9/16/2019 Lecture 3

Fundamentals of Drug Discovery

CONTENTS 3 SELECTING DRUG TARGETS

Link page The selection of a target by a drug company or research organisation — the decision to seek an agent with a biological effect that
Introduction therapeutic utility — involves many stakeholders and takes in a range of scientific, medical, financial and strategic consideration
For a detailed analysis, see this review: J. Knowles and G. Gromo, Nat. Rev. Drug Discovery, 2003, 2, 63–69 (doi: 10.1038/nrd986).
Lecture 1 chart below, taken from the review, serves to illustrate the potential complexity of the decision-making process. We will focus on
science involved.
Lecture 2
Biomolecule graphics 3.1 Choosing a disease
Small protein
DNA fragment The primary objective in drug discovery is compounds to be used as a treatment for illness or pain, and obviously there is no
Haemoglobin shortage of targets. The major drug companies generally distribute their efforts between several of broad areas (oncology, centra
nervous system, infections etc.). Commercial drug research often targets ailments that are prevalent in the 'developed' world (he
Lecture 3 disease, cancer, ulcers, migraine etc.), while the fight against malaria, one of the world's most lethal infectious diseases, has been
Captopril 2004
taken up over the years by organisations such as the World Health Organisation, The Gates Foundation and the US Army, for w
Captopril 2010
financial gain was not the primary concern. (What was? You decide.)
Lecture 4

Lecture 5
HMGR binding site
Atorvastatin 2001

Lecture 6
Cimetidine

Lecture 7
Imatinib binding site

PDFs (new window)

Appendix 1
MedChem history

Appendix 2
Glossary of terms

Appendix 3
Amino acid chart

Appendix 4
Bioisosteric groups

Appendix 5
Structure of DNA

3.2 Choosing a drug target

Identifying a medical need is relatively straightforward, but organising a biological effect that will alleviate the problem is not ea
Many early drugs were based on natural products that were found to have a biological effect, discovered through random screenin
How they worked often remained obscure for years, but recent developments provide a new situation:

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Choosing a drug target has increasingly become a ma er of identifying the proteins that are involved in the targeted condition, a
designing a molecule that will interact with them. The issue of selectivity remains crucial if side-effects are to be minimised or
avoided.

Selectivity between species can be relatively easy to achieve. For example, penicillin targets an enzyme that mediates cell wall
biosynthesis in the bacteria that cause the infection. Mammalian cells do not have a cell wall, so knocking out this enzyme does
affect humans. On the other hand, some infectious organisms have enzymes that are similar, but slightly different, to a human
equivalent, and the differences can be targeted.

Selectivity within the body is also desirable. An enzyme inhibitor should only target one enzyme, and receptor agonists/antagonis
should only target one type of receptor. But bear in mind that more complex issues might lie in wait — the body has a highly
interactive system of messengers, enzymes and receptors, and the imbalance caused by a drug can bring about unexpected (and
undesirable) responses by other parts of the 'machine.'

3.3 Enzymes as drug targets

Enzymes are a major target for drugs. Their function is to catalyse reactions that take place in the body, e.g. the hydrolysis of an
amide bond:

In the absence of the enzyme this reaction will occur, but is extremely slow (half-life many years). However, in the presence of th
enzyme it is fast. Typical rate increases for enzyme-catalysed processes are 1010 to 1012, and typical turnover numbers are 103
molecules/min (i.e. one enzyme molecule will convert 1000 molecules of substrate into product every minute).

A rough scheme of how an enzyme works is as follows:

In energetic terms we see the following reaction profile:

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The enzyme works by stabilising the transition state for the reaction (i.e. reducing the activation energy). A drug that acts on an
enzyme target blocks the active site, preventing the natural substrate from binding (and subsequently reacting). In this sense it
operates in the same way as an antagonist acts on a receptor, but it should be noted that because enzymes work in a different wa
from receptors, it is not usually possible for a drug to behave as an agonist in this context — either the drug blocks the active site
the reaction is inhibited, or it does not block the active site and has no effect.

Because enzymes are chiral (built from 'handed' amino acids), they are usually stereoselective and will generally only operate on
small range of substrates. It is important to ensure that a drug only blocks the action of the target enzyme, and no others. The active
sites of enzymes differ greatly, and as long as the differences can be exploited, this should be possible.

3.4 CASE STUDY: The development of ACE inhibitors

Angiotensin converting enzyme (ACE) is part of the renin-angiotensin system and its main role in the body is to break down
angiotensin I (decapeptide) into angiotensin II (octapeptide). It does this by cleaving the second amide bond in from the carboxyl
terminus.

The product (angiotensin II) then triggers a series of events which lead to an increase in blood pressure. In patients that suffer fr
abnormally high blood pressure (hypertension), stopping the formation of angiotensin II by blocking the action of ACE can be an
effective remedy.

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The 3-D structure of ACE has been determined by X-ray crystallography, but the first inhibitors of this enzyme were developed
while it was still unknown. The area was pioneered by Cushman, Onde i and Rubin at Squibb (now Bristol-Myers Squibb) in t
mid-1970s and led to the identification of several antihypertensive agents, including captopril (marketed as Capoten), Squibb's fir
billion-dollar drug, and one of the earliest successes of structure-based drug design. We will take a close look at two papers:

1. Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino a
[D. W. Cushman, H. S. Cheung, E. F. Sabo and M. A. Onde i, Biochemistry, 1977, 16, 5484–5491].

