An Introduction to Drug Discovery by Probing Protein–Substrate

Laboratory Experiment pubs.acs.org/jchemeduc

An Introduction to Drug Discovery by Probing Protein−Substrate Interactions Using Saturation Transfer Difference-Nuclear Magnetic Resonance (STD-NMR) Jean-Paul Guégan†,‡ and Richard Daniellou*,†,‡,§ †

Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du Général Leclerc, CS 50837, 35708 Rennes Cedex 7, France ‡ Université Européenne de Bretagne, Rennes, France § Department of Biochemistry, ICOA, Université d’Orléans, CNRS, UMR 7311, rue de Chartres, BP 6759, 45067 Orléans Cedex 2, France S Supporting Information *

ABSTRACT: NMR spectroscopy is a powerful tool for characterizing and identifying molecules and nowadays is even used to characterize complex systems in biology. In the experiment presented here, students learned how to apply this modern technique to probe interactions between small molecules and proteins. With the use of simple organic synthesis, students prepared different analogues of the well-known anxiolytic drug, nitrazepam. This study constituted the students’ first approach into the drug-discovery field and encouraged the students to think about how to improve the design of drugs in a rational way.

KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Bioorganic Chemistry, Drugs/Pharmaceuticals, Medicinal Chemistry, NMR Spectroscopy, Synthesis, Thin Layer Chromatography

N

two major drug-binding sites; site II is called the indole− benzodiazepine site.8 The STD-NMR technique relies on the transfer of saturation from the protein to the ligand and is depicted in Figure 1. A selective irradiation of the protein leads

uclear magnetic resonance (NMR) spectroscopy has become a leading tool for characterizing small compounds or assessing their purity; for studying biological macromolecules such as DNA, RNA, and proteins;1 and even for medical diagnosis through the development of magnetic resonance imaging.2 NMR was applied for the first time in 1996 in the drug discovery field using the SAR (structure−activity relationships) approach. 3 Over the last several years, applications and techniques of NMR-based screening have evolved rapidly and studies of ligand−receptor complexes have been used for rational improvement of drug leads.4,5

■

EXPERIMENT OVERVIEW A two-week experiment was introduced into an advanced organic chemistry laboratory related to chemistry and technologies for life for fourth-year undergraduate students. In an effort to expose the students to the field of drug discovery, a saturation transfer difference (STD)-NMR experiment6 was introduced to identify segments of nitrazepam, an anxiolytic drug, in direct interaction with an inexpensive protein, human serum albumin (HSA), commonly used as a model in biology. The HSA protein is well-known for its extraordinary binding capacity for a wide range of drugs.7 The crystal structures of recombinant HSA show the locations of © 2012 American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Principle of the ligand mapping using STD-NMR.

to a selective and efficient saturation of the entire protein by spin diffusion. The saturation can then be transferred to the ligand molecules that are interacting with the protein by intermolecular saturation transfer. The ligand is in fast exchange between the bound state and the free state, and therefore, the Published: June 8, 2012 1071

dx.doi.org/10.1021/ed100612k | J. Chem. Educ. 2012, 89, 1071−1073

Journal of Chemical Education

Laboratory Experiment

approximately 0.3. After purification on silica gel, fractions containing the benzodiazepine derivative were analyzed by TLC, pooled, and concentrated on a rotary evaporator. Compounds were finally obtained in overall yields of approximately 10% (roughly 300 mg). Such quantities were sufficient for fully characterizing each compound using 1H NMR and 13C NMR spectroscopy. Spectra were recorded on a 400 MHz spectrometer and students processed free induction decays offline at remote personal computers. It is noteworthy that in all NMR spectra, aromatic protons were mostly represented by overlapped signals that were resolved through 2D NMR experiments. In the second lab period, students met in the NMR laboratory. Instructors described the instrument in detail. This was the time for a complete review of the technique and the occasion to show the students the link between theory and application going from common one-dimensional NMR to COSY, HSQC, HMBC, and, finally, STD. In the laboratory, students prepared their NMR samples for the STD experiments. As this powerful method needs only a small quantity of both protein and ligand, the students were surprised to discover that only 0.3 and 1 mg, respectively, were sufficient. The compounds were dissolved in D2O, the NMR samples analyzed, and the data processed. An example of spectra obtained with derivative 3 is shown in Figure 3. Such

degree of saturation reflects directly the importance of the interaction. In Figure 1, the small protons represent protons that have no contact with the protein and large protons represent those in close proximity to the protein. The experiment exposes students to a modern NMR technique and also to essential techniques in organic chemistry. The students synthesized nitrazepam following a procedure previously described in this Journal.9 They performed thin-layer chromatography (TLC) to follow the reactions, flash chromatography for the purification, and standard 1H and 13C NMR for the characterization of the compounds (details are available in the Supporting Information). Two 4-h laboratory periods are needed so students have time to design and perform the synthesis of the nitrazepam analogues (Figure 2).

Figure 2. Nitrazepam (1) and three analogues.

Alternatively, the anxiolytic compounds can be commercially obtained and STD-NMR experiment can be carried out on two different benzodiazepines during a single 4-h laboratory period. The students found this approach exciting because they were introduced to modern NMR-based drug discovery. This challenging field is now well developed in industry but, unfortunately, rarely mentioned during organic chemistry laboratories.

