatible in terms of pressure limitations. “Organic solvents have a very low viscosity, below one centipoise (cP). MAF has a viscosity around 9 or 10 cP, which is really good for ionic liquids. This gives us better efficiency in LC if we work with a less viscous mobile phase.”
Polarity of the mobile and stationary phases, as well as that of a sample, are all involved in controlling retention of a sample compound and the separation of a mixture of sample compounds.
“We can control the polarity of the mobile phase—the amount of MAF in water. This ratio will vary the polarity, which is effective in controlling the retention of many pharmaceuticals. If we increase MAF, this lowers the retention.”
In addition, his group discovered that using low levels of MAF (1%) to replace methanol, for certain compounds like warfarin, the methylammonium ion formed an adduct and enabled easier qualitative identification by RPLC with MS detection.
When using MAF at higher concentrations (5–20%), Dr. Danielson says his group is able to control retention of water-soluble vitamins and antibacterials. “This is quite novel in that we are the first to show this ionic liquid as an organic solvent replacement in LC/MS is compatible because it’s forming volatile components in the interface.”
He adds that a compelling use of MAF is as a major mobile-phase component of LC/MS that has been shown to provide an advantage for separation of proteins as well as antibacterials. However, he says, “we’re still searching for a clear advantage for the use of MAF for small molecules.”
Better Analysis of Biofluids
In response to the current trend to develop more potent drugs with lower circulating levels, there is an ongoing effort to develop methods to increase detection sensitivity in biofluids. “Our research is focused on sensitivity in bioanalysis,” says Paul Rainville, applications chemist with Waters. One way to increase sensitivity is with smaller particles.
Waters developed the use of sub-2-micron chromatographic particles in 2004, which can increase the speed of analysis and reach up to a 10-fold increase in sensitivity. The particles consist of a patented material, bridged ethyl hybrid (BEH) that can withstand extreme pressures and operate in a wide pH range.
The advantages of using an elevated pH mobile phase include selectivity gains previously unattainable; ability to chromatograph basic analytes in a neutral state leading to improved peak shape; and the promotion of ionization of molecules that are to be detected by mass spectrometry—enabling further increases in sensitivity.
“This doesn’t eliminate steps, but it does increase the speed of running your assay without reducing chromatographic performance. Our particles maintain separation quality and are done faster, and this means increased productivity and less expense,” states Rainville.
One of the current hurdles with LC in bioanalysis is to provide a solid, inlet platform for the mass spectrometer. It must have compatible flow rates and the ability to resolve matrix interferences from the drug or metabolite undergoing quantification. “This is why we developed our sub-2-micron particles and the hardware to go around it,” he adds.
Better sensitivity also enables correct determination of the fate or pharmacokinetics of a drug molecule—both important parameters for the success of a drug. The ability of instruments to perform tasks that address regulatory concerns such as sample-matrix monitoring or metabolites is also advantageous. Rainville says that this method will soon be applied to detecting steroids in biofluids, and may lead to developing new packing material to address this. “We’ll apply the sub-2 micron particles and elevated pH for any drug that may have a challenging limit of detection.”
Phosphorylated Proteins in CSF
In-depth analysis of cerebrospinal fluid (CSF) has the potential to reveal important details and malfunctions of many nervous system diseases. Dean Stuart, from the University of Cincinnati’s department of chemistry, and colleagues, are using different instruments to obtain better information from CSF components. Samples obtained from the university’s medical school were divided into three groups to compare various phosphorylated proteins and/or peptides: patients with post-subarachnoid hemmorhage, patients with arterial vasospasms, and normal patients.
“The overall goal would be to see if there’s a difference in phosphorylated proteins across that batch, and if so whether you could say that high levels of these proteins are a precursor to stroke or vasal spasm. I can see slight differences in phosphorylation using inductively coupled plasma MS (ICPMS) as the phosphorus specific detection and then using ion-trap MS to get structural data, followed by database-search software to figure out the identity of phospholated proteins,” explains Stuart.
In addition, using size exclusion chromatography, he was able to show a slight difference in phosphorylated types. This was done using a 5 kilodalton filter, to exclude anything larger than 5 kd. “We wanted to deal with small proteins or peptides, and get rid of big macromolecules like albumin.” Then, instead of fractionating the samples first, he used a size exclusion column with a range of 100 to 7,000 daltons for molecular weight exclusion. The resulting peaks were run on ion-trap MS and run through software (Agilent’s Spectrum Mill) for identification.
