Corpora non agunt nisi fixata Compounds do not act unless bound Paul Ehrlich 1854-1915

-Dr Alicia Catabay

-Department of Pharmaceutical Chemistry

Introduction
Targets are molecular structures, chemically definable by at least a molecular mass, that will undergo a specific interaction with chemicals that we call drugs because they are administered to treat or diagnose a disease. A clinically relevant target might consist not of a single biochemical entity, but the simultaneous interference of a number of receptors. Only this will give a net clinical effect that might be considered beneficial. It is only by chance that the current in vitro screening techniques will identify drugs that work through such targets.

The number of targets presently used is still open to discussion in medicinal chemistry, but various approaches converge at a few hundred. The number of potential targets, was as estimated to be several hundred thousand in view of the manifold protein complexes, splicing variants and possible interventions with signaling pathways.

The problem with counting is mainly 2-fold: 1. the identification of the reaction partners of drug substances in the body, and 2. exactly what to define and count as the target. A target definition derived from the net effect rather than the direct chemical interaction will require input from systems biology, a nascent research field that promises to affect the drug discovery process significantly.

Mechanisms of action
An effective drug target comprises a biochemical system rather than a single molecule. Present target definitions are static. We know this to be insufficient, but techniques to observe the dynamics of drugtarget interactions are just being created. Most importantly, we are not able to gauge the interaction of the biochemical ripples that follow the drugs initial molecular effect.

It has been pointed out that two components are important to the mechanism of action The first component is the initial mass-actiondependent interaction The second component requires a coupled biochemical event to create a transition away from mass-action equilibrium and drug mechanisms that create transitions to a non-equilibrium state will be more efficient.

Although the term mechanism of action itself implies a classification according to the dynamics of drug substance effects at the molecular level, the dynamics of these interactions are only speculative models at present, and so mechanism of action can currently only be used to describe static targets.

Drug targets
Most drug targets are cellular proteins

-undergoing selective interaction with drugs to treat or diagnose a disease -are human-genome-derived proteins -or may belong to bacterial, viral, fungal or other pathogenic organisms What is a human genome?

Human genome project

The Human Genome Project (HGP) is an international

scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA and of identifying and mapping the approximately 20,00025,000 genes of the human genome from both a physical and functional standpoint Human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining. The haploid human genome occupies a total of just over 3 billion DNA base pairs.

The human genome, categorized by function of each gene product, given both as number of genes and percentage of all genes.

Key findings of the draft (2001) and complete (2004) genome sequences include : 1. There are approximately 30,000 genes in human beings, the same range as in mice and twice that of roundworms. Understanding how these genes express themselves will provide clues to how diseases are caused.[ 2. Between 1.1% to 1.4% of the genome's sequence codes for proteins 3. The human genome has significantly more segmental duplications (nearly identical, repeated sections of DNA) than other mammalian genomes. These sections may underlie the creation of new primatespecific genes 4. At the time when the draft sequence was published less than 7% of protein families appeared to be vertebrate specific

 The Human Genome Project is considered a mega

project because the human genome has approximately 3.3 billion base-pairs. If the sequence obtained was to be stored in book form, and if each page contained 1000 base-pairs recorded and each book contained 1000 pages, then 3300 such books would be needed in order to store the complete genome. However, if expressed in units of computer data storage, 3.3 billion base-pairs recorded at 2 bits per pair would equal 786 megabytes of raw data. This is comparable to a fully data loaded CD.

Benefits of the project

The work on interpretation of genome data is still in its initial

stages. It is anticipated that detailed knowledge of the human genome will provide new avenues for advances in medicine and biotechnology. Clear practical results of the project emerged even before the work was finished. For example, a number of companies, started offering easy ways to administer genetic tests that can show predisposition to a variety of illnesses, including breast cancer, hemostasis disorders, cystic fibrosis liver diseases and many others. Also, the etiologies for cancers, Alzheimers disease and other areas of clinical interest are considered likely to benefit from genome information and possibly may lead in the long term to significant advances in their management.

