Example Of How The Technique Works Term Paper

Type of paper: Term Paper

Topic: X-Ray, Crystallography, Drugs, Protein, Aliens, Technique, Ligand, Structure

Pages: 9

Words: 2475

Published: 2020/12/30

Application of X-Ray Crystallography in Drug Discovery

X-ray crystallography is a technique that is used in identification of the atomic and molecular structures of a crystal (Dziedzic et al., 2015). The crystalline atoms diffract a beam of incident X-rays to a number of specific directions. Through the measurement of the angles and the intensities of the diffracted rays, an expert in X-ray crystallography is capable of producing three-dimensional images of the density of electrons with the crystal. It is through the establishment of the electron density that the scientists are able to determine the mean positions of atoms in the crystal, their disorder, chemical bonds and all other relevant details. X-ray crystallography is important in different fields, and this is due to the ability of biological molecules, semiconductors and salts among other materials to form crystals. For quite long now, X-ray crystallography has been used determining of the “atomic-scale differences among minerals, establish the type and length of chemical bond and the size of the atom” (James, 2013). In addition to that, X-ray crystallography is also used in revealing the structure and function of biological molecules such as DNA, vitamins, nucleic acid and proteins and therefore making significant contributions to the drug discovery process (Dziedzic et al., 2015).
The history of X-ray crystallography dates back to the 17thcentury when Johannes Kepler hypothesized that hexagonal symmetry of snowflake crystals was as a result of regular packing of spherical water particles. Kepler’s hypothesis was contained in his work entitled “Strenaseu de NiveSexangula” (A New Year's Gift of Hexagonal Snow). Nicolas Steno (1669), Danish scientist was the first person to experimentally investigate crystal symmetry. However, X-rays were discovered by Wilhelm Conrad Rontgen in 1895. Earlier, the scientists were not sure of the nature of X-ray light. However, in 1912, Max von Laue confirmed that actually X-ray was a form of electromagnetic radiation (James, 2013). Hence, the knowledge of crystal symmetry and X-ray was combined to study the size of atoms and other molecular structures among others. This was done by physicist William Lawrence Bragg and his father William Henry Bragg. This discovery made the Braggs the winners of Nobel Prize in 1915. It is until the 1950s that scientists discovered that X-ray crystallography could be used in drug discovery process. For that reason, this essay focuses on the application of X-ray crystallography in drug discovery. In addition, the essay explore some of the strengths and weaknesses associated with X-ray crystallography
Application of X-Ray Crystallography in Drug Discovery

X-ray crystallography technique is used to provide detailed three-dimensional images of thousands of proteins. The major components of X-ray crystallography are a detector, source of X-ray and a protein crystal (Dziedzic et al., 2015). X-ray crystallography technique relies on the growth of solid crystals of the molecules to investigate the molecular structure. The scientists direct high-powered beam at a minute crystal that contains trillions of molecules. The tiny crystal scatters the X-ray onto an electronic detector. The electronic detector used in this case is similar to that one used on a digital camera to capture images (Casini et al., 2010). The blasting of the crystal with the beam of high-powered X-ray can last from several seconds to hours. After each blast, the crystal is rotated. The rotation of the crystal is important in helping the researchers capture three-dimensional images of how the crystal diffracts X-ray light. Thereafter, the intensity of each ray scattered by the crystal is fed into a computer. Using mathematical calculations, the position of every single atom is the molecule is established. At the end, we obtain a three-dimensional picture of the protein molecule (James, 2013).

