Welcome to Week 5. Chapter 9 - Binding, Structure, and Diversity. 9.1 Intermolecular Forces. Starting week five video. Introduction to Chapter 9

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1 Welcome to Week 5 Starting week five video Please watch the online video (49 seconds). Chapter 9 - Binding, Structure, and Diversity Introduction to Chapter 9 Chapter 9 contains six subsections. Intermolecular Forces Case Study - Stromelysin Drug-Target Complementarity Molecular Diversity Molecular Libraries Building Libraries Upon completing this chapter, you should understand how drugs bind a target and how to determine the energy of binding. You should gain a preliminary idea of how to control the shape of a molecule in order to maximize available drug-target interactions. You should realize the challenge of discovering active molecules within the immense number of possible drug molecules and what tools are available to drug companies to explore drug space. 9.1 Intermolecular Forces Binding energy video Please watch the online video (10 minutes, 26 seconds). Clarifications and corrections At approximately the 4:11 mark, the video states that 17x10-3 M is equal to 1.7x10-4 M instead of 1.7x10-2 M.

2 A condensed summary of this video can be found in the Video summary page. Henderson-Hasselbalch equation Background: Hydrogen and ionic bonds are very important for drug-target binding. These bonds can be highly dependent upon ph. Instructions: Read the passage below concerning the Henderson-Hasselbalch equation and the pk a of common functional groups. Use the information to answer the questions that follow. Learning Goals: To review usage of the Henderson-Hasselbalch equation and understand how ph influences the availability of hydrogen and ionic bonds between a target and a drug. An acid (H-A) can react reversibly with water to form a conjugate base (A - ) and hydronium ion (H 3O + ). The equilibrium constant (K) for this reaction can be expressed in the standard way - the product of the concentrations of the products divided by the product of the concentrations of the reagents. Because the concentration of water is virtually constant at around 55 M, the term [H 2O] is combined with K to define a new constant, K a. While K a is a constant, the ratio of the acid and conjugate base in solution can be affected by the ph of the medium. A lower ph, with a raised [H 3O + ], favors H-A over A -. A higher ph, with a lowered [H 3O + ], favors more A -. The Henderson-Hasselbalch equation quantitatively relates how ph affects the equilibrium ratio of A - and H-A for an acid with a known K a. Remember that pk a = log K a. Henderson-Hasselbalch equation

3 To use the Henderson-Hasselbalch equation, one needs to know the pk a of different functional groups. pk a values for a handful of common functional groups encountered in drugs are shown in the table below. A much more comprehensive list can be found here ( acid (name) conjugate base (name) pk a H 3O + (hydronium) H 2O (water) 1.7 PhCO 2H (benzoic acid) PhCO 2 (benzoate) PhNH 3 (anilinium) PhNH 2 (aniline) 4.6 CH 3CO 2H (acetic acid) CH 3CO 2 (acetate) 4.8 (pyridinium) (pyridine) 5.1 (imidazolium) (imidazole) NH 4 (ammonium) NH 3 (ammonia) 9.2 PhOH (phenol) PhO (phenolate) 10.0 H 2O (water) HO (hydroxide) 15.7 For reference, the ph of some different regions in the body are listed below. blood stomach - as low as 1 small intestine extracellular fluid intracellular fluid Please complete the online exercise.

4 Calculating binding energies Background: The binding energy of a drug-target complex can be calculated with the equation below. If kcal/mol*k is used for R, then the energy is calculated with units of kcal/mol. Temperature (T) is usually 298 K. Instructions: Use the equation above to answer the questions that follow. Learning Goal: To practice calculating binding energies between a drug and target. Please complete the online exercise. FAQ, help, and tips Binding energy video Why does the course switch from natural logarithms (ln or log e) to base-10 logarithms (log 10)? In treatments of pharmacokinetics, the relationship of C p and time is normally presented in a natural logarithm form (lnc p vs. time). In contrast, in discussions of binding energies, the use of base-10 logarithms is more standard. Why are binding energies reported in kcal/mol instead of kj/mol? Medicinal chemistry is dominated by organic chemists, who tend to favor energies in terms of kcal/mol instead of kj/mol. Is there a reference for the 0.03 kcal/mol/å 2? Yes. The original reference for that value is... Chothia, C. Hydrophobic bonding and accessible surface area in proteins. Nature 1974, 248,

