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10.1021/ed100193m Published on Web 03/30/2010

In the Laboratory

Using the β2-Adrenoceptor for Structure-BasedDrug DesignDavid T. Manallack,* David K. Chalmers, and Elizabeth YurievMedicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences,Monash University, Parkville, Victoria 3052, Australia*david.manallack@pharm.monash.edu.au

Molecular modeling is an important component of thedrug discovery process. In the past, the modeling process some-times involved physical models; however, the physical modelswere usually limited to exploring small molecules. To easilyvisualize and manipulate macromolecules, more sophisticatedcomputational graphics tools are used, many of which are nowavailable at moderate or even zero cost, particularly for educationpurposes. For example, a set of free visualization tools is avail-able from the Protein Data Bank (PDB) (1, 2) which is anarchive of experimentally determined protein and nucleic acidstructures.

It has long been recognized that molecular modeling andchemical visualization are important aids to understanding chemi-cal behavior. The use of molecular modeling software by medi-cinal chemists is now commonplace with many companiesproviding desktop tools to facilitate design work (3). As a con-sequence, it is important to teach undergraduate students thebasic principles of computational chemistry together with itsassociated strengths and limitations. Molecular visualization andcalculation of physicochemical properties helps the medicinalchemist understand the characteristics of the molecules, which inturn aids in the optimization process and makes the discoveryprocess more efficient.

G protein-coupled receptors (GPCRs) are membrane-bound proteins characterized by seven transmembrane (TM)R-helices linked by intracellular and extracellular loops. Approxi-mately 2% of the human genome encodes for GPCRs (4) com-prising around 800 sequences. By far the largest family of GPCRproteins belong to the rhodopsin class, which has 672 members(5). GPCRs function by transferring chemical signals fromoutside the cell to initiate signal transduction processes withinthe cell and are an essential component of many signalingpathways (5). Their important biochemical role means that theyare implicated in many disease states and, as a class, are the mostimportant drug target, with approximately 30% of marketeddrugs acting on GPCRs (6). GPCRs have proved to be difficultto crystallize and study by X-ray diffraction (7-9) and, untilrecently, three-dimensional (3D) structural information forGPCRs was limited to crystal structures of bovine rhodopsin(7-9). In 2007-2008, our knowledge of GPCR structurewas enhanced by the release of two structures of the humanβ2-adrenoceptor (10-12), quickly followed by a further fivestructures (13-17). These new structures have greatly added toour overall understanding of the 3D structure of GPCRs.

The binding of a drug molecule to a receptor involves arange of intermolecular interactions. Most drug binding does

not involve the formation of covalent bonds; instead, the inter-actions we are interested in are principally electrostatic in nature:ion-ion interactions, ion-dipole interactions, hydrogen bonds,dipole-dipole interactions, inducible dipoles, and van derWaalsforces. Hydrophobic interactions are substantially entropy dri-ven. Water molecules are unable to form hydrogen bonds to thehydrophobic regions of a drug or binding site. As such thesewater molecules interact with each other in an ordered manner.On binding, this ordered set of water molecules is displaced andbecomes less ordered, which results in an increase in entropyleading to a gain in binding free energy. These interactionsshould be familiar to advanced undergraduate students. Of parti-cular interest in this exercise are hydrogen bonds. These occurbetween a hydrogen attached to an electronegative atom andan electron-rich atom having a lone pair. The atom with theattached hydrogen represents the “donor”, whereas the electron-rich atom is the “acceptor” (18). In drug-protein interactions,the acceptor atoms are most commonly oxygen or nitrogen(Figure 1). Typically, hydrogen bonds made between a drug andits binding site contribute to both affinity and selectivity for theparticular macromolecular target. A typical hydrogen bond willrange in strength from 16 to 60 kJ/mol and is governed by thestrength of both the hydrogen bond acceptor and donor, as wellas the distance between the atoms. As these bonds are bothdirectional and highly distance dependent, the spatial relation-ship of the donor and acceptor is very important (Figure 2).These concepts are highlighted in this exercise during the designstep that seeks to add a further interaction between the bounddrug and the receptor.

Within the medicinal chemistry course, we provideteaching materials covering the fundamentals of computa-tional chemistry (20). Together with an understanding in thepractice of medicinal chemistry, this knowledge is used toteach the essentials of the drug discovery process. The lecturesassociated with this field introduce the principles of rationaldesign to consolidate concepts associated with structure-baseddrug design (SBDD). Given that suitable GPCR structureshave been published recently, we were keen to exploit thisinformation within our course. This article describes one ofthe molecular modeling exercises undertaken by the students

edited byAlan J. Shusterman

Reed CollegePortland, OR 97202-8199

Figure 1. Examples of hydrogen bonds (dashed lines).

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In the Laboratory

allowing them to explore a GPCR structure. The specificobjectives are

• To investigate the overall structure and function of GPCRs(i) to identify and appreciate the helical structure of the protein;(ii) to investigate interactions with other membrane molecules;and (iii) to identify and explore the location and nature of theligand binding site.

• To understand noncovalent intermolecular interactions andinvestigate their role in protein-ligand interactions particularlythe nature and role of hydrogen bonds in drug-receptor inter-actions.

• To illustrate the SBDD design process

Modeling Procedure

The β2-adrenoceptor is a GPCR that is an important com-ponent of the sympathetic nervous system. Compounds thatinteract with the β2-adrenoceptor typically comprise agonistsand antagonists. Agonists, such as salbutamol, relax the smoothmuscle of the bronchi to relieve the symptoms of asthma.β2-Adrenoceptor selectivity is required for asthma therapeuticagents to avoid cardiac side effects, which are largely mediatedthrough the β1-adrenoceptor.