This size-exclusion method mimics conventional proteomic approaches. “In my case, the two dimensions are size exclusion chromatography followed by nano-reverse phase. I would hope this could be used further to find other phospholated proteins or any other type of proteins. The methodology ought to work for anything,” Stuart summarizes.
Identifying Drug Metabolites
A lab within the department of chemistry at Purdue University is developing methods for metabolite identification. “We first started developing these as a compliment to established methods of identifying functional groups in an unknown analyte,” explains Steven Habicht, a scientist working in the lab. He says there are certain situations where identifying specific impurities is not possible by MS/MS alone, and the more additional compounds, the longer it takes for analysis and the more expensive it is.
This HPLC-MS/MS method is based on ion-molecule reactions for identifying tertiary N-oxide functional groups in unknown analytes. “This is applied early in the drug discovery phase, but it could also be used for stability-indicating assays to see if any compounds will be formed when exposed to high heat, humidity, or from storage in plastic containers. If extra products are formed from the compound, you need to identify what those are and whether they are toxic.”
In order to introduce neutral molecules into a commercial quadrupole ion trap MS, his group developed an external reagent mixing manifold. This allows tri (dimethyl amino) borane (TDMAB) to be mixed with the helium buffer gas used in the trap. The analyte of interest is isolated in the trap and reacts with TDMAB for a specific time. The reaction delivers only protonated N-oxide analytes as they elute from the HPLC column. This process was demonstrated using Clozapine N-oxide and Olanzapine-N oxide.
“We’re creating our own niche in metabolite identification with these methods,” states Habicht. “There are situations where you can’t distinguish two different types of metabolites where these ion-molecule reactions can fill the void.” He adds that the main focus is to continue to develop a library of different reagents to make functional group identification a straight-forward process.
There is little doubt that many more potential applications for LC/MS will continue to be developed as more targeted drugs become available through the efforts of genomics, proteomics, and metabolomics. LC/MS may become standard for clinicians searching to identify sources of side effects or potential toxins. Who knows where these technologies will lead medicine over the next 100 years?
Tuesday, March 31, 2009
Intel details future graphics chip at GDC
On Friday, Intel engineers are detailing the inner workings of the company's first graphics chip in over a decade at the Game Developers Conference in San Francisco--sending a signal to the game industry that the world's largest chipmaker intends to be a player.
During a conference call that served as a preview to the GDC sessions, Tom Forsyth, a software and hardware architect at Intel working on the Larrabee graphics chip project, discussed the design of Larrabee, a chip aimed squarely at Nvidia and at Advanced Micro Devices' ATI unit.
And Nvidia and AMD will no doubt be watching the progress intently. Intel's extensive and deep relationships with computer makers could give it an inside track with customers and upset the graphics duopoly now enjoyed by Nvidia and AMD. In the last decade Intel has not competed in the standalone, or "discrete" graphics chip market where Nvidia and AMD dominate. Rather, it has been a supplier of integrated graphics, a low-performance technology built into its chipsets that offers only a minimal gaming experience. (In the 1990s, Intel introduced the i740 GPU which, in relative terms, was not a success.)
Forsyth said that there is not yet a Larrabee chip to work with--it's expected late this year or early next year--and that "a lot of key developers are still being consulted on the design of Larrabee." But Intel will offer ways for developers to test the processor, he said. "On the Intel Web site there will be a C++ prototype library. It doesn't have the speed of Larrabee but has the same functionality. Developers can get a feel for the language, get a feel for the power of the machine."
Beyond games, Intel is also trying to catch a building wave of applications that run on the many-core architectures inherent to graphics chips. Nvidia and AMD graphics chips pack hundreds of processing cores that can be tapped for not only accelerating sophisticated games like Crysis but for doing scientific research and high-performance computing tasks.
One of the largest test sites for Larrabee is Dreamworks, which will use Larrabee for rendering and animation. To date, Dreamworks had to wait overnight to get a rendering project completed. "Using (the) Nehalem (processor), Dreamworks can almost do it in real time and it is only going to better with Larrabee," said Nick Knupffer, an Intel spokesperson.
Larrabee is "Intel's first many-core architecture," Forsyth said. "The first product will be very much like a GPU. It will look like a GPU. You will plug it into a machine and it will display graphics," he said. (GPU stands for graphics processing unit.)
