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10.13.5 Antisense Drugs10.13.5 Antisense Drugs

• The concept of antisense compounds The concept of antisense compounds or or sequence-defined oligonucleotides sequence-defined oligonucleotides (ONs) offers a new(ONs) offers a new specific approach specific approach to designing drugs that target nucleic to designing drugs that target nucleic acids, acids,

• The idea underlying this approachThe idea underlying this approach is is that the antisense compound that the antisense compound contains contains the sequence of the sequence of ccomplementary omplementary bases bases to those foundto those found in a short in a short section of the target nucleic acid. section of the target nucleic acid.

• This section is usually part of the This section is usually part of the genetic message being carried by an genetic message being carried by an mRNA molecule. mRNA molecule.

• The antisensThe antisensee compound binds to this compound binds to this section by hsection by hyydrogen bonding between drogen bonding between the complementary base pairs. the complementary base pairs.

• This inhibits translation of the This inhibits translation of the message carried by the mRNAmessage carried by the mRNA, which , which inhibits the production of a specific inhibits the production of a specific protein responsible for a disease state protein responsible for a disease state in a patient.in a patient.

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• Antisense compounds were originally short Antisense compounds were originally short lengths of nucleic acid chains that had lengths of nucleic acid chains that had base sequences that were complementary base sequences that were complementary to those found in their target RNA. to those found in their target RNA.

• These short lengths of nucleic acid These short lengths of nucleic acid antisense compounds were found to antisense compounds were found to be be unsuitable as drugs because of poor unsuitable as drugs because of poor binding to the target site and short half-binding to the target site and short half-lives due to enzyme actionlives due to enzyme action. However, they . However, they provided lead compounds for further provided lead compounds for further dedevvelopment (Figure 10.35). Development elopment (Figure 10.35). Development is currently taking three basic routes:is currently taking three basic routes:

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flg.re fl The bleomycin’. The drug bleomycin sulphate is a mixture of a number of bleomycins.

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• (I) modification of the backbone linking the bases (I) modification of the backbone linking the bases to increase resistance to ento increase resistance to enzymatic hydrolysiszymatic hydrolysis::

• (ii) changing the nature of the sugar residue by (ii) changing the nature of the sugar residue by either replacing some of the free either replacing some of the free hydroxyhydroxy groups groups by other substituents or forming derivatives of by other substituents or forming derivatives of these these ggrouroupsps(iii) modifying the nature of the (iii) modifying the nature of the substituent groups of the bases.substituent groups of the bases.

• Antisense compounds are able to bind to both Antisense compounds are able to bind to both RNA and DNA. In the latter case they form a triple RNA and DNA. In the latter case they form a triple helix. At present, antisense drugs are still in the helix. At present, antisense drugs are still in the early stages of their development but the concept early stages of their development but the concept has aroused considerable interest in the has aroused considerable interest in the pharmaceutical industrpharmaceutical industr

• Antisense compounds are able to Antisense compounds are able to bind to both RNA and DNA. In the bind to both RNA and DNA. In the latter case they form a triple helix. latter case they form a triple helix.

• At present, antisense drugs are still At present, antisense drugs are still in the early stages of their in the early stages of their development but the concept has development but the concept has aroused considerable interest in the aroused considerable interest in the pharmaceutical industrpharmaceutical industriyiy

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10.13.6 Chain-c10.13.6 Chain-clleeaavviing ng AgentsAgents•The interaction oThe interaction off chain-clechain-cleaaving ving

agents agents with DNA results in the with DNA results in the breaking of the nucleic acibreaking of the nucleic acidd into into fragments. fragments.

•Currently, the main cleaving Currently, the main cleaving agents are the agents are the bleomycins (bleomycins (Figure Figure 10.36) and their analogues. 10.36) and their analogues.

•However, other classes of drug However, other classes of drug are in the development stage.are in the development stage.

• The bleomycins are a group of naturally The bleomycins are a group of naturally occurring occurring glycoproteins that exhibit glycoproteins that exhibit antitumour activityantitumour activity. .

• When administered to patients they When administered to patients they tend to accumulate tend to accumulate in the squamous in the squamous cells cells and so are useful for treating and so are useful for treating cancers of the head, neck and genitalia. cancers of the head, neck and genitalia. However, the bleomycins cause pain However, the bleomycins cause pain and ulceration of areas of skin that and ulceration of areas of skin that contain a high concentration of keratin, contain a high concentration of keratin, as well as other unwanted side effects.as well as other unwanted side effects.