Identification of Drug Targets

Apart from nuclear magnetic resonance (NMR) technique, X-ray crystallography is the other modern technique used in the study of protein structure at the atomic level. X-ray crystallography has proved to be versatile, and at the moment it is being applied in different parts of the globe to study molecular structures (Dziedzic et al., 2015). One most important thing about X-ray crystallography is that it overcomes the limitations of size and complexity, and this is due to the ability of most globular macromolecules to crystallize. As mentioned earlier, the knowledge of crystal symmetry and X-ray radiation sprout many years ago. However, the study of molecular proteins began in 1950s. The first study was conducted on sperm whale myoglobin. Max Perutz and Sir John Cowdery Kendrew were awarded the Nobel Prize in 1962 for the extraordinary contributions to the study of molecular structures of proteins using X-ray crystallography (James, 2013).
Currently, X-ray crystallography is used by different scientists to identify drug targets and as well as determine how pharmaceutical drugs interact with protein target and what might be done to improve the situation. In the research study article by Wyatt et al. (2008), X-ray crystallography technique is applied in drug discovery to identify drug targets by identifying low-molecular-weight compounds with normally very low binding affinities. In this study, this technique is used to identify N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxamide (AT7519), a novel cyclin dependent kinase (CDKs) inhibitor that is being appraised as a potential site for treatment of cancers . With this technique, the researchers were able to identify multiple lead hits that bound to the CDKs and this led to the discovery of AT7519.
Figure 1; Fragment based drug discovery
According to Casini et al., X-ray crystallography has also been applied in the identification of a myriad of sugar sulfamate/sulfamide derivatives that acts as inhibitors of various carbonic anhydrase (CA) isozymes that include hCA I, hCA II and bCA IV. In this particular study, the researchers identified that isozymes are bound by a network of 7physically powerful hydrogen bonds that fix topiramate within the active site. In addition, zinc ions coordination within the ionized sulfamate moiety was found to promote structural changes that contributed to the emergence of new compounds with reduced CA inhibitory characteristics as compared to topiramate. In summary, sugar sulfamate/sulfamide derivatives were identified as one of the most effective drugs/agents in clinical development that exhibited significant CA inhibitory properties (Casini et al., 2010).
Figure 2: ionized sulfamate moiety
According to Swenson (2014), X-ray crystallography is used to identify the drug target and the role carbonic anhydrase (CA) inhibitors, most specifically acetazolamide, play in treating and managing acute mountain sickness (AMS). AMS is a chronic and debilitating condition that occurs as a result of symptomatic intolerance to altitude and hypoxia, and the affected patients exhibits a myriad of signs and symptoms that are not limited to fatigue, nausea, and headache (Swenson, 2014). Overall, the advancement in the X-ray crystallography technology has made it possible to identify new drug targets and receptors that can be can coupled to address the majority of common diseases and disorders

Structural Information of Protein-Ligand Complex/ Crystal Structure

In the concept of drug discovery, X-ray crystallography is also used to identify the structural information of protein-ligand complex. In most aspects, this is attributed to the mere fact that most of the proteins bind themselves onto small organic ligands. These types of ligands are integral to the functions of their cognate proteins. It is therefore considered important to understand the structural and dynamical aspects of binding of these small ligands in efforts to comprehend their overall structure. The term ‘ligand’ is used to refer to any molecule that interacts with a specific given molecule, for instance a protein. A ligand for that matter may include other molecules such as carbohydrates, peptides, lipids, proteins and so forth. Ligands are large and contain diverse structures of molecules. These group molecules indicate a wide range of physic-chemical properties. This nature of ligands makes it difficult to deduce information from their biophysical properties (Casini et al., 2010).
According to Nagar et al. (2010), the unintentional fusion of two ligands namely the bcr gene with the abl gene has shown that it yield produces a very active tyrosine kinase (Bcr-Abl) derivative that cause structural changes to cells leading to the cause of chronic myelogenous leukemia. Therefore, any inhibitors to these small ligands that bind to active site, most specifically in the kinase domain, can be adopted in the treatment and management chronic myelogenous leukemia. According to this article, it is important to note that Bcr-Abl ligands can interact with other molecules almost in a reversible non-covalent manner. This property makes it easy to modulate its biological role in a way that can be controlled. In terms of structural Information, X-ray crystallography identified that the ATP-binding site of the kinase domain is bound actively by hydrogen bonds to two inhibitors namely imatinib or STI-571 and PD173955, but the bind differently. STI-571 bind the active site to a conformation Abl ligard in which the activation loop mimics bound peptide substrate. On the other hand, PD173955 binds Abl via an activation loop that resembles an active kinase. In summary, the structure as shown by X-ray crystallography shows that PD173955 is more inhibitory compared to STI-571.
Figure 3. “Chemical structures of (a) STI-571 and (b) PD173955. The core compounds from which these two inhibitors were developed are shown in bold lines”.
Another good example in regard to the application of X-ray crystallography in obtaining the structural information of protein-ligand complex is in the research study article by Huai et al. (2012), which shows how the technique is used to identify the crystal structure of calcineurin-cyclophilin-cyclosporin ligand complex. From the authors, calcineurin is one of the most common targets for “two immunophilin–immunosuppressant complexes” that include “cyclophilin A–cyclosporin A” (CyPA-CsA) and “FKBP–FK506” (Huai et al., 2002). The results established that CyPA-CsA ligard binds to the catalytic and regulatory subunits of calcineurin, while the FK506 binds to calcineurin at a similar site with FKBP.
Figure 4. Stereoview of the superposition of Cα atoms of CyPA-CsA-CN (red) over unligated CNA (green) and FKBP-FK506-CN (yellow). The complex structures were overlaid with optimal overlap of the catalytic domain of CNA.
Figure 5. Calcineurin Binding
It is important to note that protein-ligand complex is fully characterized. At the simplest level, the non-covalent interaction between the protein and the ligand is represented as shown below:
P+L ↔ PL