"But at its heart are processor cores, not GPU cores. So it's bringing that x86 programmable goodness to developers," Forsyth said. Larrabee will carry the DNA of Intel's x86 architecture, the most widely used PC chip design in the world.
During a conference call that served as a preview to the GDC sessions, Tom Forsyth, a software and hardware architect at Intel working on the Larrabee graphics chip project, discussed the design of Larrabee, a chip aimed squarely at Nvidia and at Advanced Micro Devices' ATI unit.
And Nvidia and AMD will no doubt be watching the progress intently. Intel's extensive and deep relationships with computer makers could give it an inside track with customers and upset the graphics duopoly now enjoyed by Nvidia and AMD. In the last decade Intel has not competed in the standalone, or "discrete" graphics chip market where Nvidia and AMD dominate. Rather, it has been a supplier of integrated graphics, a low-performance technology built into its chipsets that offers only a minimal gaming experience. (In the 1990s, Intel introduced the i740 GPU which, in relative terms, was not a success.)
Forsyth said that there is not yet a Larrabee chip to work with--it's expected late this year or early next year--and that "a lot of key developers are still being consulted on the design of Larrabee." But Intel will offer ways for developers to test the processor, he said. "On the Intel Web site there will be a C++ prototype library. It doesn't have the speed of Larrabee but has the same functionality. Developers can get a feel for the language, get a feel for the power of the machine."
Beyond games, Intel is also trying to catch a building wave of applications that run on the many-core architectures inherent to graphics chips. Nvidia and AMD graphics chips pack hundreds of processing cores that can be tapped for not only accelerating sophisticated games like Crysis but for doing scientific research and high-performance computing tasks.
One of the largest test sites for Larrabee is Dreamworks, which will use Larrabee for rendering and animation. To date, Dreamworks had to wait overnight to get a rendering project completed. "Using (the) Nehalem (processor), Dreamworks can almost do it in real time and it is only going to better with Larrabee," said Nick Knupffer, an Intel spokesperson.
Larrabee is "Intel's first many-core architecture," Forsyth said. "The first product will be very much like a GPU. It will look like a GPU. You will plug it into a machine and it will display graphics," he said. (GPU stands for graphics processing unit.)
"But at its heart are processor cores, not GPU cores. So it's bringing that x86 programmable goodness to developers," Forsyth said. Larrabee will carry the DNA of Intel's x86 architecture, the most widely used PC chip design in the world.
Apple beats Intel to Nehalem-EP chip launch
Ponder this: Is an Intel product launch still a launch, if the product debuts very publicly in an Apple computer?
I won't presume to answer that question. But the fact is that Intel will launch Nehalem-EP server processors later this month, despite their manifestation Tuesday in the new Mac Pro under their official model names: the Xeon 3500 and 5500.
The chips--in their desktop variant known as the Core i7--are being offered in eight-core or four-core configurations and, like all Nehalam-architecture processors, come with an integrated memory controller for (theoretically) better performance. (Intel's Core architecture does not integrate the memory controller.)
Other Nehalem-architecture features include: Hyper-Threading for, according to Apple, "up to 16 virtual cores" (which improves multitasking), and Turbo Boost Technology, which dynamically increases the processor's frequency, as needed.
The Mac Pro also offers high-end Nvidia and ATI graphics. Systems can be configured with either Nvidia GeForce GT 120 or ATI Radeon HD 4870 graphics chips.
I won't presume to answer that question. But the fact is that Intel will launch Nehalem-EP server processors later this month, despite their manifestation Tuesday in the new Mac Pro under their official model names: the Xeon 3500 and 5500.
The chips--in their desktop variant known as the Core i7--are being offered in eight-core or four-core configurations and, like all Nehalam-architecture processors, come with an integrated memory controller for (theoretically) better performance. (Intel's Core architecture does not integrate the memory controller.)
Other Nehalem-architecture features include: Hyper-Threading for, according to Apple, "up to 16 virtual cores" (which improves multitasking), and Turbo Boost Technology, which dynamically increases the processor's frequency, as needed.
The Mac Pro also offers high-end Nvidia and ATI graphics. Systems can be configured with either Nvidia GeForce GT 120 or ATI Radeon HD 4870 graphics chips.
