the ligand signals rather than protein signals. Simultaneous 1D pulsed field gradient NMR methods. Lin and Shapiro observation of signals from all small molecules in a mixture [18] first reported the DECODES experiment, an experi allows the straightforward identification of the specific com- ment designed for mixture analysis of small molecule pools ponent(s) binding the target protein without a need for from combinatorial chemistry reactions. In this experi- deconvolution. More importantly, observing the ligands ment, mixtures of compounds that could not be identified directly obviates the need for isotopically labeled protein, using standard 2D techniques alone could be assigned by and allows one to examine targets with no molecular weight acquiring a series of 2D spectra at different pulsed field mit; both severe limitations of the sar by nmr method. gradient strengths, effectively separating the original 2D spectra into a third dimension modulated by molecular dif- The transferred NOE experiment is a well established fusion. This methodology was extended to detect method for determining the bound conformation of a ligand intermolecular interactions between small molecules and hich undergoes rapid exchange with a target molecule. small molecule receptors 19, 20 Due to the high ligand When a small molecule binds a large molecule, such as a concentrations required(20 mM), however, these tech even transiently, the numerical sign and magni- niques will have limited utility for screening of biological des of the NOEs change. The magnitudes of the NOEs targets. Another publication by the Novartis group [21] are dependent on the proton-proton internuclear distances describes an alternative method for examination of small and may be used to calculate approximate interproton dis- molecule interactions with proteins. The pulsed sequence tance restraints. Although the NOE information is most combines diffusion editing, to detect ligand binding, with often used to determine the structure of the ligand when 1.C isotope filtering, which removes background signals bound to the target, for screening purposes it provides a very from the protein. Although this experiment is simple to sensitive and highly reliable diagnostic for binding as well. carry out and does not rely on obtaining difference spectra veral groups have reported using the transferred Noe the method will have limited utility for higher throughput method as a screening tool. Meyer et al.[17.I demonstrated NMR screening of mixtures, due to the high costs of that this method could be used to screen a mixture of preparing the requisite amounts of i.C labeled protein oligosaccharides for binding to Aleuria aurantia agglutinin In this study, it was found that despite the complexity of the A more practical implementation of such methods is 2D spectra for two mixtures of oligosaccharides containing described by Hajduk et al. 22. In this paper, the authors six and fifteen components, a single binding ligand could be show how NMR relaxation and diffusion, well known tech readily identified and characterized. Other groups are also niques for the study of macromolecule-small molecule using the transferred NOE technique routinely for screen- interactions, may be used to screen large libraries of small ing purposes. Kline and co-workers at Lilly Research molecules against a macromolecular target. The ID meth Laboratories repo fragments derived from biotin to the throughput as experiment time is greatly reduced relative using transferred NOE methods to ods described in this paper promise much greater examine bind protein streptavidin (A Kline, personal communication). to the 2D transferred NOE or 2D 1H-15N heteronuclear ur SHAPES strategy for NMR screening, discussed below, correlation experiments used in the sar by NMR also makes extensive use of transferred NOE spectra to approach. Additional advantages of these 1D methods are identify binding components in mixtures that they do not require the use of isotopically labeled pro In and n ay be applied to targets in a much Other groups have reported on methods for screening mix- molecular weight range. Drawbacks to these methods d on relaxation and diffusion editing. Like the include the additional processing and errors associate NOE, other NMR observables that exhibit a marked use of nmr difference methods, and, for the use of line dependence on molecular mass include the transverse broadening methods, decreased sensitivity for detection of elaxation time (T2)and the translational diffusion coeffi- weaker binding compounds compared to the transferred ent (D). In relaxation editing, binding of a smal NOE experiment. It should also be noted that the group at molecule to a large protein will cause the ligand 'T2 values Bristol Myers Squibb(Wallingford, CT) also reported at a to decrease and the signals to broaden. The degree of recent meeting the use of diffusion-based techniques for broadening observed is dependent on, and increases with, high-throughput screening(DJ Detlefson et al. Abstract the affinity of the ligand for the macromolecule. This W/Th P147, 39th Experimental Nuclear Magnetic broadening is easily measured by comparing ID NMR Resonance Conference, Asilomar, CA, 1998) spectra of the ligand with and without the protein present In diffusion editing, a similar approach is used. A small The SHAPES strategy molecule diffuses in solution several orders of magnitude In our laboratory, we have developed a strategy for NMR ter than a large protein. If a small molecule binds to a screening that, like the above methods, relies on monitor macromolecule, its diffusion coefficient will be reduced ing of ligand signals to determine which compound in a compared with that for the free ligand. The extent of the mixture binds a drug target Fejzo et al. Abstract MIIA-4 reduction will be dependent on the affinity of the small XVIllth International Conference on Magnetic Resonance molecule for the target. Changes in the diffusion proper- in Biological Systems, Tokyo, Japan, 23-28 August 1998) ties of a small molecule may be measured using standard Our primary motivation for developing these methods is