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• The action of the bleomycins is not The action of the bleomycins is not fully understood. It is believed that the fully understood. It is believed that the bithiabithia role moiet (domain X in Figure role moiet (domain X in Figure 10.36) 10.36) intercalates with the DNA. intercalates with the DNA.

• In bleomycin A3 the resulting adductIn bleomycin A3 the resulting adduct to the receptor of the host cell the to the receptor of the host cell the virus—receptor complex is transported virus—receptor complex is transported into the cell by receptor-mediated into the cell by receptor-mediated endocytosis (see section 4.3.6). endocytosis (see section 4.3.6).

• In the course of this process the In the course of this process the protein capsid and any lipoprotein protein capsid and any lipoprotein envelopes may be removed. envelopes may be removed.

• Once it has entered the host cell the Once it has entered the host cell the viral nucleic acid is able to use the viral nucleic acid is able to use the host’s cellular machinery to synthesise host’s cellular machinery to synthesise the nucleic acids and proteins required the nucleic acids and proteins required to produce a number ofto produce a number of new viruses new viruses (Figure 10.38).(Figure 10.38).

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14. Viruses14. Viruses• Viruses are infective agents that are Viruses are infective agents that are

considerably smaller than bacteria. considerably smaller than bacteria. They are essentiallyThey are essentially packages, packages, known as virions, of chemicals that known as virions, of chemicals that invade host cells. invade host cells.

• However, viruses are notHowever, viruses are not independent and can only penetrate independent and can only penetrate a host cell that can a host cell that can satisfy the satisfy the specific needs specific needs of thatof that virus. virus.

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• The mode of penetration varies The mode of penetration varies considerably from virus to virus. Once considerably from virus to virus. Once inside the hostinside the host cell viruses cell viruses take over take over the metabolic machinery of the host the metabolic machinery of the host and use it to produce moreand use it to produce more virusesviruses. . Replication is often lethal to the host Replication is often lethal to the host cell, which may cell, which may undergo lysis to undergo lysis to release therelease the progeny of the virus.progeny of the virus.

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•However, in some cases the virus However, in some cases the virus may integrate into the hostmay integrate into the host chromosome chromosome and become and become dormant. The ability of viruses to dormant. The ability of viruses to reproduce means that they canreproduce means that they can be be regarded regarded as being on the as being on the borderline of being living borderline of being living organismsorganisms..

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14.1. Structure and 14.1. Structure and replicationreplication

•Viruses consist of a core of either Viruses consist of a core of either DNA or, as in the majority of cases, DNA or, as in the majority of cases, RNA RNA fully orfully or partially covered by a partially covered by a protein coating known as the protein coating known as the capsid. The capsid consists of a capsid. The capsid consists of a numbernumber of polypeptide molecules of polypeptide molecules known as capsomers (Fig.10.43).known as capsomers (Fig.10.43).

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Figure 10.37. (a) Schematic representations of the structure of a virus (a) without a lipoprotein envelope (naked virus) and (h) with a lipoprotein envelope.

• The capsid that surrounds mostThe capsid that surrounds most viruses viruses consists of a number of different consists of a number of different capsomers although some viruses will capsomers although some viruses will havehave capsids that only contain one capsids that only contain one type of capsomer. It is the type of capsomer. It is the arrangement of the capsomersarrangement of the capsomers around around the nucleic acid that determines the the nucleic acid that determines the overall shape of the virion.overall shape of the virion.

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• In the majority ofIn the majority of viruses, the viruses, the capsomers form a layer or capsomers form a layer or several layers that completely several layers that completely surround the nucleicsurround the nucleic acids. acids. However, there are some viruses However, there are some viruses in which the capsomers form an in which the capsomers form an open-ended tubeopen-ended tube that holds the that holds the nucleic acids.nucleic acids.

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• In many viruses the capsid is coated In many viruses the capsid is coated with a with a protein-containing lipid bilayer protein-containing lipid bilayer membrane.membrane.