Intel adds crush of new mobile, server chips
Intel updated its processor list Monday with new Core 2 chips for Macbook Air-class laptops and a crush of Xeon processors for workstations and servers.
The number of new processor models is 20 in all.
Intel Vice President Pat Gelsinger holds a new Xeon chip.(Credit: Intel)
As reported earlier, Intel has introduced new power-sipping low-voltage (LV) and ultra-low-voltage (ULV) processor models for laptops such as the Apple MacBook Air and Dell Adamo.
The new LV and ULV processor models include the 17-watt SL9600 (2.13GHz, $316) and 10-watt SU9600 (1.6GHz, $289). More power-hungry Intel mainstream mobile processors are typically rated at 25 watts or 35 watts.
And over a dozen new Xeon quad-core processors based on Intel's new Nehalem chip architecture were added to the Intel price list.
Processors in the Xeon 5500 series range in price from $1,600 for the 130-watt W5580 (3.2GHz) to $423 for the 60-watt L5506 (2.13GHz). Intel, for the first time, is also listing each new Xeon chip's giga-transfers-per-second rating (GT/sec). For example, the W5580 is rated at 6.40 GT/sec, while the L5506 is rated at 4.80 GT/sec.
Other Xeon 5500 series models include the 95-watt X5550 (2.66GHz, $958), the 80-watt E5520 (2.26GHz, $373), and the 60-watt L5520 (2.26GHz, $530).
Intel also debuted the Xeon 3500 series, including the 130-watt W3570 (3.2GHz, $999) and the 130-watt W3520 (2.66GHz, $284).
The number of new processor models is 20 in all.
Intel Vice President Pat Gelsinger holds a new Xeon chip.(Credit: Intel)
As reported earlier, Intel has introduced new power-sipping low-voltage (LV) and ultra-low-voltage (ULV) processor models for laptops such as the Apple MacBook Air and Dell Adamo.
The new LV and ULV processor models include the 17-watt SL9600 (2.13GHz, $316) and 10-watt SU9600 (1.6GHz, $289). More power-hungry Intel mainstream mobile processors are typically rated at 25 watts or 35 watts.
And over a dozen new Xeon quad-core processors based on Intel's new Nehalem chip architecture were added to the Intel price list.
Processors in the Xeon 5500 series range in price from $1,600 for the 130-watt W5580 (3.2GHz) to $423 for the 60-watt L5506 (2.13GHz). Intel, for the first time, is also listing each new Xeon chip's giga-transfers-per-second rating (GT/sec). For example, the W5580 is rated at 6.40 GT/sec, while the L5506 is rated at 4.80 GT/sec.
Other Xeon 5500 series models include the 95-watt X5550 (2.66GHz, $958), the 80-watt E5520 (2.26GHz, $373), and the 60-watt L5520 (2.26GHz, $530).
Intel also debuted the Xeon 3500 series, including the 130-watt W3570 (3.2GHz, $999) and the 130-watt W3520 (2.66GHz, $284).
Revitalizing Surface Plasmon Resonance
Surface plasmon resonance, or SPR, is used to measure changes in molecular weight. Thus, it has long been used to study protein-protein interactions by registering the association and dissociation of a ligand (prey) binding to an immobilized protein (bait).
SPR has recently been revitalized by its aptitude for fragment-based lead discovery. Fragments are low-molecular-weight compounds (<350 kD) that bind to targets with low affinity (µM–mM range), yet they exhibit high ligand efficiency—each atom contributes by directly contacting the target’s binding site.
Once a hit is identified, the fragment is grown or combined with other fragments, usually by structure-guided design and synthesis, to quickly generate a much more potent lead. Fragments are simple, but libraries composed of them can represent many diverse compounds and different chemistries. It is hoped that discovering drugs in this piecemeal manner will shortly yield therapeutic breakthroughs.
Biacore (now part of GE Healthcare) claims to be the first company to apply SPR to label-free protein-protein interaction analysis with its Biacore™ systems, according to Stefan Lofas, Ph.D., principle R&D scientist. The system’s various applications venture beyond protein-protein interactions to include protein-DNA interactions, peptide interactions, the binding properties of antibodies, drugs, and small molecules, and even interaction of viruses with whole cells, he adds.
