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Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals*
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals Alexandros Makriyannis and Spiro Pavlopoulos, University of Connecticut, Storrs, CT, USA & 2010 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 3, pp 2261–2271, & 1999, Elsevier Ltd., with revisions made by the Editor.
Symbols F1 F2 t1 t2 T1 T2
o1 ¼ y-axis frequency domain obtained by Fourier transformation with respect to t1 o2 ¼ x-axis frequency domain obtained by Fourier transformation with respect to t2 evolution time in a 2D pulse sequence detection period in a 2D pulse sequence spin–lattice (longitudinal) relaxation time spin–spin (transverse) relaxation time
methods allow researchers to study the interactions of a drug molecule with its site of action on the biopolymer (enzyme, receptor, nucleic acid, etc.). Such studies can lead to insights regarding the bioactive conformation of a flexible drug molecule, which constitutes invaluable information for drug design. Here we shall discuss the most commonly exploited experiments for extracting the individual NMR parameters mentioned above, and also how such parameters are utilized to obtain structural information.
Conformational Analysis of Small Molecules Scope and Applications Information on the structure of drug molecules and their interactions with their therapeutic sites of action is of critical importance in the design and development of new drugs. Of all the analytical methods, nuclear magnetic resonance spectroscopy (NMR) is the most exquisitely suited to provide such experimental results. The field of NMR is advancing continuously to include new pulse sequences and methods as well as progressively larger field instruments and improved probes. This fast progress in NMR methods and technologies has served to expand dramatically its applications in drug research. Indeed NMR, used jointly with X-ray crystallography and computational/graphical approaches, has revolutionized structure-based drug design. Currently the availability of a plethora of multidimensional/multinuclear NMR methods allows us to extract information on the structures and dynamic behaviours of a wide range of drug molecules of up to 30 kDa in size. These include the small and mediumsized traditionally used therapeutic drugs, to higher molecular weight peptides, proteins, nucleotides, nucleic acids and polysaccharide biotechnology products. Progressively, more detailed structural and dynamic information has become available because of our increased ability to measure more effectively the basic NMR parameters used in structural analysis, namely, proton and carbon chemical shifts, coupling constants, relaxation parameters (T1, T2) and the exceedingly valuable nuclear Overhauser effect. Such measurements are, in turn, used to obtain information on the three-dimensional structure of molecules as well as their conformational properties and dynamic behaviour. Additionally, the new solution NMR
Information on the structural properties of small drug molecules in solution can be obtained from a number of NMR parameters including 1H and 13C chemical shifts, 1 H–1H and 1H–13C scalar coupling constants, 1H nuclear Overhauser effects (NOEs), as well as relaxation measurements. Here, the conformational analysis of CP55,940 (Figure 1) is used to illustrate the most common experiments encountered in studying small molecules (o1000 Da) in solution. This synthetic compound is structurally related to D9-tetrahydrocannabinol (D9-THC), a psychoactive component of marijuana, and which has received much attention because it was used as the high affinity radioligand during the discovery and characterization of the G-protein coupled cannabinoid receptor (CB1). The elucidation of the conformational properties of this compound and its congeners provides information on the steric requirements for a productive interaction at the cannabinoid receptor active site. Double Quantum Filtered Correlation (DQF-COSY) and Total Correlation (TOCSY) Spectroscopy These experiments provide information on 1H chemical shifts and 1H–1H scalar coupling. Spectral assignments are made initially by an analysis based on integrated peak areas and chemical shifts in the one-dimensional spectrum. Subsequently, they are specifically assigned by analysis of scalar or spin–spin coupling connectivities observed by 1H–1H double quantum filtered correlation spectroscopy (DQF-COSY). This is a two-dimensional experiment where the information is spread onto a plane
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Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
Figure 1 The one-dimensional 1H spectrum of CP55,940 with an expanded scale of the aromatic region. The molecular numbering system for assigning the peaks is shown.
in which the diagonal is equivalent to the one-dimensional spectrum, and the scalar coupling is manifested as an off-diagonal crosspeak between the two resonances in question. Thus even though a resonance may not be visible in a one-dimensional spectrum due to overlap, its position may be identified from a crosspeak in a two-dimensional experiment. Protons that are part of a spin system often give rise to a pattern of diagonal peak–crosspeak connectivities that can be traced from a ‘starting point’ in the spin system to an ‘end point’. It is the presence of such connectivity patterns that makes this experiment such a powerful tool in assignment. For example, the 1H NMR spectrum of CP55,940 is shown in Figure 1. A logical starting point for assignment was the resonance at d 3.74 that was assigned to H9a because the size of the peak as measured by integration was consistent with one proton and because this aliphatic resonance is expected to be deshielded. In the DQFCOSY spectrum (Figure 2) vicinal coupling to H10e, H8e, H10a and H8a is observed. Assignment of H10e and H10a resonances was made with the support of the crosspeak connectivities of H9a with H10a, H10e, H8a and H8e. The DQF-COSY spectrum clearly shows three components (H10e, H8e and H11e) under the multiplet at d 2.06, in which three related strong geminal 2J couplings, H10e/a (F1 ¼ d 2.09, F2 ¼ d 1.38), H8e/a (F1 ¼ d 2.05, F2 ¼ d 1.53) and H11e/a (F1 ¼ d 1.98, F2 ¼ d 1.13) can be
discerned. This is a prime example of the improvement in spectral resolution of two-dimensional experiments over one-dimensional experiments. Complete assignment of a whole spin system may be limited because of severe spectral overlap. To overcome this, DQF-COSY data are often used in conjunction with total correlation spectroscopy (TOCSY). This experiment results in a transfer of magnetization across an entire spin system and consequently crosspeaks may be observed between each resonance of a spin system. Thus it is possible to determine whether a particular overlapped region of the spectrum contains all unidentified members of a chemical spin system. 1 H chemical shifts and scalar coupling constants can be measured directly from one-dimensional spectra if the peaks are well resolved, or, if spectra are too complex, they may be measured from DQF-COSY spectra crosspeaks. However, such measurements are often inaccurate and so are used as a basis of simulating the observed 1D spectrum to obtain more accurate values. The measurements are used as a starting point and are systematically altered until the stimulated spectrum best matches the observed spectrum. 1H–1H scalar coupling constants are especially useful in providing information on the dihedral angle within a HC–CH system and are thus one of the most important sources of conformational information.
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Figure 2 Contour plots of expanded regions from the DQF-COSY spectrum of CP55,940. The lines highlight the connectivities between the H8, H9a, H10 and H11 resonances.
Nuclear Overhauser Effect Spectroscopy (NOESY) The nuclear Overhauser effect (NOE) is another important NMR parameter used in conformational analysis because the magnitude of the NOE is inversely proportional to the sixth power of the interproton distance in space (INOE p r6). NOE spectroscopy (NOESY) is a twodimensional experiment that may be run routinely in which the NOE is manifested as a crosspeak between two resonances indicating that the two protons are near in space. For example, in the case of CP55,940, two NOE crosspeaks were assigned to the spatial coupling of H5 with H8a and H12a (Figure 3). Such crosspeaks are congruent with a conformation in which the planes of the two rings are almost perpendicular, with the Ph–OH oriented towards the a face of the cyclohexyl ring. An NOE crosspeak between the phenolic hydroxyl proton and the adjacent aromatic H2 indicates that these two protons are spatially near each other and thus coupled through a dipole–dipole interaction (Figure 3). Such a result indicates that in its preferred conformation, the
Ph–OH proton points away from the cyclohexyl ring and towards the H2 proton. The full analysis of NOESY and DQF-COSY spectra of other analogues, plus computational studies, further showed that this was typical for all congeners of CP55,940 and that the dimethylheptyl chain adopts one of four preferred conformations, in all of which the chain is almost perpendicular to the phenol ring. The most biologically active conformations were such that all hydroxyl groups were oriented towards one face of the cyclohexyl ring system (Figure 4), a feature that may be an important requirement for cannabimimetic activity.
