molecular drug targets-1

8/4/2019 Molecular Drug Targets-1

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“Corpora non agunt nisi fixata”

Compounds do not act unless bound

Paul Ehrlich 1854-1915

-Dr Alicia Catabay

-Department of Pharmaceutical Chemistry



IntroductionTargets are molecular structures, chemically definable by at

least a molecular mass, that will undergo a specificinteraction 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 beconsidered beneficial.

It is only by chance that the current in vitro screeningtechniques will identify drugs that work through suchtargets.



The problem with counting is mainly 2-fold:

1. the identification of the reaction partners of drugsubstances in the body, and

2. exactly what to define and count as the target.

A target definition derived from the net effect ratherthan the direct chemical interaction will require inputfrom systems biology, a nascent research field that

promises to affect the drug discovery processsignificantly.



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 beinsufficient, but techniques to observe the dynamics of drug–target interactions are just being created.

Most importantly, we are not able to gauge the

interaction of the biochemical “ ripples” that followthe drug’s initial molecular effect.



It has been pointed out that “ two components areimportant to the mechanism of action …

The first component is the initial mass-action-dependent interaction …

The second component requires a coupled biochemicalevent to create a transition away from mass-actionequilibrium ” and “ drug mechanisms that createtransitions to a non-equilibrium state will be moreefficient. ”



Although the term “ mechanism of action ” itself implies a classification according to the dynamics of drug

substance effects at the molecular level, the dynamicsof 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 otherpathogenic 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 whichmake up DNA and of identifying and mapping the

approximately 20,000–25,000 genes of the human genomefrom both a physical and functional standpoint

Human genome is the genome of Homo sapiens, which isstored on 23 chromosome pairs plus the smallmitochondrial DNA

22 of the 23 chromosomes are autosomal chromosomepairs, while the remaining pair is sex-determining.

The haploid human genome occupies a total of just over 3billion 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, thesame range as in mice and twice that of roundworms.Understanding how these genes express themselves willprovide clues to how diseases are caused.[

2. Between 1.1% to 1.4% of the genome's sequence codes for

proteins3. 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 primate-

specific genes4. At the time when the draft sequence was published less

than 7% of protein families appeared to be vertebratespecific



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 bookform, and if each page contained 1000 base-pairsrecorded and each book contained 1000 pages, then3300 such books would be needed in order to store thecomplete genome.

However, if expressed in units of computer datastorage, 3.3 billion base-pairs recorded at 2 bits perpair would equal 786 megabytes of raw data. This iscomparable to a fully data loaded CD.



Benefits of the project

The work on interpretation of genome data is still in its initialstages. It is anticipated that detailed knowledge of the humangenome will provide new avenues for advances in medicine andbiotechnology.

Clear practical results of the project emerged even before the work was finished.

For example, a number of companies, started offering easy ways toadminister genetic tests that can show predisposition to a variety of illnesses, including breast cancer, hemostasis disorders, cysticfibrosis liver diseases and many others.

Also, the etiologies for cancers, Alzheimer’s disease and other areasof clinical interest are considered likely to benefit from genomeinformation and possibly may lead in the long term to significantadvances 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 aparticular gene.

By visiting the human genome database on the WWW the researcher can examine what other scientists have written about this gene, including three-dimensionalstructure of its product, its function, its evolutionary relationships to other human genes, or to genes inmice or yeast or fruit flies, possible detrimentalmutations, interactions with other genes, body tissuesin which this gene is activated, diseases associated with this gene or other datatypes.



Further, deeper understanding of the disease processesat the level of molecular biology may determine newtherapeutic procedures.

Given the established importance of DNA in

molecular biology and its central role in determiningthe fundamental operation of cellular processes, it islikely that expanded knowledge in this area willfacilitate 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 amongindividuals can revolutionize the ways to diagnose,treat and even prevent a number of diseases thataffects the human beings.

It provides clues to the understanding of humanbiology.

Access to the complete human genome sequence as well as to the complete sequences of pathogenicorganisms provides information that can result in anavalanche of therapeutic targets.



The alternative method of rational drug designinvolves the design and synthesis of compounds basedon the known structure of either a specific target orone of its natural ligands.

For this reason, target identification must be followedby target validation, which confirms the likelihoodthat interfering with the target protein will impact onthe 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 theireffects may be beneficial or harmful.