5 Calculating binding energies What temperature should be used for determining binding energies? Binding energies are normally determined through in vitro tests, which are performed at room temperature. Room temperature is close to 298 K. Here is a link to an article ( that goes into much greater detail on assays that measure binding energies. 9.2 Case Study - Stromelysin Stromelysin video Please watch the online video (8 minutes, 30 seconds). A condensed summary of this video can be found in the Video summary page. More stromelysin inhibitors Background: The binding energy of a drug-target complex can be calculated with the equation below. If kcal/mol*k is used for R, then the energy is calculated with units of kcal/mol. Temperature (T) is usually 298 K. Instructions: Use the equation above to answer the questions that follow concerning stromelysin inhibitors. Learning Goal: To practice calculating binding energies between an enzyme and its inhibitor. Please complete the online exercise.

6 FAQ, help, and tips Stromelysin video How can binding be measured on compounds with such high K D values? Weak binding molecules require specialized techniques for determining their K D values. Examples include surface plasmon resonance and saturation transfer difference NMR spectroscopy. More stromelysin inhibitors Please clarify the relationship between the K values, binding energies, and IC 50. First, there are two possible K values. Both are related to the complexation equilibrium of a drug and its target (an enzyme or receptor). The equilibrium can be defined in two directions. If the drug and target are considered to be the starting materials, then the corresponding equilibrium constant is K A - the association equilibrium constant. If the drug-target complex is the starting material, then the equilibrium constant is K D - the dissociation equilibrium constant. In drug discovery, the equilibrium constant of interest is almost always K D. A low value for K D indicates that the equilibrium favors the drug-target complex, and therefore binding between the drug and target is favorable (large, negative value for ΔG). K i values also dissociation equilibrium constants. K i values specifically refer to molecules that inhibit or block an enzyme or receptor. Just as with K D values, smaller K i values indicate an inhibitor that binds strongly to the target (large, negative value for ΔG). For a full agonist that binds a receptor, the K D of the ligand-receptor complex corresponds to the EC 50 of the ligand (the concentration of ligand required to affect a 50% maximal response from the receptor). Because K D = EC 50, it can be tempting to think that K i = IC 50. The equations look the same, so perhaps they must both be true. The relationship, K i = IC 50, however is not true. IC 50 is the concentration of an inhibitor that is required to reduce a receptor's response or an enzyme's rate of reaction by 50%. IC 50, however, varies based on how much of the receptor's agonist or enzyme's substrate is present. If more agonist or substrate is present, then more

7 inhibitor will be required (IC 50 will be higher). In contrast, K i is a constant property of the inhibitortarget complex. Remember that the Cheng-Prussoff equation allows the determination of K i from an IC 50 value as long as one knows two things. First is the concentration of the agonist ([L]) used to determine the IC 50 value. Second is the K D of the agonist for the receptor. If the target is an enzyme, then one needs to know the concentration of the substrate ([S]) and the K m of the substrate for the enzyme. Can you provide a reference to the stromelysin research? Here is a link to a copy of one of the early articles from the Abbott researchers. ( 9.3 Drug-Target Complementarity Pharmacophores revisited video Please watch the online video (7 minutes, 32 seconds). A condensed summary of this video can be found in the Video summary page. Rotatable bonds Background: Rotatable bonds increase the conformational flexibility of a molecule and minimize the probability that the functional groups in a molecule match the desired pharmacophore. Instructions: Read the passage below on identifying rotatable bonds. Use the information to answer the questions that follow. Learning Goal: To learn how to identify which bonds in a molecule qualify as rotatable. No universally accepted rules exist on defining rotatable bonds. Each set of definitions has its loop holes and problems. Regardless, most methods include the rules shown below.