Identification of Drug Metabolites One major use of NMR spectroscopy in the pharmaceutical sector is as part of a concerted and integrated effort, together with mass spectrometry MS, to identify the metabolites of both candidate and marketed drugs. It is an important part of any drug development process to be able to understand the metabolism of a candidate drug in several animal species. All of the usual structural tools of NMR spectroscopy can be used, but often the very limited
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Figure 3 arrows.
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
Contour plot of the 500 MHz CP55,940 NOESY spectrum in CDCI3. The NOE interactions for CP55,940 are indicated with
Figure 4 Representation of the active biological conformation of CP55,940. According to the current hypothesis, the ligand preferentially partitions in the membrane bilayer where it assumes an orientation and location allowing for a productive collision with the active site.
availability of material, usually isolated from urine, bile or faecal material, means that NMR spectroscopy is often limited to one-dimensional 1H spectra. If a drug molecule contains a fluorine or a trifluoromethyl substituent, both quite common, then 19F NMR spectroscopy can be very useful. Rather than extract the substance of interest from its biological matrix it is now also possible to conduct HPLC separations with on-line NMR detection. The most comprehensive approach is to also couple a mass spectrometer in parallel to the NMR instrument, splitting the chromatographic eluate 95:5 to the NMR and MS, respectively.
NMR spectroscopy can also be used to investigate certain drug metabolites that show chemical reactivity, and hence might cause toxicity by interacting with proteins. NMR spectroscopy has been used extensively for this in the case of drug acyl glucuronide conjugates, where the initially formed 1-b-O-acyl glucuronide of a carboxylate containing drug can spontaneously react to form isomers in which the drug moiety has migrated around the glucuronic acid ring. These isomers have been postulated to react with proteins and cause immunological toxicity. Hence, the rate of reaction has been measured for a number of such real and model drugs, especially for non-steroidal anti-inflammatory drugs, in order to develop structure–reactivity relationships. Structure of Drug Macromolecules An increasing number of therapeutic drugs are composed of proteins or peptides and knowledge of their threedimensional structures has helped in the design of structurally modified variants with improved biological activities and pharmacokinetic properties. Such a case is insulin, which was first extracted from pancreas tissue, used in a patient in 1922, and its structure first determined in 1972. It has a molecular weight of 5.8 kDa and consists of a 21 amino acid peptide (chain A) that is connected to a 30 amino acid peptide (chain B) by two
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Figure 5 The 500 MHz 1H DQF-COSY spectrum of a shortened analogue of insulin, des-(B26–B30)-pentapeptide insulin in D2O. The spectrum was recorded on a Bruker AM 500 spectrometer in a phase-sensitive mode. 2048 complex data points were acquired in the t2 dimension, with a total of 512 free induction decays (FID) collected for transformation in the t1 dimension. Each FID was acquired with a total of 128 scans.
Figure 6 The fingerprint region of a 500 MHz 1H NOESY spectrum which contains NOEs between NH and Ha resonances of des-(B26–B30)-pentapeptide insulin. The expected intraresidue and inter-residue NOEs that form the highlighted sequential pattern of B chain backbone NH and Ha resonances are represented as solid and dashed arrows, respectively.
disulfide bonds. A third intra-subunit disulfide bond exists in chain A. The structure of insulin has been probed using X-ray crystallography, while NMR spectroscopy was used to determine its three-dimensional structure in solution. As with other similar-sized molecules, standard two-dimensional 1H homonuclear experiments can adequately provide such structural information. The use of two-dimensional experiments such as DQF-COSY, TOCSY and NOESY to assign and determine the three-dimensional conformation of peptides and proteins is well established. Briefly, the method used for the assignment of amino acid chains is not dissimilar from that of assigning small molecules. Each amino acid residue gives rise to a characteristic spin pattern that can be identified using the complementary DQF-COSY and TOCSY spectra, where connectivities between all protons within a spin system are observed in the TOCSY, and connectivities between neighbouring protons are observed in the DQF-COSY. An example of the DQFCOSY spectrum for the des-pentapeptide insulin monomer is shown in Figure 5. Of the 46 amino acids in the monomer, non-overlapped spin systems for four valine, two gylcine, one threonine and one alanine residue were distinguished. The latter two were sequence specifically assigned as threonine-8 in the A chain and alanine-14 in the B chain as these were the only alanine and threonine residues in the sequence. In addition, a further nine AMX spin systems could be clearly
distinguished that were subsequently assigned to amino acids with the aid of the TOCSY and NOESY spectra. A combination of DQF-COSY and TOCSY data were required in order to delineate the remaining overlapped amino acid spin systems. Once the spin systems arising from individual amino acids have been identified, the correct sequence of amino acids is determined from NOE data, acquired using the NOESY experiment. For molecules that fall within a particular molecular weight range (1000–3000 Da), the magnitude of the NOE is small, and a modification that is referred to as rotating Overhauser enhancement spectroscopy (ROESY) must be employed. Typically, a series of NOESY (and/or ROESY) spectra are collected in different solvents such as D2O, H2O or DMSO, with mixing times ranging from 50 to 600 ms. Different mixing times are required to gain an estimate of the magnitude of the NOE and subsequently an estimate of the distance between the protons. Care must be taken at longer mixing times, as it is possible to observe an indirect magnetization transfer between protons that are further apart than 5 A˚ via a third proton that is appropriately positioned between them. To avoid this spin diffusion effect, it is preferable to acquire NOESY experiments with the shortest mixing times possible that will result in good quality spectra. The determination of the amino acid sequence is based on the fact that NOEs will always be observed between particular protons from neighbouring residues
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regardless of the secondary structure of the protein. For example the proton attached to the a carbon (Ha) of an amino acid will always be within approximately 4.5 A˚ of the proton attached to the nitrogen (NH) of the neighbouring amino acid that is attached to the carboxyl end. In the NOESY spectrum, the NH resonances tend to occur within a particular spectral region, as do the Ha resonances. Thus, there is a particular region of crosspeaks between them in which a continuous connectivity pattern can be distinguished that begins with an NOE between the first and second residue and ends with an NOE between the terminal residue and the one preceding it (Figure 6). Such patterns reveal the sequence of amino acids in the protein. Similar patterns can also be distinguished between other groups of resonances (e.g. correlations between NH protons of neighbouring residues, Hb protons and NH protons of neighbouring residues) that can be used to confirm the sequence or resolve ambiguities. Breaks in this continuous pattern do occur in cases where resonances overlap or in some instances due to the structure of the amino acid chain, for example when a proline is present. However, once various lengths of polypeptides have been identified it is usually possible to surmise the order in which the various lengths are connected. This is achieved by considering different possibilities until a process of elimination arrives at the arrangement that best fits the data. Once the spectra have been assigned, data that contain structural information can be extracted. Nuclear Overhauser effects between non-neighbouring residues are the most revealing source of conformational information, and scalar coupling constants are important in providing information on torsion angles. All such conformational parameters are used as constraints in computational calculations that arrive at the threedimensional conformation of the protein. For small molecules, such as the non-classical cannabinoids, it is possible to infer the conformation from NMR data without the aid of a computer, given that there are a relatively small number of NOEs generated and there are limited conformational possibilities from which to choose. However, for peptides, proteins and other macromolecules such as DNA, where a large number of measured NMR parameters must be taken into consideration, computational methods are an essential tool in the determination of the vast conformational possibilities. The computational techniques utilized seek to systematically adjust the position of all nuclei in the molecule, so that all distances and bond angles derived from NOE data and coupling constants are satisfied. At the same time, the structure must not exceed set physical limits for bond lengths, bond angles, torsion angles, van der Waals contacts and Coulombic interactions between atoms. The challenge in such methods is to ensure that all possible conformations are sampled while not allowing the