Biacore makes different machines for different purposes, including screening and drug discovery. Moreover, GE Healthcare recently acquired Microcal, a market leader in calorimetry, whose instruments can detect the number of binding sites and measure the enthalpy (DH) and entropy (DS) of binding. This is a “perfect complementary technique to Biacore systems,” Dr. Lofas explains.
Tony Giannetti, Ph.D., research scientist at Genentech, uses Biacore systems to identify promiscuous binders and remove them from high-throughput screens where they would create false positives. This saves valuable time, money, and effort that might otherwise be spent crystallizing them, he notes.
Biacore systems have the ability to recognize all of the hallmarks of promiscuous binders, he insists: greater than 1:1 stoichiometry, a high Hill slope, irreversible binding, aggregation, and sensitivity to detergent. Dr. Giannetti utilized the latest methods in biosensor operation to develop a high-throughput procedure for hit identification from fragment libraries all the way to lead-generation chemistry. Key features include the ability to screen and verify thousands of compounds in a short time, the large dynamic range of the assay (kd from 200 pM to 20 mM), and the small amount of protein necessary for fragment screening (<0.5 mg protein from assay development through hit validation).
He warns of the “need to align crystallographic and SPR conditions,” lest the PEG often present in crystallography buffer destroy the binding observed in Biacore experiments; PEG should be included in the initial Biacore screen. Dr. Giannetti points out that Biacore systems can also be used to complement enzyme assays by confirming potencies. Using Biacore systems, he has identified binders to baits as varied as kinases, polymerases, cytokines, receptors, proteases, and oxidases.
Vernalis’ approach to fragment-based drug discovery is called SeeDs, for structural exploitation of experimental drug startpoints. It includes the design of a fragment library, identification of fragments that bind competitively to a target, and evolution of these hits into leads.
Unique to the SeeDs strategy is displacement of bound fragments by a potent competitor, notes Roderick Hubbard, Ph.D., of the University of York and Vernalis. This feature ensures that the fragment is binding to the target’s active site and dramatically increases the quality of the initial hits, he says.
SPR has been successfully used in two phases of the SeeDs protocol. First, it has been used to identify compounds that bind, or if NMR was used for that, to validate binding. Since it is rapid and sensitive, SPR is an especially effective way to prescreen, or triage, a library before crystallization is attempted. It also allows for the screening of more hydrophobic fragments than NMR does.
Once binding has been established, the SeeDs protocol calls for SPR to characterize binding kinetics and thermodynamics. Applying SPR to the SeeDs method has successfully ascertained why isoxazole is a much more potent growth inhibitor than pyrazole, although they both have the same IC50 against Hsp90 (pyrazole is twofold faster on, but isoxazole is 15-fold slower off).
The FujiFilm Life Sciences Affinity Screening System, or AP-3000, was specifically designed and implemented for high throughput, sensitive analysis of small molecule binding, according to the company. The target protein is immobilized on disposable sensor sticks, with a short flow path to diminish reagent use but a wide bore to alleviate clogging.
There are six parallel channels, so six compounds and their corresponding blanks are analyzed simultaneously. It offers high-throughput (3,840 compounds in 24 hours) and high sensitivity (it can detect interactions as weak as 10 mM), and it is fully automated, from protein immobilization all the way through to data analysis, according to Don Janezic, the SPR business development manager. In addition, it can find hits, run dose response curves, and obtain affinity constants in less than two weeks, he says.
Graffinity’s screening method literally turns conventional SPR methodologies upside down. Rather than immobilizing the target protein on the chip and running each compound in the library over it, it immobilizes each compound in its own sensor field and runs the target protein over them, explains Mathias Woker, CBO, who adds that this method has a number of advantages.
First, he says, it can vary the point at which the compound binds to the chip, presenting different surfaces to the target protein.
Renate Sekul, Ph.D., head of research and development at Graffinity, thinks that “it is highly important to screen an active protein,” which is feasible in this system since the protein is in solution. This also makes standardizing the binding conditions across the library much easier.
Second, it is fast, Woker notes, making it well suited to high-throughput screens. This is partially because chip regeneration is not as much of an issue, and partially because they measure light spectrum shifts rather than light angle shifts to minimize errors, Dr. Sekul said.
Woker claims that “a Graffinity screen of 110,000 compounds takes 10 days where usually an experiment would take more than three months with strong limitations concerning the solubility of the compounds screened and the conformity of the results concerning consistent protein quality over the experiment time frame.”