• These are known as enveloped viruses. These are known as enveloped viruses. Their lipid bilayers are often Their lipid bilayers are often derived derived from thefrom the plasma membrane of the host plasma membrane of the host cell and are formed when the virus cell and are formed when the virus leaves the host cell by aleaves the host cell by a process known process known as budding.as budding.

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• Budding is a mechanism by which a Budding is a mechanism by which a virus leaves a host cellvirus leaves a host cell without killing without killing that cell. that cell. It provides the virus with a It provides the virus with a membrane whose lipid components membrane whose lipid components areare identical to those of the host (Fig. identical to those of the host (Fig. 10.43). This 10.43). This allows the virus to allows the virus to penetrate new host cellspenetrate new host cells without without activating the host’s, immune activating the host’s, immune systems.systems.

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• Viruses bind to host cells at specific Viruses bind to host cells at specific receptor sites on the host’s cell receptor sites on the host’s cell envelope.envelope.

• The binding sites on the virus are The binding sites on the virus are polypeptides in its capsid or polypeptides in its capsid or lipoprotein envelope.lipoprotein envelope. Once the virus Once the virus has bound to the receptor of the host has bound to the receptor of the host cell cell the virus–receptor complexthe virus–receptor complex is is transported into the cell by receptor-transported into the cell by receptor-mediated endocytosis.mediated endocytosis.

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• InIn the course of this process the protein the course of this process the protein capsid and any lipoprotein envelopes capsid and any lipoprotein envelopes may bemay be removed. removed.

• Once it has entered the host cell the Once it has entered the host cell the viral nucleic acid viral nucleic acid is able to use the is able to use the host’shost’s cellular machinery to synthesise cellular machinery to synthesise the nucleic acids and proteins required the nucleic acids and proteins required to replicateto replicate a number of new viruses a number of new viruses (Fig. 10.44).(Fig. 10.44).

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•A great deal of information is A great deal of information is availableavailable concerning the details of concerning the details of the mechanism of virus replication the mechanism of virus replication but this text will onlybut this text will only outline the outline the main points. For greater detail the main points. For greater detail the reader is referred to specialist reader is referred to specialist texts ontexts on virology.virology.

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14.2. Classification14.2. Classification

• RNA-viruses can be broadly classified RNA-viruses can be broadly classified into two general types, namely: RNA-into two general types, namely: RNA-viruses andviruses and RNA-retroviruses.RNA-retroviruses.

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• Figure 10.44 A schematic representation of the replication of Figure 10.44 A schematic representation of the replication of RNA-virusesRNA-viruses

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RNA-virusesRNA-viruses

•RNA-virus replication usually RNA-virus replication usually occurs entirely in the occurs entirely in the cytoplasm. The viral mRNA cytoplasm. The viral mRNA eithereither forms part of the RNA forms part of the RNA carried by the virion or is carried by the virion or is synthesised by an enzyme synthesised by an enzyme already presentalready present in the virion. in the virion.

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•This viral mRNA is used to This viral mRNA is used to produce the necessary viral produce the necessary viral proteins by translation using proteins by translation using the host cell’s ribosomes and the host cell’s ribosomes and enzyme systems.enzyme systems.

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•Some of the viral proteins areSome of the viral proteins are enzymes that are used to catalyse enzymes that are used to catalyse the reproduction of more viral the reproduction of more viral mRNA. The new viralmRNA. The new viral RNA and RNA and viral proteins are assembled into viral proteins are assembled into a number of new virions that are a number of new virions that are ultimatelyultimately released from the host released from the host cell by either lysis or buddingcell by either lysis or budding..

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RetrovirusesRetroviruses• Retroviruses Retroviruses synthesise viral DNA synthesise viral DNA

using their viral RNA as a using their viral RNA as a templatetemplate. .

• This process isThis process is catalysed by enzyme catalysed by enzyme systems known as systems known as reverse reverse transcriptasestranscriptases that form part of the that form part of the virion. Thevirion. The viral DNA is incorporated viral DNA is incorporated into the host genome to form a so-into the host genome to form a so-called called provirus.provirus.

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•Transcription ofTranscription of the provirus the provirus produces new ‘genomic’ viral produces new ‘genomic’ viral RNA and viral mRNA. The viral RNA and viral mRNA. The viral mRNA is used tomRNA is used to produce viral produce viral proteins, which together with the proteins, which together with the ‘genomic’ viral RNA are ‘genomic’ viral RNA are assembled into newassembled into new virions. virions.