molecule to exceed the set physical limits. Various algorithms and methods for calculation exist; however, the most common are the distance geometry and/or restrained molecular dynamics methods. Using this basic methodology, the three-dimensional conformation of insulin was determined, and a significant amount of structure–activity information was gained by the study of insulin analogues and insulins purified from different sources. Structural Analysis of Drug-Binding Domains Macromolecules such as proteins and nucleic acids form the sites at which drugs interact. Knowledge of threedimensional conformations assists in the design of analogues that are more potent and have improved pharmacokinetic properties. Furthermore, the structural analyses of protein receptors and enzymes add to the knowledge of biological systems, and therefore assist in identifying novel types of therapeutic agents. Because of their large molecular weights, most macromolecular therapeutic targets cannot be studied using exclusively 1 H homonuclear methods. The advent of three-dimensional and heteronuclear pulse techniques has greatly expanded the ability to study macromolecules of up to 30 kDa in size. Three-dimensional techniques are a natural progression of the twodimensional experiments. The pulse sequence is altered so that a vertical domain is introduced and the information is spread into a third dimension, so that the spectrum now is projected into a cube instead of a plane. The diagonal of the cube is equivalent to the one-dimensional spectrum, and crosspeaks that may be overlapped in a two-dimensional spectrum can be resolved in the third dimension. A case in point is the structure determination of the insulin receptor substrate-1 (IRS-1). Insulin binds to a membrane-bound receptor that is a ligand-activated protein tyrosine kinase. Upon insulin binding there is an autophosphorylation of several tyrosine residues on the cytosolic side of the receptor. This enhances the tyrosine kinase activity of the insulin receptor towards other substrates and is required for signal transduction. A cascade of events is initiated, the first of which is the phosphorylation of IRS-1. This occurs when IRS-1 binds to the insulin receptor via a specific domain of the protein that is termed the phosphotyrosine binding (PTB) domain. The structure of this domain was determined while interacting with a tyrosine-containing peptide derived from a receptor. As such, this study is also an example of the use of NMR to study the interactions between molecules. The three-dimensional NMR experiment was coupled with heteronuclear techniques to increase the level of resolution of the spectra. For larger proteins such as IRS-1, homonuclear experiments are limited because
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
proton resonances become broader, and the efficiency of magnetization transfer between protons is decreased. With the advent of recombinant DNA technology, this problem can be overcome by producing the proteins to contain isotopic labels, such as 13C, 15N or 2H, either at specific sites or uniformly throughout the protein. The magnetic properties of these nuclei offer significant advantages over protons. Carbon and nitrogen nuclei resonate over a much larger spectral width or range of frequencies, and as such are less likely to suffer from overlap. Also their scalar couplings to each other and to protons are higher in magnitude, and a more efficient magnetization transfer takes place than for 1H–1H couplings. Thus, for macromolecules, heteronuclear experiments that correlate 15N to 13C nuclei or to protons offer significant gains in sensitivity and resolution compared to homonuclear experiments. The most often used heteronuclear experiments are the heteronuclear single quantum coherence experiment (HSQC) and the related heteronuclear multiple quantum coherence experiment. These experiments allow the measurement of one- and two-bond heteronuclear couplings (and homonuclear 13 C–13C couplings). They are most often combined with traditional two-dimensional experiments such as NOESY and TOCSY to yield a three-dimensional experiment. For example, in the case of an HSQC-NOESY spectrum of a protein, two of the axes represent the heteronuclei such as 15N and the protons which are directly attached to the nitrogen nuclei, while the third axis contains chemical shifts of protons which share an NOE effect with the amide proton. This offers a significant increase in resolution compared to a traditional two-dimensional NOESY. A large array of these types of three-dimensional, heteronuclear-edited experiments have been designed to extract structural information in various situations, and are described in other articles in this encyclopedia. Using these methods, the structure of the PTB domain of the IRS-1 protein was found to be similar to phosphopeptide-binding regions of several other proteins. Once the structural details were known, the different binding specificities could be compared and rationalized based on the interactions with their substrates. Drug Interactions with Macromolecular Targets NMR spectra are capable of supplying information about molecular interactions in solution. When a drug interacts with a receptor in a reversible manner, a number of effects may be observed in the spectra due to the exchange of the molecules between free and bound states. These effects will be discussed in relation to studies of antitumour antibiotics binding to short sequences of oligonucleotides. Compounds such as adriamycin (Figure 7) are currently in use as chemotherapeutic agents,
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Figure 7 Chemical structures of intercalating and minor groove DNA binding ligands.
and an understanding of factors involved in binding specificity may lead to more effective drugs to combat cancer. Chemical exchange effects have been exploited to great effect in obtaining information about these interactions. The free and bound states of the molecules represent two different chemical environments in which participating molecules may be found. Thus, the same nucleus of a particular molecule may be characterized by different values for NMR parameters, such as chemical shift, when located in each different environment and may give rise to different sets of resonances. The ability to measure the parameters that characterize each environment is dependent on the rate at which the nucleus exchanges between them. The exchange rate therefore has a significant effect on the appearance of NMR spectra, and the exchange rate is dependent upon the affinity of the drug for the receptor. The kinetics of the interaction can be examined by acquiring a series of spectra in which the concentrations of the reactants or the temperature are modulated. Figure 8 shows an expansion of the aromatic region of a series of 1H NMR spectra of the oligonucleotide duplex d(GGTAATTACC)2 to which has been added increasing amounts of a terephthalamide derivative (Figure 7). It is clear that the addition of the ligand
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Figure 8 Expanded aromatic regions from the 300 MHz 1H NMR spectra of complexes between a terephthalamide derivative and d(GGTAATTACC)2, recorded at 101C. Increasing ligand concentrations cause perturbations to chemical shifts of nuclei located at or near the binding site. All perturbations are upfield except for adenine H2 resonances that are perturbed downfield and are located on the floor of the minor groove.
causes significant perturbations of the free DNA resonances, and these indicate that the molecules are interacting. At ligand:DNA ratios between 0:1 and 1:1 there is a mixture of DNA molecules that are bound and unbound. The DNA resonances observed are averages of resonances arising from each of these states, and this is characteristic of fast exchange between the bound and unbound state due to a low affinity of the ligand for the binding site. There is a point in the titration in which the DNA resonances are no longer perturbed, and this identifies the point at which all binding sites have been occupied and there are only bound DNA molecules present in solution. By noting the resonances that are most perturbed and the direction of the perturbation (upfield or downfield), it was determined that the ligand was bound at the ‘ATTA’ binding site. The observed perturbations were consistent with the ligand being inserted edge-on into the minor groove. This places protons on the floor of the groove in the same plane as the aromatic drug which are then deshielded due to ring current effects. Protons above and below the plane of the ring experience shielding ring current effects. An example of a ligand that has a high affinity for the binding site and exhibits slow exchange characteristics is shown in Figure 9, where the antitumour antibiotic hedamycin (see Figure 7) was titrated into a solution of the oligonucleotide duplex d(CACGTG)2. Upon addition of hedamycin, the free DNA resonances diminish in
Figure 9 Expanded regions from the 300 MHz 1H NMR spectra of complexes between hedamycin and d(CACGTG)2, showing (a) aromatic resonances and (b) methyl resonances. Spectra up to 0.8:1 ligand:DNA ratio were recorded at 21C and the complex allowed to equilibrate for 24 h at this temperature. Subsequent spectra were recorded to 101C as the resonances were sharper. The dotted arrows highlight resonances that disappear after the 24 h equilibration period.
intensity and new peaks appear in the spectrum. These new peaks do not correspond to chemical shifts of the free duplex or ligand resonances and increase in intensity with increasing ligand concentration. This suggests that they arise from the bound form of these molecules, and that slow exchange conditions exist due to the high affinity of the ligand for the binding site. In this particular case it became apparent that time-dependent changes in the spectra were taking place. Allowing the mixture to equilibrate for 24 h resulted in certain resonances disappearing from the spectrum and sharpening of the remainder of the resonances. Given that hedamycin was subsequently shown to intercalate and alkylate, the transient peaks may represent reaction intermediates prior to alkylation where the ligand is intercalating reversibly to sites other than the most favoured binding site, and the sharpening of the spectra is caused by alkylation of the DNA by the ligand. In cases where the binding affinity is high, or the binding is irreversible, a detailed model of the interaction between the molecules can be constructed based on intermolecular NOEs. A NOESY spectrum of the 1:1
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Figure 10 Contour plot of the 500 MHz 1H NOESY spectrum of the 1:1 hedamycin:DNA complex recorded at 101C with a mixing time of 300 ms. Spectra were acquired on a Bruker 500AMX spectrometer with 2048 complex data points in the t2 dimension and a total of 512 free induction decays (FID) collected for transformation in the t1 dimension. Each FID was acquired with a total of 128 scans.