 There are also many tangible benefits for biological

scientists. For example, a researcher investigating a certain form of cancer may have narrowed down his/her search to a particular gene. By visiting the human genome database on the WWW the researcher can examine what other scientists have written about this gene, including three-dimensional structure of its product, its function, its evolutionary relationships to other human genes, or to genes in mice or yeast or fruit flies, possible detrimental mutations, interactions with other genes, body tissues in which this gene is activated, diseases associated with this gene or other datatypes.

 Further, deeper understanding of the disease processes

at the level of molecular biology may determine new therapeutic procedures. Given the established importance of DNA in molecular biology and its central role in determining the fundamental operation of cellular processes, it is likely that expanded knowledge in this area will facilitate medical advances in numerous areas of clinical interest that may not have been possible without them.

Advantages of Human Genome Project:

Knowledge of the effects of variation of DNA among

individuals can revolutionize the ways to diagnose, treat and even prevent a number of diseases that affects the human beings. It provides clues to the understanding of human biology. Access to the complete human genome sequence as well as to the complete sequences of pathogenic organisms provides information that can result in an avalanche of therapeutic targets.

 The alternative method of rational drug design

involves the design and synthesis of compounds based on the known structure of either a specific target or one of its natural ligands. For this reason, target identification must be followed by target validation, which confirms the likelihood that interfering with the target protein will impact on the disease.

Drug targets
Drugs are substances that exert some kind of

physiological or biochemical effect on our bodies. They may be single compounds or mixtures, and their effects may be beneficial or harmful. All drugs interact with specific targets, which are usually proteins but in some cases DNA or RNA. Drugs work either by stimulating or blocking the activity of their targets.

 For the past decade, the number of molecular targets

for approved drugs has been debated. Surprisingly, for an industry that spends in excess of US$50 billion on research and development each year, there is a lack of knowledge of the set of molecular targets that the modern pharmacopoeia acts on. If we are to develop predictive methods to identify potential new drug targets, it is essential that we establish with confidence the number, characteristics and biological diversity of targets of approved drugs.

Number of drug targets

Based on the mapping of the human genome in 2002 an

estimated 8000 targets were identified In 2006 Wishart et al reported 6,000 targets on the Drug Data Base Website - only a small part of these relates to approved drugs In 2003 Golden proposed that all then approved drugs acted through 273 proteins In 2006 Zheng et al disclose 268 successful targets in Therapeutics Target Database and Imming etal cataloged 218 targets for approved drugs A consensus of 324 drug targets for all classes of approved therapeutic drugs was proposed by Overington et al: 266 are HGD-proteins and 58 bacterial, viral, fungal or other organism targets.

What accounts for the discrepancies?

Criteria chosen by the authors

- to include or not drugs under clinical trials but not yet approved - considering or not multiple relevant targets for a unique drug including isoenzymes or different members of a receptor family Overingtons study Identifies 21,000 marketed drugs (US) , of these 1,357 are unique drugs, 1,204 are small molecules (incl 192 prodrug) and 166 are biological drugs

Overingtons study (2006)

27% binds to G-protein-coupled receptor 13% to nuclear receptors 7.9% to ligands-gated ion channels 5.5% to voltage-gated ion chanels A selected target may have a unique approved drug or a

large number of me-too molecules

Imming etal study

218 listed targets: 66 human enzymes, 20 bacterial, viral and
fungal or parasital enzymes; 20 families of GPCR; 12 nuclear receptors, 7 cytokine receptors and about 10 ion channels and 10 transport proteins of the plasma membrane

Conclusions from these studies

Confirm that a very large number of putative drug targets

remains to be explored. The introduction of genomics, proteomics and metabolomics has paved the way for biology-driven process, leading to plethora of drug targets. The list of potential drug targets encoded in a genome includes most natural choice of virulent genes and speciesspecific genes. Other options include targeting RNA, enzymes of the intermediary metabolism, systems for DNA replication, translation apparatus or repair and membrane proteins

Biochemical classes of drug targets

Common dug targets

Species-specific genes as drug targets

An interesting approach to the prediction of potential drug targets designated as the differential genome display has been proposed by Bork and co-workers. This approach relies on the fact that genome of parasitic microorganisms are generally much smaller and code for fewer proteins than the genomes of free-living organisms. The genes that are present in the genome of a parasitic bacterium, but absent in a closely related genome of free living bacterium, are therefore likely to be important for pathogenecity and can be considered as potential drug targets.