2. High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhib
and antihypertensive drugs [M. Akif, D. Georgiadis, A. Mahajan, V. Dive, E. D. Sturrock, R. E. Isaac and K. R. Acharya, J. Mol. Bi
2010, 400, 502–517].

The Squibb group started out with a clue from a natural product. The toxic effects of venom from a Brazilian viper (Bothrops jara
were found to be due to a sudden, massive drop in blood pressure. It was established that the compound responsible, a nonapep
that was later named SQ20881, was a potent ACE inhibitor.

In rationalising their search for drugs with the same property, the Squibb team advanced a hypothetical model of the active site o
ACE based on comparisons with another zinc metalloproteinase, carboxypeptidase A. This enzyme, present in the human diges
system, is able to cleave the C-terminal amino acid from a peptide chain:

The structure of carboxypeptidase A was established in the 1960s and it was known that (R)-2-benzylsuccinic acid functioned as
inhibitor of this enzyme. The Squibb rationale is summarised in the following figures (adapted from ref. 1).

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The chemists at Squibb made a series of amino acid derivatives which were tested as ACE inhibitors. The following tables show
some of the structure-activity relationships (SARs) they found, and reveal the logic of the drug design process, whereby a structu
systematically modified until it possesses the optimum properties (N.B. only a few of the many structures tested are shown here

The best terminal unit is proline (cf. the first 'hit' compound SQ20881) and it must have the (S)-configuration. A 3-
mercaptopropanoyl side-chain is a more effective Zn binder than carboxyl.

The most effective spacer for a mercaptoacyl Zn binder is –(CH2)2– whereas for a carboxyl binder the –(CH2)3– is be er (glutaroy
more effective than succinyl):

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For optimum activity, the mercaptoacyl Zn binder should have a free –SH group, the proline should have a free carboxyl, and a
central amide link with the correct orientation is needed.

The inhibitor constants of the most potent of the compounds tested, including the snake venom peptide SQ20881, are shown in T
4. Using Lineweaver-Burk plots it was confirmed that all were competitive inhibitors of ACE.

The competitive nature of the inhibition with respect to substrate is fully consistent with the binding of these inhibitors to the act
site of the angiotensin converting enzyme.

REMINDER: If a reversible inhibitor can bind to the enzyme active site in place of the substrate, it is described as a competitive inhibitor. In pure competitiv
inhibition, the inhibitor is assumed to bind to the free enzyme but not to the enzyme-substrate (ES) complex. The binding is described thus:

Here Ki is the dissociation constant for the enzyme-inhibitor (EI) complex. EI does not react to form E + P, and the enzyme is unable to bind both S and I a
same time.

3.5 ACE Inhibitors: Mechanism of action

The mechanism by which ACE is now believed to function is shown below. In the complete scheme, the natural substrate
(angiotensin I) is cleaved at the second peptide bond in from the C-terminus to give two smaller peptides. The process is facilita
by a nearby basic group that effectively 'holds' a proton during the hydrolytic step.

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Captopril binds strongly to the active site and thereby prevents the processing of the natural substrate, angiotensin I (see Section
for the X-ray analysis of the binding site).

The drug structure maintains all the key binding interactions, but does not participate in the reaction. Because it binds so strong
the enzyme active site, none of the natural substrate can bind, and so no angiotensin II can be produced — this results in a lower
of blood pressure. Captopril was marketed in 1981, and since that time a number of other ACE-inhibitors have been developed:

The adverse effect and pharmacokinetic limitations of captopril stimulated the development of enalapril and subsequent ACE
inhibitors (discussed later). The Bristol-Myers Squibb patent for captopril expired in 1996.

3.6 X-Ray analysis of the captopril binding site

For interactive 3D molecular models of the ACE-captopril complex, follow the links in the Contents panel on the left.

High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors an
antihypertensive drugs (M. Akif, D. Georgiadis, A. Mahajan, V. Dive, E. D. Sturrock, R. E. Isaac and K. R. Acharya, J. Mol. Biol., 2
400, 502–517).
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Captopril is the smallest orally active tight binding peptide analogue inhibitor of ACE. In the ACE–captopril structure the thiol
group of the inhibitor makes a direct interaction with the catalytic Zn2+ ion (distance, 2.15 Å). The captopril molecule is also held
8 H-bonds, including three with water molecules. The central carbonyl oxygen positioned between the thiol and the terminal pr
moiety of captopril is anchored by two strong H-bonds with His337 and His497. One of the oxygen atoms from the proline moie
carboxylate group is held by interactions with side-chains of residues Gln265, Lys495 and Tyr504. The second oxygen of the pro
moiety's carboxylate group in the present is held indirectly by water-molecule-mediated interactions with Asn261 and Glu150.
Furthermore, a network of water molecules makes indirect interactions between the carboxylate oxygen and with Arg356, Asp36
and Gln361 in the S1' and S2' subsites.

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