complex of hedamycin with d(CACGTG)2 is shown in Figure 10. Assignment of the resonances was achieved using a combination of COSY, TOCSY and NOESY spectra. As in the case of proteins, the NOESY spectra of oligonucleotides yield characteristic crosspeak patterns that allow the sequential assignment of residues. Once the spectrum had been assigned and intramolecular NOEs arising from the DNA duplex and hedamycin had been eliminated, a total of 61 intermolecular NOEs from each of the oligonucleotide strands to protons located on the chromophore, epoxide chain and sugar groups of hedamycin were identified. These intermolecular NOEs identified the binding site and allowed the orientation of the ligand within the binding site to be determined. For example, a number of intermolecular NOEs, summarized in Figure 11, showed that the molecule was intercalated between the central CG basepairs. Similarly, intermolecular contacts showed that the sugar groups of the molecule were located in the minor groove (Figure 12) and the epoxide chain in the major groove. Furthermore, the epoxide chain was shown to be in an appropriate location, so that the terminal carbon was capable of alkylating to the N7 of the guanine. Direct evidence for this was observed in that the guanine H8 proton became labile (i.e. disappeared when spectra were acquired in D2O) following complexation. Another example involving proteins is the interaction of the PTB domain of the IRS-1 protein with a tyrosine
Figure 11 Schematic representation of NOEs observed between the central GC basepairs and the hedamycin ligand. Contacts to major groove protons are shown as dotted arrows and contacts to the minor groove are shown as solid arrows.
phosphorylated peptide in which the peptide was found to bind in a surface-exposed pocket of the PTB domain. More specifically, the peptide was bound along one strand of the b sheet structure of the PTB domain and interacted with an a helix. Hydrogen bonding, van der Waals contacts and hydrophobic interactions stabilized the interaction. The peptide was found to be in a type I b turn, and the N-terminal residues of the peptide were in an extended conformation that formed an additional strand of the PTB domains b sheet. Combinatorial Methods Traditional methods of drug discovery involved a search amongst a range of diverse compounds that were ultimately derived from nature. This was a long and arduous process in which advances were due more often to serendipity rather than scientific thought and technique. The means to study the three-dimensional conformation of drugs and their receptors opened the door to a more rational approach in which compounds could be synthesized to better interact with a receptor. Along with the better understanding of drug/receptor interactions came improvements in biochemical techniques to isolate
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This method relies heavily on the use of NMR to assess the success of each reaction step. As a non-destructive technique, a sample of the resin may be removed, placed in the NMR machine and then returned to the column. However, the fact that the compound is attached to the resin results in broad resonances if solution NMR techniques are utilized. This difficulty is circumvented by using solid-state NMR methods, including magic angle spinning (MAS). These experiments lead to narrow linewidths and high quality spectra, so that even two-dimensional experiments can be performed.
Combinatorial Methods in Drug Discovery
Figure 12 Schematic representation of contacts between the sugar rings of hedamycin and the DNA duplex. Contacts are observed only to minor groove protons and each ring is associated predominately with one DNA strand.
receptors and test compounds for their affinity towards these receptors. The ability to test thousands of compounds in a relatively short amount of time using high throughput screening methods resulted in pressure to produce large numbers of diverse compounds to be tested.
Analysis of Solid-State Intermediates Combinatorial chemistry is a term used to describe the production of a large number (thousands) of diverse compounds, and different methods are employed to achieve this. One such method is solid-phase synthesis where the chemistry occurs on a solid support that may be packed into a column. The solid support may be a material such as crosslinked polystyrene and the compounds to be produced generally consist of a number of basic pharmacologically relevant structures (pharmacophores) that are to be linked in different ways. The method requires that one of the pharmacophores be linked to the solid support, then each subsequent pharmacophore can be delivered to the column with appropriate reagents, allowed to react, and then washed off the column. By varying the order in which the pharmacophores are delivered, different compounds can be produced.
One of the most interesting applications of NMR in drug research is in the field of high throughput screening. One such described approach bridges combinatorial chemistry with biochemical screening and was named ‘SAR by NMR’. The first step in this method is to identify the ability of ligands to bind to a target preparation of the molecular therapeutic target (e.g. an enzyme) in solution. Thus, batches of ligand mixtures (e.g. one batch containing 20 compounds) are allowed to interact with the enzyme preparation in solution while following changes in the protein 15N and 1H NMR frequencies. In this manner, batches that produce a response can be identified quickly and the compounds within each batch further tested to identify specific compounds that bind to adjacent but different sites on the protein. These ligands are then further optimized using rational design techniques to improve the binding at each respective site. The second generation ligands are then linked together to produce a compound that has a higher affinity than either of the two lead compounds. Using this method it was possible to identify two ligands that bound with micromolar affinities to the FK506 binding protein, which is involved in immunosuppression when activated. Linking these two individual ligands resulted in a compound that bound to FK506 binding protein with a nanomolar affinity. Variations of this technique, which is applicable only to biomolecules of less than 30 kDa, indicates that it can have wide-ranging usefulness and is a potentially valuable tool in drug research.
See also: Drug Metabolism Studied Using NMR Spectroscopy, High Resolution Solid State NMR, 13C, High Resolution Solid State NMR, 1H, 19F, NMR Pulse Sequences, Nuclear Overhauser Effect, Nucleic Acids Studied by NMR Spectroscopy, Small Molecule Applications of X-Ray Diffraction, Structural Chemistry Using NMR Spectroscopy, Peptides, Structural Chemistry Using NMR Spectroscopy, Organic Molecules.
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
Further Reading Anderson RC, Stokes JP, and Shapiro MJ (1995) Structure determination in combinatorial chemistry: Utilization of magic angle spinning HMQC and TOCSY NMR spectra in the structure determination of wang-bound lysine. Tetrahedron Letters 36: 5311--5314. Boelens R, Ganadu ML, Verheyden P, and Kaptein R (1990) Twodimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis. European Journal of Biochemistry 191: 147--153. Brange J, Ribel U, Hansen JF, et al. (1988) Monomeric insulins obtained by protein engineering and their medical applications. Nature 333: 679--682. Craik DJ (1996) NMR in Drug Design. Boca Raton: CRC Press. Davis SN and Granner DK (1996) Insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, and Gilman AG (eds.) The Pharmacological Basis of Therapeutics, 9th edn., pp. 1487--1517. New York: McGraw-Hill. Hajduk PJ (2006) SAR by NMR: Putting the pieces together. Molecular Interventions 6: 266–272.
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Lindon JC, Nicholson JK, and Wilson ID (2000) Directly Coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development. Journal of Chromatography B 748: 233–258. Pavlopoulos S, Bicknell W, Craik DJ, and Wickham G (1996) Structural characterization of the 1:1 adduct formed between the antitumor antibiotic hedamycin and the oligonucleotide duplex d(CACGTG)2 by 2D NMR spectroscopy. Biochemistry 35: 9314--9324. Shuker SB, Hajduk PJ, Meadows RP, and Fesik SW (1996) Discovering high affinity ligands for proteins: SAR by NMR. Science 274: 1531--1534. Wilson SR (1997) Introduction to combinatorial libraries: Concepts and terms. In: Wilson SR and Czarnick AW (eds.) Combinatorial Chemistry, Synthesis and Applications, pp. 1--23. New York: Wiley Interscience. Xie XQ, Melvin LS, and Makriyannis A (1996) The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. The Journal of Biological Chemistry 271: 10640--10647. Zhou MM, Huang B, Olejniczak ET, et al. (1996) Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nature Structural Biology 3: 388--393.