Nucleic acid as drug targets

Nucleic acids are the repository of genetic information.

DNA itself has been shown to be the receptor for many drugs used in cancer and other diseases. These work through a variety of mechanisms including chemical modification and cross linking of DNA (cisplatin) or cleavage of the DNA (bleomycin). Much work either by intercalation of a polyaromatic ring system into the double stranded helix (actinomycin D, ethidium) or by binding to the major and minor grooves of DNA (e.g., netropsin) has been reported.

 DNA has been shown to be

the target for chemotherapy with efforts to design sequence-specific reagents for gene therapy. Netropsin molecule. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove

RNA as drug target

Recent advances in the determination of RNA structure

and function have led to new opportunities that will have a significant impact on the pharmaceutical industry. RNA, which, among other functions, serves as a messenger between DNA and proteins, was thought to be an entirely flexible molecule without significant structural complexity. However, recent studies have revealed a surprising intricacy in RNA structure. This observation unlocks opportunities for the pharmaceutical industry to target RNA with small molecules.

 Drugs that bind to RNA might produce effects that

cannot be achieved by drugs that bind to proteins.

Proof of the principle has already been provided by

success of several classes of drugs obtained from natural sources that bind to RNA or RNA-protein complexes.

Membranes as drug targets

Membranes are significant structural elements, both in

defining the boundaries of a cell as well as providing interior compartments within the cell associated with particular functions. Cell membranes themselves can also act as targets for molecular recognition. An understanding of the structural and dynamic functions of the membranes (e.g., plasma membranes and intercellular membranes) may add to a more rational design of drug molecules with improved permeation characteristics or specific membrane effects.

 Many general anesthetics are believed to work by their

physical effects when dissolved in membranes. Several classes of antibiotics like gramicidin A, antifungals like alamethicin and toxins such as mellitin found in bee venoms have direct effects on planar lipid bilayers, causing transmembrane pores.

Proteins as drug targets

Proteins continue to assume significant attention from

the pharmaceutical and biotechnology industries as a valuable source of potential drug targets. Proteins provide the critical link between genes and disease, and as such are the key to the understanding of basic biological processes including disease pathology, diagnosis, and treatment. Researchers have discovered many potential therapeutic targets, and there are currently more than 700 products in various phases of development.

 However, translating the study of proteins into

validated drug targets poses substantial challenges. Genome sequences instruct cells on how and when to make proteins. The proteins in turn are the active players in the cell. Proteins form the machinery of cells, allow cells to communicate, and can control growth or death of an organism. Because of their role in cells, most of the drug targets are proteins. Drugs work by binding specifically to a protein.

 Small molecules such as drugs, insecticides or

herbicides usually exert their effects by binding to protein targets. In the past, many of these molecules were found empirically with little or no knowledge of the mechanism of action involved. In many cases, the targets that are modified by these substances were identified in retrospect. Interestingly, the majority of drugs currently in use modulate either enzymes or receptors, most of them G-protein-coupled receptors.

A. Enzymes
The macromolecule responsible for the catalysis of

biochemical reactions are an obvious target when a disease state is associated with production of a biologically active species. Enzymes are a classic target for therapeutic intervention and numerous well-studied examples exist. Traditional medicinal chemistry enzyme targets include kinases, phosphodiesterases, proteases and phosphotases.