In this exercise, students commence by downloading thecrystal structure of theGPCR for this exercise (PDB code 2RH1)(10) from the PDB (1, 2). All of the molecular modelingmanipulations are undertaken using the Sybyl software package(Tripos, St. Louis, MO). In our case, the students have had priorexposure to Sybyl, which they readily assimilate; however, othermodeling packages could be adapted for this task, for example,HyperChem (21), MacroModel (22), or the freely availablepackage PyMOL (23). Once the protein complex has been readinto Sybyl, the students identify each of the molecules present inthe crystal structure, that is, the protein, associated lipid mole-cules (cholesterol and palmitic acid), and the ligand (carazolol),using atom or molecule coloring. To visualize the protein moreeffectively, one protein domain needs to be deleted (residues1002-1161). This particular domain, T4 phage lysozyme(T4L), was used by Cherezov and co-workers (10) to helpaccomplish the difficult task of crystallizing the β2-adrenoceptor.Having removed the relevant amino acids, the transmembranesection is rendered more apparent and can be viewed clearly byrepresenting the backbone of the protein as a tube (see slides 5and 6 in the supporting information). Thus, the focus is placedon the protein structure as a whole and sections where ligandsand other proteins generally interact with GPCRs.

Additional components of the crystal complex include a setof cholesterol molecules and palmitic acid, which bind to the

transmembrane region of the protein. This TM region is a seriesof lipophilic amino acid residues that reside in a band around theGPCR where the protein interacts with the cell membrane. Thehydrophobic amino acids involved in these interactions can bevisualized by the students by coloring the hydrophobic residuesof the protein.

Having investigated the protein structure, the next step ofthe exercise is to focus on the active site and the binding ofcarazolol (Figure 3). This molecule makes numerous intermole-cular interactions with the binding site. Students are asked toidentify many of these interactions such as charge-assistedhydrogen bonds, hydrogen bonds, and hydrophobic interactions.Finally, the students are asked to look at regions of close contactbetween the protein and carazolol. With help from the instruc-tor, they are directed to the aromatic “C” ring (Figure 3) ofcarazolol. The object of this inspection is to note that a tyrosineresidue (Tyr308) sits close to the aromatic ring (about 3.5 Å)and that the tyrosine hydroxyl group could readily interactwith a substituent placed on the aromatic group of carazolol(6-position, Figure 3 and supporting information). Having beenguided to this possibility, students are required to suggest one ormore functional groups that could hydrogen bond to the hydroxylgroup on the tyrosine side chain (remembering that the hydroxylcould act either as a hydrogen bond donor or acceptor). Simplesuggestions include an amino or hydroxyl group. Although thedistancemay bemarginally short for a hydrogen bond (about 2.7Å),the important aspect of the exercise is to draw on other materialtaught in the course to result in a “eureka” moment for thestudent when they design a new molecule. The exercise alsoprovides an opportunity to discuss the equilibrium between thefree and bound states of the ligand and receptor and theimportance of solvation of both the ligand and receptor.

The students may work alone or in pairs while the instructorintroduces the topic and guides progress, particularly at the finalstages to help foster deeper learning outcomes.

Experiment

Before the laboratory session, students research the structureof carazolol. In the laboratory, the students download the 2RH1protein structure from the PDB (1, 2) and visualize the proteincomplex. The T4L subdomain is removed. Diagrams are gene-rated viewing the protein structure from the side and from above.Students explore the interactions made with one of the choles-terol molecules and interpret this observation with regard to howthe GPCR interacts with cell membranes.

A thorough examination of the interaction between cara-zolol and the protein structure is made and all significant con-tacts are noted and classified (and illustrated by generateddiagrams). The instructor assists the design process to highlightthe proximity of Tyr308 to the aromatic ring and individualdiscussion takes place to help design small substituents that couldfavorably interact with the tyrosine hydroxyl group.

Figure 3. Structure of carazolol. The aromatic ring of interest is indi-cated by the arrow and the 6-position on the ring is shown.

Figure 2. Geometric parameters for a strong hydrogen bond. D is thedonor heavy atom,H the hydrogen and A the acceptor atom. DD is thedonor antecedent (atom that is two covalent bonds away from thehydrogen), and AA the acceptor antecedent. The distance from A to His typically 1.8 Å and to avoid a significant steric clash needs to begreater than 1.5 Å. The angleD-H-A is most frequently found between150 and 180�, whereas the angle H-A-AA usually ranges between120 and 180� (18, 19).

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In the Laboratory

Results

Most students suggested that the -OH and -NH2 sub-stituents could be attached to the 6-position of carazolol. Othersuggestions included -F, -NO2, -SH, -OCH3, -COOH,-CONH2, and-SO2NH2 (not an exhaustive list). If a hydroxylgroup is attached to the 6-position, then the oxygen-oxygendistance is 2.7 Å, which is acceptable given that we have not takeninto account any protein or ligand flexibility that could occurupon binding.

Conclusion

The proposed procedure gives the medicinal chemistrystudents an opportunity to investigate a GPCR structure usingmolecular modeling software. They are able to explore theprotein as well as two interacting small molecules (carazololand cholesterol). The final part of the session enables students todraw on other aspects of the course material to design a newmolecule that can make further favorable interactions with theprotein. The exercise builds on and consolidates the concepts ofchemical interactions that are taught within the course. Overall,the procedure reinforces the course material covering GPCRsand associated drugs, intermolecular interactions (particularlyhydrogen bonds), and the principles of SBDD.

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Supporting Information Available

Instructors' notes; handouts for students. This material is availablevia the Internet at http://pubs.acs.org.