Since 2002, Graffinity has screened over 80 targets, including kinases, phospodiesterases, proteases, nuclear hormone receptors, and even RNAs, and found hits for all of them, Woker concludes.
Protein Interaction Studies
Bernhard Geierstanger, Ph.D., group leader for NMR at the Genomics Institute of the Novartis Research Foundation, incorporates unnatural amino acids into proteins as site-specific NMR-active labels. This is “a unique way of putting an NMR active label at a chosen site in a protein,” he says, which is essential because in a “reasonable size protein—30 kD, for example—the number of signals is tremendous and impossible to resolve.”
Dr. Geierstanger, in collaboration with Peter Schultz, Ph.D., head of the institute, has created over 50 different unnatural amino acids with different purposes, along with their cognate orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Some are fluorescent, some are photoactive, and some are labeled with fluorine or 15N or 13C. They can be used in lieu of any natural amino acid and placed exactly where the investigator chooses, which is a particular boon in large systems.
A potential limitation is that only one label can be incorporated into each sample. Monitoring the chemical shift change in a series of such single resonances, however, allows site-directed screening for binders. This can reduce the number of false positives currently seen in drug development by ensuring that the drug is binding to the correct site.
In an exciting twist, Dr. Geierstanger has made photocaged serine, cysteine, and tyrosine. Once incorporated at the desired site, the NMR label can be cut off of these residues. Thus, for the first time, individual amino acids can be labeled without altering the protein’s primary sequence, which alleviates any fears about changing the protein’s conformation or interactions. This allows him to use NMR for label-free binding, much like the other investigators use SPR.
SPR has recently been revitalized by its aptitude for fragment-based lead discovery. Fragments are low-molecular-weight compounds (<350 kD) that bind to targets with low affinity (µM–mM range), yet they exhibit high ligand efficiency—each atom contributes by directly contacting the target’s binding site.
Once a hit is identified, the fragment is grown or combined with other fragments, usually by structure-guided design and synthesis, to quickly generate a much more potent lead. Fragments are simple, but libraries composed of them can represent many diverse compounds and different chemistries. It is hoped that discovering drugs in this piecemeal manner will shortly yield therapeutic breakthroughs.
Biacore (now part of GE Healthcare) claims to be the first company to apply SPR to label-free protein-protein interaction analysis with its Biacore™ systems, according to Stefan Lofas, Ph.D., principle R&D scientist. The system’s various applications venture beyond protein-protein interactions to include protein-DNA interactions, peptide interactions, the binding properties of antibodies, drugs, and small molecules, and even interaction of viruses with whole cells, he adds.
Biacore makes different machines for different purposes, including screening and drug discovery. Moreover, GE Healthcare recently acquired Microcal, a market leader in calorimetry, whose instruments can detect the number of binding sites and measure the enthalpy (DH) and entropy (DS) of binding. This is a “perfect complementary technique to Biacore systems,” Dr. Lofas explains.
Tony Giannetti, Ph.D., research scientist at Genentech, uses Biacore systems to identify promiscuous binders and remove them from high-throughput screens where they would create false positives. This saves valuable time, money, and effort that might otherwise be spent crystallizing them, he notes.
Biacore systems have the ability to recognize all of the hallmarks of promiscuous binders, he insists: greater than 1:1 stoichiometry, a high Hill slope, irreversible binding, aggregation, and sensitivity to detergent. Dr. Giannetti utilized the latest methods in biosensor operation to develop a high-throughput procedure for hit identification from fragment libraries all the way to lead-generation chemistry. Key features include the ability to screen and verify thousands of compounds in a short time, the large dynamic range of the assay (kd from 200 pM to 20 mM), and the small amount of protein necessary for fragment screening (<0.5 mg protein from assay development through hit validation).
He warns of the “need to align crystallographic and SPR conditions,” lest the PEG often present in crystallography buffer destroy the binding observed in Biacore experiments; PEG should be included in the initial Biacore screen. Dr. Giannetti points out that Biacore systems can also be used to complement enzyme assays by confirming potencies. Using Biacore systems, he has identified binders to baits as varied as kinases, polymerases, cytokines, receptors, proteases, and oxidases.
Vernalis’ approach to fragment-based drug discovery is called SeeDs, for structural exploitation of experimental drug startpoints. It includes the design of a fragment library, identification of fragments that bind competitively to a target, and evolution of these hits into leads.