Symbols F1 F2 t1 t2 T1 T2
o1 ¼ y-axis frequency domain obtained by Fourier transformation with respect to t1 o2 ¼ x-axis frequency domain obtained by Fourier transformation with respect to t2 evolution time in a 2D pulse sequence detection period in a 2D pulse sequence spin–lattice (longitudinal) relaxation time spin–spin (transverse) relaxation time
methods allow researchers to study the interactions of a drug molecule with its site of action on the biopolymer (enzyme, receptor, nucleic acid, etc.). Such studies can lead to insights regarding the bioactive conformation of a flexible drug molecule, which constitutes invaluable information for drug design. Here we shall discuss the most commonly exploited experiments for extracting the individual NMR parameters mentioned above, and also how such parameters are utilized to obtain structural information.
Conformational Analysis of Small Molecules Scope and Applications Information on the structure of drug molecules and their interactions with their therapeutic sites of action is of critical importance in the design and development of new drugs. Of all the analytical methods, nuclear magnetic resonance spectroscopy (NMR) is the most exquisitely suited to provide such experimental results. The field of NMR is advancing continuously to include new pulse sequences and methods as well as progressively larger field instruments and improved probes. This fast progress in NMR methods and technologies has served to expand dramatically its applications in drug research. Indeed NMR, used jointly with X-ray crystallography and computational/graphical approaches, has revolutionized structure-based drug design. Currently the availability of a plethora of multidimensional/multinuclear NMR methods allows us to extract information on the structures and dynamic behaviours of a wide range of drug molecules of up to 30 kDa in size. These include the small and mediumsized traditionally used therapeutic drugs, to higher molecular weight peptides, proteins, nucleotides, nucleic acids and polysaccharide biotechnology products. Progressively, more detailed structural and dynamic information has become available because of our increased ability to measure more effectively the basic NMR parameters used in structural analysis, namely, proton and carbon chemical shifts, coupling constants, relaxation parameters (T1, T2) and the exceedingly valuable nuclear Overhauser effect. Such measurements are, in turn, used to obtain information on the three-dimensional structure of molecules as well as their conformational properties and dynamic behaviour. Additionally, the new solution NMR
Information on the structural properties of small drug molecules in solution can be obtained from a number of NMR parameters including 1H and 13C chemical shifts, 1 H–1H and 1H–13C scalar coupling constants, 1H nuclear Overhauser effects (NOEs), as well as relaxation measurements. Here, the conformational analysis of CP55,940 (Figure 1) is used to illustrate the most common experiments encountered in studying small molecules (o1000 Da) in solution. This synthetic compound is structurally related to D9-tetrahydrocannabinol (D9-THC), a psychoactive component of marijuana, and which has received much attention because it was used as the high affinity radioligand during the discovery and characterization of the G-protein coupled cannabinoid receptor (CB1). The elucidation of the conformational properties of this compound and its congeners provides information on the steric requirements for a productive interaction at the cannabinoid receptor active site. Double Quantum Filtered Correlation (DQF-COSY) and Total Correlation (TOCSY) Spectroscopy These experiments provide information on 1H chemical shifts and 1H–1H scalar coupling. Spectral assignments are made initially by an analysis based on integrated peak areas and chemical shifts in the one-dimensional spectrum. Subsequently, they are specifically assigned by analysis of scalar or spin–spin coupling connectivities observed by 1H–1H double quantum filtered correlation spectroscopy (DQF-COSY). This is a two-dimensional experiment where the information is spread onto a plane
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Figure 1 The one-dimensional 1H spectrum of CP55,940 with an expanded scale of the aromatic region. The molecular numbering system for assigning the peaks is shown.
in which the diagonal is equivalent to the one-dimensional spectrum, and the scalar coupling is manifested as an off-diagonal crosspeak between the two resonances in question. Thus even though a resonance may not be visible in a one-dimensional spectrum due to overlap, its position may be identified from a crosspeak in a two-dimensional experiment. Protons that are part of a spin system often give rise to a pattern of diagonal peak–crosspeak connectivities that can be traced from a ‘starting point’ in the spin system to an ‘end point’. It is the presence of such connectivity patterns that makes this experiment such a powerful tool in assignment. For example, the 1H NMR spectrum of CP55,940 is shown in Figure 1. A logical starting point for assignment was the resonance at d 3.74 that was assigned to H9a because the size of the peak as measured by integration was consistent with one proton and because this aliphatic resonance is expected to be deshielded. In the DQFCOSY spectrum (Figure 2) vicinal coupling to H10e, H8e, H10a and H8a is observed. Assignment of H10e and H10a resonances was made with the support of the crosspeak connectivities of H9a with H10a, H10e, H8a and H8e. The DQF-COSY spectrum clearly shows three components (H10e, H8e and H11e) under the multiplet at d 2.06, in which three related strong geminal 2J couplings, H10e/a (F1 ¼ d 2.09, F2 ¼ d 1.38), H8e/a (F1 ¼ d 2.05, F2 ¼ d 1.53) and H11e/a (F1 ¼ d 1.98, F2 ¼ d 1.13) can be
discerned. This is a prime example of the improvement in spectral resolution of two-dimensional experiments over one-dimensional experiments. Complete assignment of a whole spin system may be limited because of severe spectral overlap. To overcome this, DQF-COSY data are often used in conjunction with total correlation spectroscopy (TOCSY). This experiment results in a transfer of magnetization across an entire spin system and consequently crosspeaks may be observed between each resonance of a spin system. Thus it is possible to determine whether a particular overlapped region of the spectrum contains all unidentified members of a chemical spin system. 1 H chemical shifts and scalar coupling constants can be measured directly from one-dimensional spectra if the peaks are well resolved, or, if spectra are too complex, they may be measured from DQF-COSY spectra crosspeaks. However, such measurements are often inaccurate and so are used as a basis of simulating the observed 1D spectrum to obtain more accurate values. The measurements are used as a starting point and are systematically altered until the stimulated spectrum best matches the observed spectrum. 1H–1H scalar coupling constants are especially useful in providing information on the dihedral angle within a HC–CH system and are thus one of the most important sources of conformational information.
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Figure 2 Contour plots of expanded regions from the DQF-COSY spectrum of CP55,940. The lines highlight the connectivities between the H8, H9a, H10 and H11 resonances.
Nuclear Overhauser Effect Spectroscopy (NOESY) The nuclear Overhauser effect (NOE) is another important NMR parameter used in conformational analysis because the magnitude of the NOE is inversely proportional to the sixth power of the interproton distance in space (INOE p r6). NOE spectroscopy (NOESY) is a twodimensional experiment that may be run routinely in which the NOE is manifested as a crosspeak between two resonances indicating that the two protons are near in space. For example, in the case of CP55,940, two NOE crosspeaks were assigned to the spatial coupling of H5 with H8a and H12a (Figure 3). Such crosspeaks are congruent with a conformation in which the planes of the two rings are almost perpendicular, with the Ph–OH oriented towards the a face of the cyclohexyl ring. An NOE crosspeak between the phenolic hydroxyl proton and the adjacent aromatic H2 indicates that these two protons are spatially near each other and thus coupled through a dipole–dipole interaction (Figure 3). Such a result indicates that in its preferred conformation, the
Ph–OH proton points away from the cyclohexyl ring and towards the H2 proton. The full analysis of NOESY and DQF-COSY spectra of other analogues, plus computational studies, further showed that this was typical for all congeners of CP55,940 and that the dimethylheptyl chain adopts one of four preferred conformations, in all of which the chain is almost perpendicular to the phenol ring. The most biologically active conformations were such that all hydroxyl groups were oriented towards one face of the cyclohexyl ring system (Figure 4), a feature that may be an important requirement for cannabimimetic activity.