8 Bonds that are not rotatable... non single bonds bonds to hydrogen and other monovalent atoms (halogens) ring bonds bonds to terminal atoms, including CH 3, NH 2, and OH the C-N bond between a carbonyl and amide nitrogen (also goes for the C-N bond in thioamides and the S-N bond in sulfonamides) bonds connecting two aromatic rings with collectively three or more ortho substituents bonds connected to terminal triple bonds, including bonds to cyano groups Under these rules, naproxen has only three rotatable bonds (bolded below). Duloxetine has six (bolded below). Please complete the online exercise. FAQ, help, and tips Pharmacophores revisited video How can researchers possibly know what groups are required in the pharmacophore? Determining the pharmacophore of a drug may seem like an insurmountable organic chemistry puzzle, but researchers often have considerable information at their disposal. The biology group will have intensely studied any target, whether an enzyme or receptor. For enzymes, the substrate will almost certainly be known. The same can be said for the endogenous ligand of a receptor. If the substrate or natural ligand is known, then the medicinal chemistry group has a handle on the type of structure that has a high affinity for a binding pocket on the target. That information might be enough for the discovery team to search intelligently for promising compounds.

9 Drug discovery teams often also have x-ray structural information on a target. An x-ray structure allows the team to visualize in three-dimensions the contours of different binding pockets of the target. If an x-ray structure of the target is available, often an x-ray structure of the target complexed with a second molecule will also be at hand. An x-ray of a target that has co-crystallized with another molecule allows the discovery group to see precisely how the functional groups of the bound molecule interact with the target. Potential hydrophobic interactions and hydrogen bonding interactions can be clear. Knowing these interactions can allow the discovery group to hypothesize the structure of the pharmacophore and begin to plan how binding might be improved in subsequent candidate molecules. 9.4 Molecular Diversity Numbers game video Please watch the online video (7 minutes, 55 seconds). A condensed summary of this video can be found in the Video summary page. Privileged structures Background: Molecular space for potential drug molecules is indescribably diverse. Instructions: Read the passage below about certain types of structures and substructures that repeatedly appear in many different types of drug. Learning Goal: To understand the concept of privileged structures. While drug space is incredibly diverse, a handful of common structures and molecular fragments can be found regularly among active sets of molecules. These compounds and fragments have become known as privileged structures because of their seemingly universal ability to bind protein targets.

10 One such privileged structure is the diphenylmethane subunit. The subunit can be seen in numerous compounds that bind a variety of different targets. Privileged structures are both good and bad for drug discovery. On the good side, privileged structures help researchers focus on molecular scaffolds that are more likely to show activity. The drug discovery team can focus on promising compounds and hopefully avoid the less interesting structures. On the bad side, single compounds that contain privileged structures may show activity against multiple targets. Compounds that bind multiple targets often cause side effects. Such compounds are sometimes labeled as promiscuous. Another issue with privileged structures is that they have received considerable research attention. Patenting a compound with a privileged structure can be a challenge because so many similar structures have already been patented. Promiscuity revealed Background: Privileged structures are molecular scaffolds that tend to show activity against a broad range of biological targets. Such compounds are sometimes said to have promiscuous activity. Instructions: Read about Molinspiration's Predict Bioactivity function for JSME and use this function to rank the promiscuity of a set of privileged scaffolds. Learning Goal: To gain exposure to emerging biological activity prediction tools. Molinspiration, a site that we have used for determining whether a compound satisfies Lipinski's rules, has coupled a Predict Bioactivity function with the JSME tool. With this functionality, a user may predict the activity of a structure against six different types of commonly encountered drug targets, including GPCRs, ion channels, kinases, nuclear receptors, proteases.

11 Molinspiration's page is shown below with a structure of a known protease inhibitor, captopril, drawn in the JSME tool. Captopril is used to manage high blood pressure. Once the "Predict Bioactivity" button is clicked, the Molinspiration engine checks the properties of the structure against a library of molecules with known biological activity and predicts the activity of the submitted structure. The predicted activities are then shown to the user. Activities are predicted through a numerical score, larger and positive values indicate higher predicted activity. If a molecule is predicted to have activity against a target, then the target is shaded light green (moderate activity) or dark green (strong activity). In the case of captopril, the Molinspiration tool successfully identifies the known protease activity of structure. With this webpage, one can make educated predictions on what type of targets might be bound by molecule. Please complete the online exercise.

12 FAQ, help, and tips Numbers game video What is the original Guida reference? Here is a link to the original article. ( The full text is unfortunately not freely available. Behind the numbers Can edx accept answers in scientific notation in other formats? The question suggests the following format. 6.8*10^2 Another valid and simpler format is shown below. 6.8e2 Privileged structures Are there reviews on privileged structures? One review on privileged structures can be found through this link. ( CurrOpinChemBiol_2010.pdf) 9.5 Molecular Libraries Molecule collections video Please watch the online video (6 minutes 56 seconds). A condensed summary of this video can be found in the Video summary page.