Unique to the SeeDs strategy is displacement of bound fragments by a potent competitor, notes Roderick Hubbard, Ph.D., of the University of York and Vernalis. This feature ensures that the fragment is binding to the target’s active site and dramatically increases the quality of the initial hits, he says.
SPR has been successfully used in two phases of the SeeDs protocol. First, it has been used to identify compounds that bind, or if NMR was used for that, to validate binding. Since it is rapid and sensitive, SPR is an especially effective way to prescreen, or triage, a library before crystallization is attempted. It also allows for the screening of more hydrophobic fragments than NMR does.
Once binding has been established, the SeeDs protocol calls for SPR to characterize binding kinetics and thermodynamics. Applying SPR to the SeeDs method has successfully ascertained why isoxazole is a much more potent growth inhibitor than pyrazole, although they both have the same IC50 against Hsp90 (pyrazole is twofold faster on, but isoxazole is 15-fold slower off).
The FujiFilm Life Sciences Affinity Screening System, or AP-3000, was specifically designed and implemented for high throughput, sensitive analysis of small molecule binding, according to the company. The target protein is immobilized on disposable sensor sticks, with a short flow path to diminish reagent use but a wide bore to alleviate clogging.
There are six parallel channels, so six compounds and their corresponding blanks are analyzed simultaneously. It offers high-throughput (3,840 compounds in 24 hours) and high sensitivity (it can detect interactions as weak as 10 mM), and it is fully automated, from protein immobilization all the way through to data analysis, according to Don Janezic, the SPR business development manager. In addition, it can find hits, run dose response curves, and obtain affinity constants in less than two weeks, he says.
Graffinity’s screening method literally turns conventional SPR methodologies upside down. Rather than immobilizing the target protein on the chip and running each compound in the library over it, it immobilizes each compound in its own sensor field and runs the target protein over them, explains Mathias Woker, CBO, who adds that this method has a number of advantages.
First, he says, it can vary the point at which the compound binds to the chip, presenting different surfaces to the target protein.
Renate Sekul, Ph.D., head of research and development at Graffinity, thinks that “it is highly important to screen an active protein,” which is feasible in this system since the protein is in solution. This also makes standardizing the binding conditions across the library much easier.
Second, it is fast, Woker notes, making it well suited to high-throughput screens. This is partially because chip regeneration is not as much of an issue, and partially because they measure light spectrum shifts rather than light angle shifts to minimize errors, Dr. Sekul said.
Woker claims that “a Graffinity screen of 110,000 compounds takes 10 days where usually an experiment would take more than three months with strong limitations concerning the solubility of the compounds screened and the conformity of the results concerning consistent protein quality over the experiment time frame.”
Since 2002, Graffinity has screened over 80 targets, including kinases, phospodiesterases, proteases, nuclear hormone receptors, and even RNAs, and found hits for all of them, Woker concludes.
Protein Interaction Studies
Bernhard Geierstanger, Ph.D., group leader for NMR at the Genomics Institute of the Novartis Research Foundation, incorporates unnatural amino acids into proteins as site-specific NMR-active labels. This is “a unique way of putting an NMR active label at a chosen site in a protein,” he says, which is essential because in a “reasonable size protein—30 kD, for example—the number of signals is tremendous and impossible to resolve.”
Dr. Geierstanger, in collaboration with Peter Schultz, Ph.D., head of the institute, has created over 50 different unnatural amino acids with different purposes, along with their cognate orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Some are fluorescent, some are photoactive, and some are labeled with fluorine or 15N or 13C. They can be used in lieu of any natural amino acid and placed exactly where the investigator chooses, which is a particular boon in large systems.
A potential limitation is that only one label can be incorporated into each sample. Monitoring the chemical shift change in a series of such single resonances, however, allows site-directed screening for binders. This can reduce the number of false positives currently seen in drug development by ensuring that the drug is binding to the correct site.
In an exciting twist, Dr. Geierstanger has made photocaged serine, cysteine, and tyrosine. Once incorporated at the desired site, the NMR label can be cut off of these residues. Thus, for the first time, individual amino acids can be labeled without altering the protein’s primary sequence, which alleviates any fears about changing the protein’s conformation or interactions. This allows him to use NMR for label-free binding, much like the other investigators use SPR.
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