However, two scientists, Westwell and Williams proposed that the interaction between protein-ligand should be written as follows:

P + L ↔ P’L’
The two equations are same. The only difference is formulism. In the second equation, the protein and ligand have undergone interaction making them non-existent. Instead, the protein and ligand is replaced with modified entities of the protein/ligand (P’ L’). Tight complexes are formed when protein-ligand interactions are stronger than protein-solvent and ligand-solvent interactions. A well described equilibrium molar association is represented as follows:
Ka = [P’L’] / ([P].[L]) (Dziedzic et al., 2015).

Design and Improving Drugs

X-ray crystallography and the concept of three-dimensional structures are also used by pharmaceutical companies to produce and improve drugs. Most of these pharmaceutical companies have the crystallographic technology. In the article by Dziedzic et al. (2015), this technology is used to identify the complex design of “Biaryltriazoles that is identified as potent inhibitors of Macrophage Migration Inhibitory Factor (MIF) associated with cancer and other inflammatory diseases among humans” (Dziedzic et al., 2015). In this study, the binding sites of receptors tend to overlap and the inhibitors of the Macrophage Migration Inhibitory Factor (MIF) exhibits their mechanism of action by binding the active site (Dziedzic et al., 2015).
Figure 6. Three-dimensional structure of the binding of Biaryltriazoles Macrophage Migration Inhibitory Factor (MIF).
In addition, X-ray crystallography is used to design drugs that inhibit Histone Methyltransferase DOT1L. According to Yao et al. (2011), this is technique is used to identify the structure and mechanism based design that establishes key inhibitors of DOT1L. From the results obtained, X-ray crystal structure of a DOT1L–inhibitor reveals that its selectivity and high affinity is attributed to the N6-methyl group that constitutes the inhibitor (Yao et al., 2011).
Figure 7. The X-ray crystal structure of a DOT1L–inhibitor complex