13 High-throughput screening Background: Potential drug space is immense with an estimated number of molecules of Compound libraries, although nowhere near the same size as drug space, are regardless very large and may include 1,000,000 or more compounds. Instructions: Read the text below and the accompanying Wikipedia entry concerning the rapid testing of molecular libraries. Learning Goal: To understand how the large number of molecules in compound libraries can be quickly and inexpensively tested for biological activity. For molecular libraries to be useful to a drug company, there must exist a method for quickly and inexpensively testing the activity of each compound in the library. Fortunately there is. That method is called high-throughput screening or HTS. HTS involves the automated testing of molecules in a quick, inexpensive, in vitro assay. The process relies upon robotic equipment to perform the screen reproducibly. With such a method, a pharmaceutical company can screen an entire large library, which may include a million or more compounds, in around a week in a particular screen. Therefore, in a fairly short period of time, hits for a specific target can be identified. The entry for high-throughput screening in Wikipedia provides some more details on precisely how the process is automated. The statistical and emerging technology discussions in the article are beyond the scope of this course, but they do reveal interesting facets of HTS. HTS and academia Background: High-throughput screening (HTS) is an automated, quick method for gaining preliminary activity information on molecules in a compound library. HTS has traditionally been a technique only available to pharmaceutical companies. Instructions: Read the article below. Use the information in the article to answer the questions that follow. Learning Goal: To understand how the drug discovery process is becoming increasingly available to groups outside the traditional pharmaceutical industry. A recent report in the journal Nature Methods notes the growing movement of academic laboratories screening their own molecular libraries. ( Please complete the online exercise.

14 FAQ, help, and tips HTS and academia Exactly how does HTS make drug discovery faster? In drug discovery, sometimes the research team has little or no information to help find a molecule that binds the desired target. The only option is to test individual molecules for binding a target in an assay. Traditional biochemical assays, when performed individually, can be very time intensive. When performed in an automated fashion with robotic assistance, the time required for the assays can be greatly reduced. In this manner, the automated assays, called high-throughput screens, can accelerate the discovery of molecules with promising target binding 9.6 Building Libraries Combinatorial chemistry video Please watch the online video (7 minutes 10 seconds). A condensed summary of this video can be found in the Video summary page. Ugi reaction Background: Reactions that can quickly and easily assemble large numbers of diverse molecules are highly sought after in combinatorial chemistry. Instructions: Read passage below on the Ugi reaction and answer the question that follows. Learning Goal: To learn about a reaction that is commonly exploited in combinatorial chemistry. The Ugi reaction involves the reaction of four different starting materials in a single reaction vessel. The starting materials are a carboxylic acid (1), primary amine (2), aldehyde (3), and isocyanide (4).

15 The simplicity of the Ugi reaction and the widespread availability of the starting materials make the reaction very popular starting point in preparing molecular scaffolds in combinatorial chemistry. Please complete the online exercise. Combichem challenges Background: In the early 1990s combinatorial chemistry was hoped to accelerate greatly the rate at which new drugs could be discovered. The benefits of combinatorial chemistry did not however materialize as expected. Instructions: Read the Chemistry and Engineering News article linked below. Use the information to answer the questions that follow. Learning Goal: To learn about a reaction that is commonly exploited in combinatorial chemistry. Although over 15 years old, an article from Chemistry and Engineering News, a trade magazine published by the American Chemical Society, captures the early sentiments of a booming field called combinatorial chemistry. Even in 1998 in the face of great enthusiasm, the promises of combinatorial chemistry were already being questioned. The article nicely captures both sides of the story. ( Please complete the online exercise. FAQ, help, and tips Combinatorial chemistry video Please provide an additional reference on high-throughput screening. The Wikipedia article on HTS is very dense. A freely available article ( provides a good, although long summary of the field. The language of the article fits well with the terminology that has been covered in this course.

16 Combi chem challenges Is there any other literature that more fully describes limitations of combinatorial chemistry libraries for drug discovery? Here is an article by Lipinski that goes into some detail about combi chem library problems. (