Identification of Drug Metabolites One major use of NMR spectroscopy in the pharmaceutical sector is as part of a concerted and integrated effort, together with mass spectrometry MS, to identify the metabolites of both candidate and marketed drugs. It is an important part of any drug development process to be able to understand the metabolism of a candidate drug in several animal species. All of the usual structural tools of NMR spectroscopy can be used, but often the very limited
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Figure 3 arrows.
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
Contour plot of the 500 MHz CP55,940 NOESY spectrum in CDCI3. The NOE interactions for CP55,940 are indicated with
Figure 4 Representation of the active biological conformation of CP55,940. According to the current hypothesis, the ligand preferentially partitions in the membrane bilayer where it assumes an orientation and location allowing for a productive collision with the active site.
availability of material, usually isolated from urine, bile or faecal material, means that NMR spectroscopy is often limited to one-dimensional 1H spectra. If a drug molecule contains a fluorine or a trifluoromethyl substituent, both quite common, then 19F NMR spectroscopy can be very useful. Rather than extract the substance of interest from its biological matrix it is now also possible to conduct HPLC separations with on-line NMR detection. The most comprehensive approach is to also couple a mass spectrometer in parallel to the NMR instrument, splitting the chromatographic eluate 95:5 to the NMR and MS, respectively.
NMR spectroscopy can also be used to investigate certain drug metabolites that show chemical reactivity, and hence might cause toxicity by interacting with proteins. NMR spectroscopy has been used extensively for this in the case of drug acyl glucuronide conjugates, where the initially formed 1-b-O-acyl glucuronide of a carboxylate containing drug can spontaneously react to form isomers in which the drug moiety has migrated around the glucuronic acid ring. These isomers have been postulated to react with proteins and cause immunological toxicity. Hence, the rate of reaction has been measured for a number of such real and model drugs, especially for non-steroidal anti-inflammatory drugs, in order to develop structure–reactivity relationships. Structure of Drug Macromolecules An increasing number of therapeutic drugs are composed of proteins or peptides and knowledge of their threedimensional structures has helped in the design of structurally modified variants with improved biological activities and pharmacokinetic properties. Such a case is insulin, which was first extracted from pancreas tissue, used in a patient in 1922, and its structure first determined in 1972. It has a molecular weight of 5.8 kDa and consists of a 21 amino acid peptide (chain A) that is connected to a 30 amino acid peptide (chain B) by two
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
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Figure 5 The 500 MHz 1H DQF-COSY spectrum of a shortened analogue of insulin, des-(B26–B30)-pentapeptide insulin in D2O. The spectrum was recorded on a Bruker AM 500 spectrometer in a phase-sensitive mode. 2048 complex data points were acquired in the t2 dimension, with a total of 512 free induction decays (FID) collected for transformation in the t1 dimension. Each FID was acquired with a total of 128 scans.
Figure 6 The fingerprint region of a 500 MHz 1H NOESY spectrum which contains NOEs between NH and Ha resonances of des-(B26–B30)-pentapeptide insulin. The expected intraresidue and inter-residue NOEs that form the highlighted sequential pattern of B chain backbone NH and Ha resonances are represented as solid and dashed arrows, respectively.
disulfide bonds. A third intra-subunit disulfide bond exists in chain A. The structure of insulin has been probed using X-ray crystallography, while NMR spectroscopy was used to determine its three-dimensional structure in solution. As with other similar-sized molecules, standard two-dimensional 1H homonuclear experiments can adequately provide such structural information. The use of two-dimensional experiments such as DQF-COSY, TOCSY and NOESY to assign and determine the three-dimensional conformation of peptides and proteins is well established. Briefly, the method used for the assignment of amino acid chains is not dissimilar from that of assigning small molecules. Each amino acid residue gives rise to a characteristic spin pattern that can be identified using the complementary DQF-COSY and TOCSY spectra, where connectivities between all protons within a spin system are observed in the TOCSY, and connectivities between neighbouring protons are observed in the DQF-COSY. An example of the DQFCOSY spectrum for the des-pentapeptide insulin monomer is shown in Figure 5. Of the 46 amino acids in the monomer, non-overlapped spin systems for four valine, two gylcine, one threonine and one alanine residue were distinguished. The latter two were sequence specifically assigned as threonine-8 in the A chain and alanine-14 in the B chain as these were the only alanine and threonine residues in the sequence. In addition, a further nine AMX spin systems could be clearly
distinguished that were subsequently assigned to amino acids with the aid of the TOCSY and NOESY spectra. A combination of DQF-COSY and TOCSY data were required in order to delineate the remaining overlapped amino acid spin systems. Once the spin systems arising from individual amino acids have been identified, the correct sequence of amino acids is determined from NOE data, acquired using the NOESY experiment. For molecules that fall within a particular molecular weight range (1000–3000 Da), the magnitude of the NOE is small, and a modification that is referred to as rotating Overhauser enhancement spectroscopy (ROESY) must be employed. Typically, a series of NOESY (and/or ROESY) spectra are collected in different solvents such as D2O, H2O or DMSO, with mixing times ranging from 50 to 600 ms. Different mixing times are required to gain an estimate of the magnitude of the NOE and subsequently an estimate of the distance between the protons. Care must be taken at longer mixing times, as it is possible to observe an indirect magnetization transfer between protons that are further apart than 5 A˚ via a third proton that is appropriately positioned between them. To avoid this spin diffusion effect, it is preferable to acquire NOESY experiments with the shortest mixing times possible that will result in good quality spectra. The determination of the amino acid sequence is based on the fact that NOEs will always be observed between particular protons from neighbouring residues
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regardless of the secondary structure of the protein. For example the proton attached to the a carbon (Ha) of an amino acid will always be within approximately 4.5 A˚ of the proton attached to the nitrogen (NH) of the neighbouring amino acid that is attached to the carboxyl end. In the NOESY spectrum, the NH resonances tend to occur within a particular spectral region, as do the Ha resonances. Thus, there is a particular region of crosspeaks between them in which a continuous connectivity pattern can be distinguished that begins with an NOE between the first and second residue and ends with an NOE between the terminal residue and the one preceding it (Figure 6). Such patterns reveal the sequence of amino acids in the protein. Similar patterns can also be distinguished between other groups of resonances (e.g. correlations between NH protons of neighbouring residues, Hb protons and NH protons of neighbouring residues) that can be used to confirm the sequence or resolve ambiguities. Breaks in this continuous pattern do occur in cases where resonances overlap or in some instances due to the structure of the amino acid chain, for example when a proline is present. However, once various lengths of polypeptides have been identified it is usually possible to surmise the order in which the various lengths are connected. This is achieved by considering different possibilities until a process of elimination arrives at the arrangement that best fits the data. Once the spectra have been assigned, data that contain structural information can be extracted. Nuclear Overhauser effects between non-neighbouring residues are the most revealing source of conformational information, and scalar coupling constants are important in providing information on torsion angles. All such conformational parameters are used as constraints in computational calculations that arrive at the threedimensional conformation of the protein. For small molecules, such as the non-classical cannabinoids, it is possible to infer the conformation from NMR data without the aid of a computer, given that there are a relatively small number of NOEs generated and there are limited conformational possibilities from which to choose. However, for peptides, proteins and other macromolecules such as DNA, where a large number of measured NMR parameters must be taken into consideration, computational methods are an essential tool in the determination of the vast conformational possibilities. The computational techniques utilized seek to systematically adjust the position of all nuclei in the molecule, so that all distances and bond angles derived from NOE data and coupling constants are satisfied. At the same time, the structure must not exceed set physical limits for bond lengths, bond angles, torsion angles, van der Waals contacts and Coulombic interactions between atoms. The challenge in such methods is to ensure that all possible conformations are sampled while not allowing the