All drugs interact with specific targets, which areusually 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 weestablish 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 drugsacted through 273 proteins

In 2006 Zheng et al disclose 268 successful targets inTherapeutics Target Database and Imming etal cataloged218 targets for approved drugs

A consensus of 324 drug targets for all classes of approvedtherapeutic drugs was proposed by Overington et al: 266are HGD-proteins and 58 bacterial, viral, fungal or otherorganism 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 aunique drug including isoenzymes or differentmembers of a receptor family

Overington’s study

Identifies 21,000 marketed drugs (US) , of these 1,357 areunique drugs, 1,204 are small molecules (incl 192prodrug) and 166 are biological drugs



Overington’s 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 alarge number of “me-too” molecules

Imming etal study

218 listed targets: 66 human enzymes, 20 bacterial, viral andfungal or parasital enzymes; 20 families of GPCR; 12 nuclearreceptors, 7 cytokine receptors and about 10 ion channels and 10transport 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-drivenprocess, leading to plethora of drug targets.

The list of potential drug targets encoded in a genomeincludes most natural choice of virulent genes and species-

specific 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



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 includingchemical 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 majorand minor grooves of DNA (e.g., netropsin) has beenreported.



DNA has been shown to bethe target for

chemotherapy with effortsto design sequence-specificreagents for gene therapy.

Netropsin molecule. Thenarrowness of the groove

forces the netropsinmolecule to sitsymmetrically in thecenter, with its two pyrrolerings slightly non-coplanarso that each ring is parallelto the walls of itsrespective region of thegroove

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891708/figure/F2/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891708/figure/F2/



RNA as drug target Recent advances in the determination of RNA structure

and function have led to new opportunities that will have asignificant impact on the pharmaceutical industry.

RNA, which, among other functions, serves as a messengerbetween 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 smallmolecules.



Drugs that bind to RNA might produce effects thatcannot be achieved by drugs that bind to proteins.

Proof of the principle has already been provided by

success of several classes of drugs obtained fromnatural sources that bind to RNA or RNA-proteincomplexes.



Membranes as drug targets Membranes are significant structural elements, both in

defining the boundaries of a cell as well as providinginterior 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 andintercellular membranes) may add to a more rationaldesign of drug molecules with improved permeationcharacteristics or specific membrane effects.



Many general anesthetics are believed to work by theirphysical 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 onplanar 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 anddisease, and as such are the key to the understandingof basic biological processes including diseasepathology, diagnosis, and treatment.

Researchers have discovered many potentialtherapeutic targets, and there are currently more than700 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 tomake proteins.

The proteins in turn are the active players in the cell.Proteins form the machinery of cells, allow cells tocommunicate, and can control growth or death of anorganism.

Because of their role in cells, most of the drug targetsare proteins.

Drugs work by binding specifically to a protein.



Small molecules such as drugs, insecticides orherbicides usually exert their effects by binding toprotein targets.

In the past, many of these molecules were foundempirically 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 themG-protein-coupled receptors.



A. Enzymes

The macromolecule responsible for the catalysis of biochemical reactions are an obvious target when adisease state is associated with production of a

biologically active species. Enzymes are a classic target for therapeutic

intervention and numerous well-studied examplesexist.

Traditional medicinal chemistry enzyme targetsinclude kinases, phosphodiesterases, proteases andphosphotases.



B. Receptor proteins G-protein–coupled 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 andsmooth muscle contraction as well as visual andchemosensory perception.

In view of their widespread distribution and importance inhealth and disease, it is not surprising that GPCRs are themost successful class of target proteins for drug discovery research.



Some currently marketed drugs that target

GPCRsGPCR Indication(s) Drug(s)

Histamine Allergies, ulcers Cimetidine,Ranitidine,Terfenadine

β-adrenergic Hypertension, asthma Atenolol, Albuterol, Salmeterol

α -adrenergic Benign prostatichypertrophy

Terazosin , doxazosin

Dopamine Psychosis, Parkinson's Aripiprazole, Ropinerole

Serotonin Migraine, anxiety Zolmitriptan, clozapine, buspirone

Opiod Pain Butarphanol

Angiotensin Hypertension Losartan, Eprosartan

Muscarinic acetylcoline Alzheimer' s disease Bethanechol, dicyclomine

Leukotriene Asthma Pranlukast



The goal in developing drugs against the targets listedabove is often to modulate the function of the humanprotein while the goal in developing drugs againstpathogenic organisms is total inhibition, leading tothe death of the pathogen.

Antimicrobial drugs should be essential to thepathogen, have a unique function in the pathogen, bepresent only in the pathogen, and be able to beinhibited by a small molecule.



The target should be essential, in that it is a part of acrucial cycle in the cell, and its elimination should leadto the pathogen's death.

The target should be unique: no other pathway shouldbe 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 humancells as well as in a pathogen, specificity can often be

engineered into the inhibitor by exploiting structuralor biochemical differences between the pathogenicand human forms.



Finally, the target molecule should be capable of inhibition by binding of a small molecule.

Enzymes are often excellent drug targets becausecompounds are designed to fit within the active sitepocket.