Strength and Weakness of X-ray Crystallography

X-ray crystallography as a technique in drug discovery has a number of strengths. The technique is well established. The steps involved are also well defined. They include producing a sufficient quantity of pure macromolecule, analysis of conditions for crystallization and production of crystals of suitable quality. The steps that follow include collection data on X-ray diffraction, solving the problem using the multi-wavelength anomalous diffraction and finally the calculation of the electron density (James, 2013). Another important thing about X-ray crystallography is that it requires more mathematical image construction. The production of the three-dimensional molecular structure is based on mathematical formulations. The determining of the positions of atoms in the molecule also depends on mathematical formulae. This aspect increases accuracy (Drenth & Mesters, 2010).
Objective interpretation of data is easier compared to other methods such as nuclear magnetic resonance (NMR). There is software such as PDB developed to help in analysis of data and development of molecular structure models (James, 2013). Another advantage of X-ray crystallography is highly automated processing of raw data. Modern software and hardware have been developed to facilitate the drug discovery process using this modern technique. In addition to that, the X-ray crystallography has quality indicators such as resolution and R-factor which increase accuracy and reliability of the results obtained (Drenth & Mesters, 2010).
The flexibility of proteins is a major weakness for this technique. Proteins tend to change from time to time depending on the conditions they are subjected to. Therefore, obtaining an accurate molecular structure of the protein-ligand complex is challenging (James, 2013). This property of proteins has forced researchers to look for other softer models that will take into account the conformations of the proteins. Another weakness of this X-ray crystallography technique is scoring functions (James, 2013). A number of scoring functions are done by weighing in different physical terms such as van der Waals interaction energy, hydrogen bonding, solvation electro statistics and ligand deformation energy. As a result, it has been found that many scoring functions have been optimized for one protein which means it’s only appropriate for use when working with proteins from that family (Dziedzic et al., 2015).


In conclusion, X-ray crystallography is a technique that uses X-ray light to study molecular structure in crystals. The technique has been used to measure the size of the atoms and even locate their positions in the molecule. This technology has also been employed in field of pharmaceuticals. Crystallographers have crystallized biological structures with an aim of identifying the active and binding sites. This technology has led to the discovery of drugs. Through the X-ray crystallography technology, high –resolution images of important protein-coupled receptors (GPCRs) have been developed. The images have helped in understanding of ligand recognition ad receptor activation. Through this move, drugs such as chemotypes have been developed. The X-ray crystallography technique has several benefits. All the steps in crystallography are well defined beginning with creation of pure macromolecule to calculating the density of the electrons. The process is also highly automated. The limitations of size and weight of molecules in drug designing have been overcome by the use of X-ray crystallography. However, the conforming nature of proteins and scoring functions remain the main challenge in X-ray crystallography technology. All in all, the X-ray crystallography has proved useful in designing and improving drugs.


Casini, A.; Antel, J,; Abbate, F.; Scozzafav, A.; David, S.; Waldeck, H,; Supurn, C.T. Carbonic anhydrase inhibitors: SAR and X-ray crystallographic study for the interaction of sugar sulfamates/sulfamides with isozymes I, II and IV. Bioorg Med Chem Lett. 2010 10;13(5):841-5.
Drenth, J.; Mesters, J. Principles of protein x-ray crystallography (3rd ed.). New York: Springer, 2010.
Dziedzic P; Cisneros J.;, Robertso, M.; Hare A.; Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor. J Am Chem Soc. 2015 137(8):2996-3003.
Huai, Q.; Kim, H.; Liu, Y.; Zhao, Y.; Mondragon, A.; Liu, J.; Ke, H. Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. Proc Natl Acad Sci 2012 17;99(19):12037-42
James, R. X-ray crystallography (5th ed.). London: Methuen, 2012.
Nagar, B,; Bornmann, W.G.; Pellicena, P.; Kuriyan, J. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI- 571). Res. 2010 62(15):4236-43.
Swenson, E. Carbonic Anhydrase Inhibitors and High Altitude Illnesses. Subcellular Biochemistry 75, 2014, pp 361-386
Yao, Y.; Chen, P.; Diao, J.; Cheng, G.; Deng, L.; Anglin, J.L.; Prasad, B.V.; Song, Y. Selective inhibitors of histone methyltransferase DOT1L: design, synthesis, and crystallographic studies. J Am Chem Soc. 2011; 133(42):16746-9.
Wyatt, P.G., Woodhead, A.J., Berdini V. et al. Identification of N-(4-piperidinyl)-4-(2,6- dichlorobenzoylamino)-1H-pyrazole-3-carboxamide (AT7519), a novel cyclin dependent kinase inhibitor using fragment-based X-ray crystallography and structure based drug design. J Med Chem. 2008 51(16):4986-99.

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