molecule to exceed the set physical limits. Various algorithms and methods for calculation exist; however, the most common are the distance geometry and/or restrained molecular dynamics methods. Using this basic methodology, the three-dimensional conformation of insulin was determined, and a significant amount of structure–activity information was gained by the study of insulin analogues and insulins purified from different sources. Structural Analysis of Drug-Binding Domains Macromolecules such as proteins and nucleic acids form the sites at which drugs interact. Knowledge of threedimensional conformations assists in the design of analogues that are more potent and have improved pharmacokinetic properties. Furthermore, the structural analyses of protein receptors and enzymes add to the knowledge of biological systems, and therefore assist in identifying novel types of therapeutic agents. Because of their large molecular weights, most macromolecular therapeutic targets cannot be studied using exclusively 1 H homonuclear methods. The advent of three-dimensional and heteronuclear pulse techniques has greatly expanded the ability to study macromolecules of up to 30 kDa in size. Three-dimensional techniques are a natural progression of the twodimensional experiments. The pulse sequence is altered so that a vertical domain is introduced and the information is spread into a third dimension, so that the spectrum now is projected into a cube instead of a plane. The diagonal of the cube is equivalent to the one-dimensional spectrum, and crosspeaks that may be overlapped in a two-dimensional spectrum can be resolved in the third dimension. A case in point is the structure determination of the insulin receptor substrate-1 (IRS-1). Insulin binds to a membrane-bound receptor that is a ligand-activated protein tyrosine kinase. Upon insulin binding there is an autophosphorylation of several tyrosine residues on the cytosolic side of the receptor. This enhances the tyrosine kinase activity of the insulin receptor towards other substrates and is required for signal transduction. A cascade of events is initiated, the first of which is the phosphorylation of IRS-1. This occurs when IRS-1 binds to the insulin receptor via a specific domain of the protein that is termed the phosphotyrosine binding (PTB) domain. The structure of this domain was determined while interacting with a tyrosine-containing peptide derived from a receptor. As such, this study is also an example of the use of NMR to study the interactions between molecules. The three-dimensional NMR experiment was coupled with heteronuclear techniques to increase the level of resolution of the spectra. For larger proteins such as IRS-1, homonuclear experiments are limited because
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
proton resonances become broader, and the efficiency of magnetization transfer between protons is decreased. With the advent of recombinant DNA technology, this problem can be overcome by producing the proteins to contain isotopic labels, such as 13C, 15N or 2H, either at specific sites or uniformly throughout the protein. The magnetic properties of these nuclei offer significant advantages over protons. Carbon and nitrogen nuclei resonate over a much larger spectral width or range of frequencies, and as such are less likely to suffer from overlap. Also their scalar couplings to each other and to protons are higher in magnitude, and a more efficient magnetization transfer takes place than for 1H–1H couplings. Thus, for macromolecules, heteronuclear experiments that correlate 15N to 13C nuclei or to protons offer significant gains in sensitivity and resolution compared to homonuclear experiments. The most often used heteronuclear experiments are the heteronuclear single quantum coherence experiment (HSQC) and the related heteronuclear multiple quantum coherence experiment. These experiments allow the measurement of one- and two-bond heteronuclear couplings (and homonuclear 13 C–13C couplings). They are most often combined with traditional two-dimensional experiments such as NOESY and TOCSY to yield a three-dimensional experiment. For example, in the case of an HSQC-NOESY spectrum of a protein, two of the axes represent the heteronuclei such as 15N and the protons which are directly attached to the nitrogen nuclei, while the third axis contains chemical shifts of protons which share an NOE effect with the amide proton. This offers a significant increase in resolution compared to a traditional two-dimensional NOESY. A large array of these types of three-dimensional, heteronuclear-edited experiments have been designed to extract structural information in various situations, and are described in other articles in this encyclopedia. Using these methods, the structure of the PTB domain of the IRS-1 protein was found to be similar to phosphopeptide-binding regions of several other proteins. Once the structural details were known, the different binding specificities could be compared and rationalized based on the interactions with their substrates. Drug Interactions with Macromolecular Targets NMR spectra are capable of supplying information about molecular interactions in solution. When a drug interacts with a receptor in a reversible manner, a number of effects may be observed in the spectra due to the exchange of the molecules between free and bound states. These effects will be discussed in relation to studies of antitumour antibiotics binding to short sequences of oligonucleotides. Compounds such as adriamycin (Figure 7) are currently in use as chemotherapeutic agents,
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Figure 7 Chemical structures of intercalating and minor groove DNA binding ligands.
and an understanding of factors involved in binding specificity may lead to more effective drugs to combat cancer. Chemical exchange effects have been exploited to great effect in obtaining information about these interactions. The free and bound states of the molecules represent two different chemical environments in which participating molecules may be found. Thus, the same nucleus of a particular molecule may be characterized by different values for NMR parameters, such as chemical shift, when located in each different environment and may give rise to different sets of resonances. The ability to measure the parameters that characterize each environment is dependent on the rate at which the nucleus exchanges between them. The exchange rate therefore has a significant effect on the appearance of NMR spectra, and the exchange rate is dependent upon the affinity of the drug for the receptor. The kinetics of the interaction can be examined by acquiring a series of spectra in which the concentrations of the reactants or the temperature are modulated. Figure 8 shows an expansion of the aromatic region of a series of 1H NMR spectra of the oligonucleotide duplex d(GGTAATTACC)2 to which has been added increasing amounts of a terephthalamide derivative (Figure 7). It is clear that the addition of the ligand
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Figure 8 Expanded aromatic regions from the 300 MHz 1H NMR spectra of complexes between a terephthalamide derivative and d(GGTAATTACC)2, recorded at 101C. Increasing ligand concentrations cause perturbations to chemical shifts of nuclei located at or near the binding site. All perturbations are upfield except for adenine H2 resonances that are perturbed downfield and are located on the floor of the minor groove.
causes significant perturbations of the free DNA resonances, and these indicate that the molecules are interacting. At ligand:DNA ratios between 0:1 and 1:1 there is a mixture of DNA molecules that are bound and unbound. The DNA resonances observed are averages of resonances arising from each of these states, and this is characteristic of fast exchange between the bound and unbound state due to a low affinity of the ligand for the binding site. There is a point in the titration in which the DNA resonances are no longer perturbed, and this identifies the point at which all binding sites have been occupied and there are only bound DNA molecules present in solution. By noting the resonances that are most perturbed and the direction of the perturbation (upfield or downfield), it was determined that the ligand was bound at the ‘ATTA’ binding site. The observed perturbations were consistent with the ligand being inserted edge-on into the minor groove. This places protons on the floor of the groove in the same plane as the aromatic drug which are then deshielded due to ring current effects. Protons above and below the plane of the ring experience shielding ring current effects. An example of a ligand that has a high affinity for the binding site and exhibits slow exchange characteristics is shown in Figure 9, where the antitumour antibiotic hedamycin (see Figure 7) was titrated into a solution of the oligonucleotide duplex d(CACGTG)2. Upon addition of hedamycin, the free DNA resonances diminish in
Figure 9 Expanded regions from the 300 MHz 1H NMR spectra of complexes between hedamycin and d(CACGTG)2, showing (a) aromatic resonances and (b) methyl resonances. Spectra up to 0.8:1 ligand:DNA ratio were recorded at 21C and the complex allowed to equilibrate for 24 h at this temperature. Subsequent spectra were recorded to 101C as the resonances were sharper. The dotted arrows highlight resonances that disappear after the 24 h equilibration period.
intensity and new peaks appear in the spectrum. These new peaks do not correspond to chemical shifts of the free duplex or ligand resonances and increase in intensity with increasing ligand concentration. This suggests that they arise from the bound form of these molecules, and that slow exchange conditions exist due to the high affinity of the ligand for the binding site. In this particular case it became apparent that time-dependent changes in the spectra were taking place. Allowing the mixture to equilibrate for 24 h resulted in certain resonances disappearing from the spectrum and sharpening of the remainder of the resonances. Given that hedamycin was subsequently shown to intercalate and alkylate, the transient peaks may represent reaction intermediates prior to alkylation where the ligand is intercalating reversibly to sites other than the most favoured binding site, and the sharpening of the spectra is caused by alkylation of the DNA by the ligand. In cases where the binding affinity is high, or the binding is irreversible, a detailed model of the interaction between the molecules can be constructed based on intermolecular NOEs. A NOESY spectrum of the 1:1
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Figure 10 Contour plot of the 500 MHz 1H NOESY spectrum of the 1:1 hedamycin:DNA complex recorded at 101C with a mixing time of 300 ms. Spectra were acquired on a Bruker 500AMX spectrometer with 2048 complex data points in the t2 dimension and a total of 512 free induction decays (FID) collected for transformation in the t1 dimension. Each FID was acquired with a total of 128 scans.