Some enzyme as drug targets

Enzyme Dihydrofolate reductase HIV-1 protease ACE Neuraminidase Drug Methotrexate Saquinavir, Indinavir Captopril Oseltamivir

Cyclin dependent Kinase(CDKs)

Cyclooxygenase Thymidylate synthase Guanine phosphoribosyltranseferase (GPRT) Inosine5'-monophosphate dehydrogense

Flavopiridol
Diclofenac, Indomethacin Tomudex Allopurinol Tiazopurin

B. Receptor proteins
G-proteincoupled receptors are a super family of seven

transmembrane spanning proteins that are activated by a wide range of extracellular ligands and are expressed in virtually all tissues. Signaling through these receptors regulates a wide variety of physiological processes such as neurotransmission, chemotaxis, inflammation, cell proliferation, cardiac and smooth muscle contraction as well as visual and chemosensory perception. In view of their widespread distribution and importance in health and disease, it is not surprising that GPCRs are the most successful class of target proteins for drug discovery research.

 The sequencing of human genome has led to the

prediction of as many as 1000 GPCRs, of which 400 are non-chemosensory receptors and can therefore be considered as potential drug-targets. It has been estimated that up to 50 % of all marketed drugs directly target this family of receptors, some of which are listed in next slide

Some currently marketed drugs that target GPCRs

GPCR
Histamine -adrenergic -adrenergic Dopamine Serotonin

Indication(s)
Allergies, ulcers Hypertension, asthma Benign prostatic hypertrophy Psychosis, Parkinson's Migraine, anxiety

Drug(s)
Cimetidine,Ranitidine,Terfenadine Atenolol, Albuterol, Salmeterol Terazosin , doxazosin Aripiprazole, Ropinerole Zolmitriptan, clozapine, buspirone

Opiod Angiotensin
Muscarinic acetylcoline Leukotriene

Pain Hypertension
Alzheimer' s disease Asthma

Butarphanol Losartan, Eprosartan

Bethanechol, dicyclomine Pranlukast

 The goal in developing drugs against the targets listed

above is often to modulate the function of the human protein while the goal in developing drugs against pathogenic organisms is total inhibition, leading to the death of the pathogen. Antimicrobial drugs should be essential to the pathogen, have a unique function in the pathogen, be present only in the pathogen, and be able to be inhibited by a small molecule.

 The target should be essential, in that it is a part of a

crucial cycle in the cell, and its elimination should lead to the pathogen's death. The target should be unique: no other pathway should be able to supplement the function of the target and overcome the presence of the inhibitor. If the macromolecule satisfies all the outlined criteria to be a drug target but functions in healthy human cells as well as in a pathogen, specificity can often be engineered into the inhibitor by exploiting structural or biochemical differences between the pathogenic and human forms.

 Finally, the target molecule should be capable of

inhibition by binding of a small molecule. Enzymes are often excellent drug targets because compounds are designed to fit within the active site pocket.

Protein as drug targets

Drug action at proteins

Types of proteins which drug interacts
a. Carrier proteins

b. Structural proteins

Carrier proteins
Cells smugglers smuggling the important

chemical building blocks of amino acids, sugars, and nucleic acids across the cell membrane such that the cell can synthesize its proteins, carbohydrates and nucleic acids Also transmit important neurotransmitters back into the nerve that release them to limit its activity. Carrier proteins are not identical; specific carriers for different molecules that need to be carried across membranes

Carrier proteins
Have recognition site that allows them to bind and

encapsulate their specific cargo Some carrier proteins can be fooled and it is possible to design drugs that are mistaken as the usual cargo molecules: carries it across membrane into the cell Carrier proteins can be viewed as drug targets when drugs prevent them from functioning properly: drugs tightly bound to the carrier once stowed away they remain permanent lodgers - function of carrier is blocked Example: cocaine and TCA: prevent neurotransmitter i.e. Noradrenaline from reentering nerve cells

Structural proteins
Structural proteins do not normally act as drug targets
-

except tubulin Tubulin molecules polymerize to fom small tubes called microtubules in the cytoplasm Microtubules function: maintenance of shape exocytosis, and release of neurotansmitters Also involved in the mobility of cells