Protein as drug

targets



Drug action at proteins Types of proteins which drug interacts

a. Carrier proteinsb. Structural proteins



Carrier proteins Cell’s “smugglers” – smuggling the important

chemical building blocks of amino acids, sugars, andnucleic acids across the cell membrane such that the

cell can synthesize it’s proteins, carbohydrates andnucleic acids

Also transmit important neurotransmitters back intothe nerve that release them to limit its activity.

Carrier proteins are not identical; specific carriers fordifferent molecules that need to be carried acrossmembranes



Carrier proteins

Have recognition site that allows them to bind andencapsulate their specific cargo

Some carrier proteins can be fooled and it is possibleto 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: drugstightly 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 tubescalled microtubules in the cytoplasm

Microtubules function:

- maintenance of shape

- exocytosis, and- release of neurotansmitters

- Also involved in the mobility of cells



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 todepolymerize

Once mictotubules are broken down neutrophils losesmobility 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 weresolved.

In 1897, Ehrlich suggested a theory called the sidechain theory wherein he proposed that specific groupson the cells combine with the toxin.

Ehrlich coined these side chains as receptors.

Structure-based drug design of protein ligands hasemerged as a new tool in medicinal chemistry.



The central assumption

of structure-based drugdesign is an iterativeone as shown in theright and often

proceeds throughmultiple cycles beforean optimized lead goesinto clinical trials.



The first cycle includes the cloning, purification andstructure determination of the target protein ornucleic acid by one of three principal methods: X-ray crystallography, NMR or comparative modeling.

Using computer algorithms, compounds or fragmentsof compounds from a database are positioned into aselected region of the structure.



These compounds are scored and ranked based on their

steric and electrostatic interactions with the target site andthe best compounds are tested further with biochemicalassays.

In the second cycle, structure determination of the targetin complex with a promising lead from the first cycle, one

with at least micromolar inhibition in vitro, reveals sites onthe 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, theoptimized compounds usually show marked improvementin 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, cardiovascularprocesses)



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 challengethis principle it remains valid for the immediate future



Drug effects

3. Other mechanisms of drug action may occur atcellular sites and may involve macromolecularcomponents, but the biological effects produced are

non-specific consequences of the chemicalproperties of the drugs.

Example: Detergents, alcohol, oxidizing agents, phenolderivatives act by destroying the integrity of the cell

through disrupting the cellular constituents



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 notact unless bound), has been subject to refinement butis still valid.

The term “ receptor” should be now restricted to the

target of endogenous mediators but is often extentedto 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 targetdrug interaction:

1. Hydrophobic interactions plays an important role instabilizing the conformation of proteins and in theassociation of hydrophobic structure between the drugand its target.

2. Hydrogen bonding is strongly directional and hasconsiderable importance both in the maintaining thesecondary and tertiary structure of the target itself andin the target–drug interaction.



3. Charge transfer complexes formed between electron

rich donor molecules and electron-deficient acceptorsare also often involved in drug–target 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 thatoccurs between oppositely charged ions;

most targets have a number of ionizable groups (COO-

, -O-,NH3+ ) at physiological pH that are available forthe binding with charged drug



Covalent bonds resulting in the formation of a long

lasting complex are less important in drug–targetinteraction.

Although most drug–target interactions are readily

reversible, some drugs, such as anticancer nitrogenmustards 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 theaffinity of the drug for its target



Various ligands for a single drug target

Most of target types can be stimulated or inhibiteddepending of the ligand chosen.

This leads to opposite regulations of related cellular

functions.

Terms used to characterize these different ligand typesdiffer according to the biochemical nature of thetargets.



Enzyme ligands more often lead to the inhibition of

the enzyme activity, binding the active site withcompetition with the substrate (competitive inhibitors)or to allosteric sites ( non-competitive inhibitors ).

Activation of an enzyme is more difficult to proceedunless giving or generating an excess of substrate orco-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 (orblockers ), respectively.

However, such ligands are too often improperly referredto 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, orphysiological, agonists of their receptors.

Some exceptions to this concept are now known, somecouples 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 relatedfunction 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 bindingsite.



Receptors of mediators including an intrinsic ion

channels (ligand-gated ion channels such as nicotinicreceptors), or an enzyme activity (i.e. a tyrosine kinaseactivity such as insulin receptors, or ganylyl cyclaseactivity such as ANF receptors) have ligands for their

receptor part (agonists and antagonists) as well as fortheir 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 proteinkinases or dephosphorylation involving proteinphosphatases, or by protein–protein interactions such

as regulations induced by interaction with thecalciprotein calmoduline.

This offers large alternatives to modify the status of aputative target through indirect ways when directtargeting has been unsuccessful.

molecular drug targets-1

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