complex of hedamycin with d(CACGTG)2 is shown in Figure 10. Assignment of the resonances was achieved using a combination of COSY, TOCSY and NOESY spectra. As in the case of proteins, the NOESY spectra of oligonucleotides yield characteristic crosspeak patterns that allow the sequential assignment of residues. Once the spectrum had been assigned and intramolecular NOEs arising from the DNA duplex and hedamycin had been eliminated, a total of 61 intermolecular NOEs from each of the oligonucleotide strands to protons located on the chromophore, epoxide chain and sugar groups of hedamycin were identified. These intermolecular NOEs identified the binding site and allowed the orientation of the ligand within the binding site to be determined. For example, a number of intermolecular NOEs, summarized in Figure 11, showed that the molecule was intercalated between the central CG basepairs. Similarly, intermolecular contacts showed that the sugar groups of the molecule were located in the minor groove (Figure 12) and the epoxide chain in the major groove. Furthermore, the epoxide chain was shown to be in an appropriate location, so that the terminal carbon was capable of alkylating to the N7 of the guanine. Direct evidence for this was observed in that the guanine H8 proton became labile (i.e. disappeared when spectra were acquired in D2O) following complexation. Another example involving proteins is the interaction of the PTB domain of the IRS-1 protein with a tyrosine
Figure 11 Schematic representation of NOEs observed between the central GC basepairs and the hedamycin ligand. Contacts to major groove protons are shown as dotted arrows and contacts to the minor groove are shown as solid arrows.
phosphorylated peptide in which the peptide was found to bind in a surface-exposed pocket of the PTB domain. More specifically, the peptide was bound along one strand of the b sheet structure of the PTB domain and interacted with an a helix. Hydrogen bonding, van der Waals contacts and hydrophobic interactions stabilized the interaction. The peptide was found to be in a type I b turn, and the N-terminal residues of the peptide were in an extended conformation that formed an additional strand of the PTB domains b sheet. Combinatorial Methods Traditional methods of drug discovery involved a search amongst a range of diverse compounds that were ultimately derived from nature. This was a long and arduous process in which advances were due more often to serendipity rather than scientific thought and technique. The means to study the three-dimensional conformation of drugs and their receptors opened the door to a more rational approach in which compounds could be synthesized to better interact with a receptor. Along with the better understanding of drug/receptor interactions came improvements in biochemical techniques to isolate
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This method relies heavily on the use of NMR to assess the success of each reaction step. As a non-destructive technique, a sample of the resin may be removed, placed in the NMR machine and then returned to the column. However, the fact that the compound is attached to the resin results in broad resonances if solution NMR techniques are utilized. This difficulty is circumvented by using solid-state NMR methods, including magic angle spinning (MAS). These experiments lead to narrow linewidths and high quality spectra, so that even two-dimensional experiments can be performed.
Combinatorial Methods in Drug Discovery
Figure 12 Schematic representation of contacts between the sugar rings of hedamycin and the DNA duplex. Contacts are observed only to minor groove protons and each ring is associated predominately with one DNA strand.
receptors and test compounds for their affinity towards these receptors. The ability to test thousands of compounds in a relatively short amount of time using high throughput screening methods resulted in pressure to produce large numbers of diverse compounds to be tested.
Analysis of Solid-State Intermediates Combinatorial chemistry is a term used to describe the production of a large number (thousands) of diverse compounds, and different methods are employed to achieve this. One such method is solid-phase synthesis where the chemistry occurs on a solid support that may be packed into a column. The solid support may be a material such as crosslinked polystyrene and the compounds to be produced generally consist of a number of basic pharmacologically relevant structures (pharmacophores) that are to be linked in different ways. The method requires that one of the pharmacophores be linked to the solid support, then each subsequent pharmacophore can be delivered to the column with appropriate reagents, allowed to react, and then washed off the column. By varying the order in which the pharmacophores are delivered, different compounds can be produced.
One of the most interesting applications of NMR in drug research is in the field of high throughput screening. One such described approach bridges combinatorial chemistry with biochemical screening and was named ‘SAR by NMR’. The first step in this method is to identify the ability of ligands to bind to a target preparation of the molecular therapeutic target (e.g. an enzyme) in solution. Thus, batches of ligand mixtures (e.g. one batch containing 20 compounds) are allowed to interact with the enzyme preparation in solution while following changes in the protein 15N and 1H NMR frequencies. In this manner, batches that produce a response can be identified quickly and the compounds within each batch further tested to identify specific compounds that bind to adjacent but different sites on the protein. These ligands are then further optimized using rational design techniques to improve the binding at each respective site. The second generation ligands are then linked together to produce a compound that has a higher affinity than either of the two lead compounds. Using this method it was possible to identify two ligands that bound with micromolar affinities to the FK506 binding protein, which is involved in immunosuppression when activated. Linking these two individual ligands resulted in a compound that bound to FK506 binding protein with a nanomolar affinity. Variations of this technique, which is applicable only to biomolecules of less than 30 kDa, indicates that it can have wide-ranging usefulness and is a potentially valuable tool in drug research.
See also: Drug Metabolism Studied Using NMR Spectroscopy, High Resolution Solid State NMR, 13C, High Resolution Solid State NMR, 1H, 19F, NMR Pulse Sequences, Nuclear Overhauser Effect, Nucleic Acids Studied by NMR Spectroscopy, Small Molecule Applications of X-Ray Diffraction, Structural Chemistry Using NMR Spectroscopy, Peptides, Structural Chemistry Using NMR Spectroscopy, Organic Molecules.
Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals
Further Reading Anderson RC, Stokes JP, and Shapiro MJ (1995) Structure determination in combinatorial chemistry: Utilization of magic angle spinning HMQC and TOCSY NMR spectra in the structure determination of wang-bound lysine. Tetrahedron Letters 36: 5311--5314. Boelens R, Ganadu ML, Verheyden P, and Kaptein R (1990) Twodimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis. European Journal of Biochemistry 191: 147--153. Brange J, Ribel U, Hansen JF, et al. (1988) Monomeric insulins obtained by protein engineering and their medical applications. Nature 333: 679--682. Craik DJ (1996) NMR in Drug Design. Boca Raton: CRC Press. Davis SN and Granner DK (1996) Insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, and Gilman AG (eds.) The Pharmacological Basis of Therapeutics, 9th edn., pp. 1487--1517. New York: McGraw-Hill. Hajduk PJ (2006) SAR by NMR: Putting the pieces together. Molecular Interventions 6: 266–272.
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Lindon JC, Nicholson JK, and Wilson ID (2000) Directly Coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development. Journal of Chromatography B 748: 233–258. Pavlopoulos S, Bicknell W, Craik DJ, and Wickham G (1996) Structural characterization of the 1:1 adduct formed between the antitumor antibiotic hedamycin and the oligonucleotide duplex d(CACGTG)2 by 2D NMR spectroscopy. Biochemistry 35: 9314--9324. Shuker SB, Hajduk PJ, Meadows RP, and Fesik SW (1996) Discovering high affinity ligands for proteins: SAR by NMR. Science 274: 1531--1534. Wilson SR (1997) Introduction to combinatorial libraries: Concepts and terms. In: Wilson SR and Czarnick AW (eds.) Combinatorial Chemistry, Synthesis and Applications, pp. 1--23. New York: Wiley Interscience. Xie XQ, Melvin LS, and Makriyannis A (1996) The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. The Journal of Biological Chemistry 271: 10640--10647. Zhou MM, Huang B, Olejniczak ET, et al. (1996) Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nature Structural Biology 3: 388--393.