Space-filling model of a microtubule segment derived from cryoelectron microscopy. The protofilaments are seen running along the axis of the segment. The microtubule (+) end is towards the top of the image.[

Function
Inflammatory cells called neutrophils are moble cells

which normally protects the body against infection They can also enter joints leading to inflammation and arthritis One way of treatment is with colchicine which binds to tubulin and causes the microtubules to depolymerize Once mictotubules are broken down neutrophils loses mobility and can no longer migrate to the joints

Structure based drug design

Drug discovery referred to, as rational did not take

flight until the first structures of the targets were solved. In 1897, Ehrlich suggested a theory called the side chain theory wherein he proposed that specific groups on the cells combine with the toxin. Ehrlich coined these side chains as receptors. Structure-based drug design of protein ligands has emerged as a new tool in medicinal chemistry.

 The central assumption

of structure-based drug design is an iterative one as shown in the right and often proceeds through multiple cycles before an optimized lead goes into clinical trials.

 The first cycle includes the cloning, purification and

structure determination of the target protein or nucleic acid by one of three principal methods: X-ray crystallography, NMR or comparative modeling. Using computer algorithms, compounds or fragments of compounds from a database are positioned into a selected region of the structure.

 These compounds are scored and ranked based on their

steric and electrostatic interactions with the target site and the best compounds are tested further with biochemical assays. In the second cycle, structure determination of the target in complex with a promising lead from the first cycle, one with at least micromolar inhibition in vitro, reveals sites on the compound that can be optimized to increase potency. Additional cycles include synthesis of the optimized lead, structure determination of the new target: lead complex, and further optimization of the lead compound. After several cycles of the drug design process, the optimized compounds usually show marked improvement in binding, and often, specificity for the target.

Drug action
Effects induced by the drug on the biological system

Steps:
a. Binding to cellular molecular target b. Signaling events leading to a cellular response

(secretion, contraction, metabolism) c. Integration at the level of tissues and organs: corresponding to a modification of physiological function ( digestion, motricity, cardiovascular processes)

Drug action
Drugs act by increasing or decreasing normal function

but do not endow the organism with new functions.

Future direction: Gene therapy may soon challenge

this principle it remains valid for the immediate future

Drug effects
The vast majority of drugs produce their effects by : 1. Interacting with proteins, either with those on the surface of the cell comprising the plasma membrane, such as receptors of mediators, ionic channels and transporters (about 60% of drugs), or with components of the interior of the cell, such as enzymes and nuclear receptor 2. Some others act extracellularly at non-cellular constituents of the body without involving a drug receptor interaction. Example: the neutralization of gastric acid by antacid drugs. In this reaction the excess of gastric acid is neutralized by a base such as sodium bicarbonate. This reaction is not considered as a drugreceptor interaction, since no macromolecular component is involved.

Drug effects
3. Other mechanisms of drug action may occur at cellular sites and may involve macromolecular components, but the biological effects produced are non-specific consequences of the chemical properties of the drugs. Example: Detergents, alcohol, oxidizing agents, phenol derivatives act by destroying the integrity of the cell through disrupting the cellular constituents

Drug effects
4. A number of other molecular interactions between

drugs and the components of the biological system may occur, such as the binding of drugs to plasma albumin. Serum albumin can transport drugs in the circulation to organs, and it can hold drugs up, preventing them from binding to their site of action. Those interactions affect the duration of the drug action or its rate of actions. Albumin might then be considered as an acceptor site for the drug rather than a target or receptor.

Drug binding, affinity and selectivity

The receptor concept was formulated by Langley and the term receptor was proposed by Ehrlich. The concept of target binding or receptor binding, Corpora non agunt nisi fi xata (compounds do not act unless bound), has been subject to refinement but is still valid. The term receptor should be now restricted to the target of endogenous mediators but is often extented to the targets of exogenous compounds endowing various biochemical functions

Drug binding, affinity and selectivity

The various physicochemical interactions between a ligand and the target cooperate to establish the target drug interaction: 1. Hydrophobic interactions plays an important role in stabilizing the conformation of proteins and in the association of hydrophobic structure between the drug and its target. 2. Hydrogen bonding is strongly directional and has considerable importance both in the maintaining the secondary and tertiary structure of the target itself and in the targetdrug interaction.

