Accelerating NMR in Pharmaceutical Research and Development
Introduction Recent developments in biological and chemical sciences have equipped researchers with new tools allowing for an accelerated pace in pharmaceutical research and development. While these advances create public optimism for the discovery of new and better medicines, they also challenge the analytical methods that are integral components in the process creating a critical need for industrial analytical sciences to advance and evolve. One such analytical method that is particularly challenged is NMR spectroscopy, known for the wealth of detail it provides and the mountain of resources it consumes. Overall, it seems clear that NMR techniques are underutilized in modern pharmaceutical research as a consequence of resource barriers. Novatia believes the accessibility of NMR results can be improved by accelerating results generation along with lowering the resource barriers for NMR studies and believe this can be accomplished by developing question-specific methods keeping a few simple guidelines in mind. First, develop methods for rapid qualitative analysis: sometimes maybe / maybe not a thousand times a day is more useful than absolutely every couple of days. Second, provide unique and useful data that requires minimal interpretation: more researchers will be able to get quality results and may find new and better uses for NMR. Third, conserve resources by minimizing the amount of sample required, limiting the use of isotopic labels and lowering instrumentation field requirements: these will increase the chance that NMR can be used and useful to more researchers. Fourth, increase automation at all levels and make NMR data and methods easily accessed by and integrated into the enterprise: the farther from the spectrometer these get, the more useful they will be. Finally, don't provide similar information that can be obtained by more sensitive analytical methods: NMR will lose every time when sensitivity matters so don't make it an issue. These simple guidelines, combined with viewing the NMR spectrometer not for what it is but for what relevant questions it can answer, focus application development increasing the likelihood that appropriate and useful tools will result. Two examples of NMR applications for accelerating pharmaceutical research and development are described below including Molecule Structure Analysis and Ligand Binding Assays. These are representative of Novatia's ongoing efforts to modernize NMR ensuring that it remains a relevant spectroscopic tool in pharmaceutical research and development.
Molecular Structure Analysis Knowledge and proof of chemical structure is of central importance in drug discovery efforts. Structural information is required for decisions ranging from mechanism of action and compound stability through metabolic fate of lead drug compounds. NMR is uniquely suited to provide detailed covalent, stereochemical and tertiary structural insight. Unfortunately, this information is unavailable on a high throughput basis. Novatia believes that it is possible to develop automated qualitative approaches for NMR structure confirmation and has prototyped a Molecular Structure Analysis application that starts with a molecular structural proposal and utilizes a NMR software package (data acquisition, processing and reporting) to provide a qualitative answer.
Specifically, our approach consists of 1D 1H TOCSY NMR experiments to produce scalar connectivity patterns of a molecule that are compared to predicted patterns for an analyte of interest. Development initially focused on software to accept structural queries in the form a Simplified Molecular Input Line Entry Specification (SMILES) string. SMILES, a valence model description of molecular structure that is designed to be parsed, is well suited for being the basis of structural query syntax. Novatia has completed initial development of a spin system parser in Python and developed code to make this application callable as a webservice (over IP using XML-RPC) directly from the spectrometer software. In addition, a third party has developed a 1H chemical shift estimation software. These two functions are packaged together into Novatia's ASEprediction package. Additional development has centered on data acquisition and processing software. While this could be implemented on any modern NMR spectrometer, Novatia is using the Varian NMR platform using VNMR (the acquisition and processing software) with its MAGICAL control language for automated 1) collection 1D 1H NMR data, 2) peak lists for selective excitation and 1D 1H TOCSY, 3) preparation of shaped pulse array using Pandora's Box and 4) collection of a series of 1D 1H TOCSY experiments for each. The final phase of the project, already in progress, involves integration of the spin system prediction routine with VNMR code on the spectrometer into a package that accepts SMILES structural query and a sample as input and outputs a prediction that describes whether or not the structure may be present in the sample. As development of this package proceeds, new ideas are being uncovered that can either increase the utility and accuracy of this application or point towards new applications for small molecules. Novatia strongly feels that NMR can make other contributions in a more rapid manner and that this Molecular Structure Analysis application is only a beginning. Novatia will be making the entire package available (Automated Structure Elucidator ) later in 2003
Ligand Binding Assay A key first step in drug discovery is the identification of potential lead compounds. Often, the function of the target molecule is known and it is possible to screen for new leads using high throughput function-based assays. The development of these assays into a format for screening is a critical and time-consuming process: they are crucial to the fidelity and success of the high throughput screens. However, as targets gleaned from genomics efforts are increasingly considered, there will be more situations where the function is unknown or poorly understood. Developing functional based screens in these situations is unlikely. In the absence of functional assays, methods that characterize binding will increase in importance as a substitute for primary or secondary assays. The idea is that if a compound binds tightly and specifically, it has a good chance of being a drug as binding becomes a proxy for pharmacological effect. The availability of rapid and robust Ligand Binding Assays would serve as a useful secondary screen to verify and provide more detailed information compared to the higher throughput assays described above.
To address this need, Novatia has developed methods to rapidly characterize interactions of small molecule pharmaceuticals and target receptors based on diffusion coefficient measurements derived from NMR. A more detailed description of these experiments can be found here. Diffusion is a molecular transport process driven by Brownian motion where larger molecules move more slowly than smaller molecules. Intermolecular interactions between a small molecule and macromolecule can significantly modulate D (the diffusion coefficient) of the small molecule. A small molecule ligand in a free state diffuses faster than if it binds to a macromolecule where its D changes proportional to how tightly it binds. In a titration where increasing amounts of ligand are added to a fixed amount of receptor, the response of the ligand D as a function of changing molar ratios of ligand:receptor yield data that can be used to determine if and how tightly a ligand binds. The Dissociation Constant Estimation figure shows an example of data derived from NMR diffusion experiments on a titration of vancomycin with increasing concentration of peptides containing D-Ala where the diffusion coefficient of the ligand is plotted against the ligand:peptide molar ratio. The results in the figure show the agreement between the expected response based on extracted binding values and that diffusion indeed can indeed be used to characterize and quantify receptor-ligand binding with the experimental data points (denoted by a, b & c in the figure). In addition to diffusion, other NMR techniques to study ligand receptor interaction, such as Saturation Transfer Detection (STD) methods are being explored that have advantages over diffusion based methods most notably in their overall sample and concentration requirements. Overall, NMR Binding assay methods show promise as a general lead identification tool and may be particularly useful in screening genomics targets where functional assays are not available.
Conclusions Novatia's approach to NMR blends new NMR technology, strategies for streamlined sample preparation and delivery, automated data acquisition and processing and results reporting software to make NMR derived results more accessible. By adopting this analytical application engineering approach pioneered by researchers at Novatia, prospects for increased participation and utility of NMR in pharmaceutical discovery and development efforts are promising. The initial applications (Molecular Structural Analysis and Ligand Binding Assays) described illustrate the process and product of Novatia's NMR approach to answer pharmaceutically relevant questions in a timely manner. They also provide a road map for additional NMR based applications to address other key needs for pharmaceutical research efforts. Novatia plans to continue the development of NMR-based analytical applications, explore and develop applications and field these with end-users and instrument vendors in the hopes helping to deliver on the promise of new and better medicines in the future.
 
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