welcome to week 5 - edx · calculating binding energies background: the binding energy of a...

Welcome to Week 5

Starting week five video Please watch the online video (49 seconds).

OPTIONAL-Please participate in the online discussion forum.

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.

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 pKa

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

(H3O+). 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 [H2O] is

combined with K to define a new constant, Ka.

While Ka 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 [H3O+], favors H-A over A-. A higher pH, with a lowered

[H3O+], 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 Ka. Remember that pKa = −log Ka.

Henderson-Hasselbalch equation

To use the Henderson-Hasselbalch equation, one needs to know the pKa of different functional

groups. pKa 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

(http://research.chem.psu.edu/brpgroup/pKa_compilation.pdf)

acid (name) conjugate base (name) pKa

H3O+ (hydronium) H2O (water) −1.7

PhCO2H (benzoic acid) PhCO2− (benzoate) 4.2

PhNH3+ (anilinium) PhNH2 (aniline) 4.6

CH3CO2H (acetic acid) CH3CO2− (acetate) 4.8

(pyridinium) (pyridine) 5.1

(imidazolium) (imidazole) 7.0

NH4+ (ammonium) NH3 (ammonia) 9.2

PhOH (phenol) PhO− (phenolate) 10.0

H2O (water) HO− (hydroxide) 15.7

For reference, the pH of some different regions in the body are listed below.

blood - 7.4

stomach - as low as 1

small intestine - 7-8

extracellular fluid - 7.4

intracellular fluid - 6.8

Please complete the online exercise.


http://research.chem.psu.edu/brpgroup/pKa_compilation.pdf

Calculating binding energies Background: The binding energy of a drug-target complex can be calculated with the equation

below. If 0.00199 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.



FAQ, help, and tips

Binding energy video

Why does the course switch from natural logarithms (ln or loge) to base-10 logarithms (log10)?

In treatments of pharmacokinetics, the relationship of Cp and time is normally presented in a natural

logarithm form (lnCp 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/Å2?

Yes. The original reference for that value is...

Chothia, C. Hydrophobic bonding and accessible surface area in proteins. Nature 1974, 248, 338-

339.

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

(http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.2009.00604.x/pdf) 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).



More stromelysin inhibitors Background: The binding energy of a drug-target complex can be calculated with the equation

below. If 0.00199 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.



http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.2009.00604.x/pdf

FAQ, help, and tips

Stromelysin video

How can binding be measured on compounds with such high KD values?

Weak binding molecules require specialized techniques for determining their KD 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 IC50.

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 KA - the association equilibrium constant. If the drug-target complex is the starting material, then

the equilibrium constant is KD - the dissociation equilibrium constant.

In drug discovery, the equilibrium constant of interest is almost always KD. A low value

for KD indicates that the equilibrium favors the drug-target complex, and therefore binding between

the drug and target is favorable (large, negative value for ΔG). Ki values also dissociation equilibrium

constants. Ki values specifically refer to molecules that inhibit or block an enzyme or receptor. Just

as with KD values, smaller Ki values indicate an inhibitor that binds strongly to the target (large,

negative value for ΔG).

For a full agonist that binds a receptor, the KD of the ligand-receptor complex corresponds to

the EC50 of the ligand (the concentration of ligand required to affect a 50% maximal response from

the receptor). Because KD = EC50, it can be tempting to think that Ki = IC50. The equations look the

same, so perhaps they must both be true. The relationship, Ki = IC50, however is not true.

IC50 is the concentration of an inhibitor that is required to reduce a receptor's response or an

enzyme's rate of reaction by 50%. IC50, 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

http://en.wikipedia.org/wiki/Surface_plasmon_resonance

http://nmrwiki.org/wiki/index.php?title=Saturation_transfer_difference_spectroscopy

inhibitor will be required (IC50 will be higher). In contrast, Ki is a constant property of the inhibitor-

target complex.

Remember that the Cheng-Prussoff equation allows the determination of Ki from an IC50 value as

long as one knows two things. First is the concentration of the agonist ([L]) used to determine

the IC50 value. Second is the KD 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 Km 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.

(http://classes.soe.ucsc.edu/bme280b/Spring04/journalClubArticleApr30BretBarnes.pdf)


9.3 Drug-Target Complementarity

Pharmacophores revisited video Please watch the online video (7 minutes, 32 seconds).



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.

http://classes.soe.ucsc.edu/bme280b/Spring04/journalClubArticleApr30BretBarnes.pdf

Bonds that are not rotatable...

non single bonds

bonds to hydrogen and other monovalent atoms (halogens)

ring bonds

bonds to terminal atoms, including CH3, NH2, 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).



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.

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).



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.

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.

http://www.molinspiration.com/cgi-bin/properties

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.



FAQ, help, and tips

Numbers game video

What is the original Guida reference?

Here is a link to the original article. (http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291098-

1128%28199601%2916:1%3C3::AID-MED1%3E3.0.CO;2-6/abstract). 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.

(http://www.columbia.edu/cu/biology/faculty/stockwell/StockwellLab/index/publications/Welsch_

CurrOpinChemBiol_2010.pdf)


9.5 Molecular Libraries

Molecule collections video Please watch the online video (6 minutes 56 seconds).



http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291098-1128%28199601%2916:1%3C3::AID-MED1%3E3.0.CO;2-6/abstract

http://www.columbia.edu/cu/biology/faculty/stockwell/StockwellLab/index/publications/Welsch_CurrOpinChemBiol_2010.pdf

High-throughput screening Background: Potential drug space is immense with an estimated number of molecules of 1063.

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.

(http://www.nature.com/nmeth/journal/v4/n6/full/nmeth0607-523.html)


http://en.wikipedia.org/wiki/High-throughput_screening

http://www.nature.com/nmeth/journal/v4/n6/full/nmeth0607-523.html


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).



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).

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.



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. (http://pubs.acs.org/cen/hotarticles/cenear/980406/comb.html)



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

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962124/) 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.

http://pubs.acs.org/cen/hotarticles/cenear/980406/comb.html

http://en.wikipedia.org/wiki/High-throughput_screening

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962124/

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.

(http://www.molecularmedicineireland.ie/uploads/files/MMIcourses/Lipinski.pdf)


http://www.molecularmedicineireland.ie/uploads/files/MMIcourses/Lipinski.pdf

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