3. Charge transfer complexes formed between electron rich donor molecules and electron-deficient acceptors are also often involved in drugtarget interaction. 4. Ionic bonds are of importance in the actions of ionizable drugs since they act across long distances; ionic bonds result from the electrostatic attraction that occurs between oppositely charged ions; most targets have a number of ionizable groups (COO, -O-,NH3+ ) at physiological pH that are available for the binding with charged drug

Covalent bonds resulting in the formation of a long lasting complex are less important in drugtarget interaction. Although most drugtarget interactions are readily reversible, some drugs, such as anticancer nitrogen mustards and alkylating compounds form reactive cationic intermediates (i.e. aziridinium ion) that can react with electron donor groups on the target. These chemical interactions are related to the affinity of the drug for its target

Various ligands for a single drug target

Most of target types can be stimulated or inhibited depending of the ligand chosen. This leads to opposite regulations of related cellular functions. Terms used to characterize these different ligand types differ according to the biochemical nature of the targets.

Enzyme ligands more often lead to the inhibition of the enzyme activity, binding the active site with competition with the substrate (competitive inhibitors) or to allosteric sites ( non-competitive inhibitors ). Activation of an enzyme is more difficult to proceed unless giving or generating an excess of substrate or co-substrate. However some drugs are known to activate enzymes by direct binding, that is, forskolin for adenylyl cyclase

Membrane transporters and ion channels permeability can be increased or decreased by direct binding of selected drugs termed openers and inhibitors (or blockers ), respectively. However, such ligands are too often improperly referred to as agonists and antagonists. Receptors of mediators are able to interact with a large diversity of ligand types:

 Agonists mimic the effects of endogenous mediators

(neurotransmitters, hormones, cytokines ). Thus, mediators are considered the endogenous, or physiological, agonists of their receptors. Some exceptions to this concept are now known, some couples of mediators acting through the binding to a single receptor with agonist or antagonist properties respectively, i.e. interleukin 1 and IRAP, RANK-L and OPG, MSH and AGRP. Full agonists elicit a maximal response of the organism, usually similar to that of the mediator. Partial agonists elicit a partial response of the organism, and prevent the binding of the mediator. Thus the related function of the organism is decreased.

Neutral antagonists prevent the binding of the mediator and thus abolish downstream signaling biochemical events and physiological responses. Most neutral antagonists bind to the agonist binding site.

Inverse agonists also termed negative antagonists have been found among antagonists. Like neutral antagonists, they prevent the binding of agonists, including mediators, but elicit a response inverse to that of agonists. This has been first shown for ligands of the benzodiazepine binding site of GABA-A ( -aminobutyric-acid) receptors, whose agonists (classical benzodiazepines) potentiate the opening of the intrinsic chloride channel elicited by GABA, whereas inverse agonists decrease it with opposite reponses, i.e. anxiolytic effect for agonists and anxiogenic effect for inverse agonists. This concept of inverse agonism has been first extended to opioid receptors and then to others GPCR, showing that such ligands decreased the constitutive activity of the receptor, e.g. its activity noticable in the absence of mediator.

Receptors of mediators including an intrinsic ion channels (ligand-gated ion channels such as nicotinic receptors), or an enzyme activity (i.e. a tyrosine kinase activity such as insulin receptors, or ganylyl cyclase activity such as ANF receptors) have ligands for their receptor part (agonists and antagonists) as well as for their ion channel (openers and inhibitors or blockers) or enzyme part (inhibitors).

The activity of these various target types can also be modulated indirectly through intracellular signaling, for instance by phosphorylation elicited by protein kinases or dephosphorylation involving protein phosphatases, or by proteinprotein interactions such as regulations induced by interaction with the calciprotein calmoduline. This offers large alternatives to modify the status of a putative target through indirect ways when direct targeting has been unsuccessful.