■

RESULTS AND DISCUSSION Before the experiment, students were asked to design analogues of nitrazepam 1 (Figure 2). Instructions were given to (i) follow the published synthetic route, 9 (ii) use only commercially available reagents, (iii) find affordable derivatives, and (iv) rationalize the chemical modifications. The analogues shown in Figure 2 probe the influence on the binding of the ligand through the substitution of the phenyl group by an oxygene atom in compound 2 (the group in the dashed box) or the substitution of the nitro group by a fluorine, a chlorine, or an hydrogen atom in 2, 3, and 4, respectively (the dashed circle). The experiment was divided in two parts: (i) the synthesis, the purification, and the characterization of compounds (1−4) and (ii) the STD-NMR analysis with HSA. During the first laboratory period, reactions were performed following the procedure described by Babin and Devaux9 on 3 g scale of the corresponding starting material. TLC was used to monitor the reaction and also to find a suitable solvent system for the flash chromatography.10 For the four compounds, a solvent composed of CH2Cl2/acetone 9:1 (v/v) gave an Rf of

Figure 3. STD-NMR spectra (red = 1H NMR, blue = STD) and saturation effects of 4 (in percent relative to the highest effect). The peak close to 5 ppm is the residual HOD of the deuterated water. Note that the spectra are not on the same scale (as indicated by the noise).

experiments were also performed on compounds 1, 2, and 4. Unfortunately, comparisons between the prepared molecules were difficult as signals were overlapping, and no conclusions could be obtained concerning the importance of the NO2 group. However, compounds 1, 3, and 4 appeared to be binding in a similar manner with HSA, thus, leading to the conclusion that the nitro group is not required for the binding. All nonexchangeable protons of these molecules appeared to be in close contact with the protein except those of the methylene group. Such observation should lead students to the conclusion 1072

dx.doi.org/10.1021/ed100612k | J. Chem. Educ. 2012, 89, 1071−1073

Journal of Chemical Education

Laboratory Experiment

(2) Steinmetz, W. E.; Maher, M. C. J. Chem. Educ. 2007, 84, 1830− 1831. (3) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531−1534. (4) Lepre, C. A.; Moore, J. M.; Peng, J. W. Chem. Rev. 2004, 104, 3641−3675. (5) Peltier, P.; Guégan, J.-P.; Daniellou, R.; Nugier-Chauvin, C.; Ferrières, V. Eur. J. Org. Chem. 2008, 5988−5994. (6) (a) Mayer, M.; Meyer, B. Angew. Chem., Int. Ed. 1999, 38, 1784− 1788. (b) Viegas, A.; Manso, J.; Nobrega, F. L.; Cabrita, E. J. J. Chem. Educ. 2011, 88, 990−994. (7) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17−53. (8) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Protein Eng. 1999, 12, 439−446. (9) Babin, P.; Devaux, G. J. Chem. Educ. 1989, 66, 522−523. (10) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923− 2925. (11) Darrouzain, F.; André, C.; Ismaili, L.; Matoga, M.; Guillaume, Y. C. J. Chromatogr., B 2005, 820, 283−288.

that this particular methylene group of the molecule may be chemically modified to improve the binding. More significantly, the benzodiazepine 2 exhibited no signal during the STD-NMR experiment. Such effect suggests the importance of the phenyl ring for the interaction. A careful integration of the STD-NMR spectra was finally conducted and a relative value of the STD effect was attributed for each signal, starting from the highest signal that was given 100% (on structure in Figure 3). A proton in close contact with the protein is given a high percentage. Thus, a full map of interactions for 4 with HSA was obtained from the STD-NMR.

■

HAZARDS Students should wear gloves and goggles and work in a chemical fume hood. Particular attention should be paid when using bromoacetyl bromide and sodium azide. Bromoacetyl bromide reacts violently with water and is corrosive and may cause burns. Sodium azide is considered as a poison and may be fatal if swallowed or absorbed through skin. The chemically prepared benzodiazepines constitute drugs currently used in many therapies and should therefore be manipulated with precautions. The solvents (toluene, acetone, dichloromethane, ether) are flammable; are harmful or fatal if swallowed; and cause irritation to skin, eyes and respiratory tract. Triphenylphosphine may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Students are required to obtain the toxicological forms from either INRS (French National Research and Scientific Institute) files or MSDS (Material Safety Data Sheets).

■

SUMMARY This experiment has been performed for two years and represents a nice experience for both students and instructor. Compounds were easily synthesized in sufficient quantities. All students were able to analyze the NMR data and enjoyed being able to see the direct interaction between drugs and a human protein. In most cases, this was the first time the students carried out drug-design experiments. The NMR spectroscopy represents only one tool for the instructor and such experiments can lead to further discovery of fields such as molecular docking, bio-organic chemistry, and QSAR studies. The binding of benzodiazepines to HSA can also be studied using other techniques and especially chromatography.11

■

ASSOCIATED CONTENT

S Supporting Information *

Notes for the instructor. This material is available via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

■

ACKNOWLEDGMENTS The authors wish to thank the Ecole Nationale Supérieure de Chimie de Rennes for financial support and fourth-year students for their contribution to practical optimizations of this experiment. We are also indebted to Pr Vincent Ferrières for helpful discussions.

■

REFERENCES

(1) Veeraraghavan, S. J. Chem. Educ. 2008, 85, 537−540. 1073

dx.doi.org/10.1021/ed100612k | J. Chem. Educ. 2012, 89, 1071−1073