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•These virions are released by These virions are released by budding , which in many budding , which in many cases does not kill the host cases does not kill the host cell. Retroviruses are cell. Retroviruses are responsible for some forms of responsible for some forms of cancer and AIDScancer and AIDS

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DNA-virusesDNA-viruses

• Most DNA-viruses enter the host cell’s Most DNA-viruses enter the host cell’s nucleus where formation of viral mRNA nucleus where formation of viral mRNA byby transcription from the viral DNA is transcription from the viral DNA is brought about by the host cell’s brought about by the host cell’s polymerases. This viralpolymerases. This viral mRNA is used mRNA is used to produce viral proteins by translation to produce viral proteins by translation using the host cell’s ribosomes andusing the host cell’s ribosomes and enzyme systems.enzyme systems.

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• Some of these proteins will be Some of these proteins will be enzymes that can catalyse the enzymes that can catalyse the synthesis ofsynthesis of more viral DNA. more viral DNA.

• This DNA and the viral proteins This DNA and the viral proteins synthesised in the host cell are synthesised in the host cell are assembledassembled into a number of new into a number of new virions that are ultimately released virions that are ultimately released from the host by either cell lysisfrom the host by either cell lysis or or buddingbudding

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14.3. Viral diseases14.3. Viral diseases

• Viral infection of host cells is a common Viral infection of host cells is a common occurrence. Most of the time this occurrence. Most of the time this infection doesinfection does not result in illness as not result in illness as the body’s immune system can usually the body’s immune system can usually deal with such viral invasiondeal with such viral invasion..

• When illness occurs it is often short When illness occurs it is often short lived and leads to long-term immunity.lived and leads to long-term immunity.

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• However, aHowever, a number of viral infections can lead to serious number of viral infections can lead to serious medical conditions (. Somemedical conditions (. Some viruses like HIV, the aetiological viruses like HIV, the aetiological agent of AIDS, are able to remain dormant in the host foragent of AIDS, are able to remain dormant in the host for a a number of years before becoming active, whilst others such as number of years before becoming active, whilst others such as herpes zoster (shingles)herpes zoster (shingles) can give rise to recurrent bouts of the can give rise to recurrent bouts of the illness. Both chemotherapy and preventativeillness. Both chemotherapy and preventative

• vaccination are used to treat vaccination are used to treat patients. The latter is the main patients. The latter is the main clinical approach since it hasclinical approach since it has

• been difficult to design drugs that only target the virus. been difficult to design drugs that only target the virus. However, a number of antiviralHowever, a number of antiviral

• drugs have been developed and are in clinical use.drugs have been developed and are in clinical use.

• AIDSAIDS

• AIDSAIDS

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• Both chemotherapy and preventativeBoth chemotherapy and preventative vaccination are used to treat vaccination are used to treat patients. The latter is the main patients. The latter is the main clinical approach since it hasclinical approach since it has been been difficult to design drugs that only difficult to design drugs that only target the virus. However, a number target the virus. However, a number of antiviralof antiviral drugs have been drugs have been developed and are in clinical use.developed and are in clinical use.

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AIDSAIDS

• AIDS is a disease that progressively AIDS is a disease that progressively destroys the human immune system. destroys the human immune system. It is caused by theIt is caused by the human human immunodeficiency virus (HIV), which immunodeficiency virus (HIV), which is a retrovirus. This virus is a retrovirus. This virus enters and enters and destroysdestroys human T4 lymphocyte cellshuman T4 lymphocyte cells. . These cells are a vital part of the These cells are a vital part of the human immune system.human immune system.

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•Their destruction reduces the Their destruction reduces the body’s resistance to other body’s resistance to other infectious diseases, such asinfectious diseases, such as pneumonia, and some rare forms pneumonia, and some rare forms of cancer.of cancer.

•..

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• The entry of the virus into the body The entry of the virus into the body usually causes an initial period of usually causes an initial period of acute ill health with the patient acute ill health with the patient suffering from headaches, fevers and suffering from headaches, fevers and rashes, amongst other symptoms.rashes, amongst other symptoms.

• This is followed by a period of This is followed by a period of relatively good healthy where the relatively good healthy where the virus replicates in the lymph nodes.virus replicates in the lymph nodes.

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• This relatively healthy period This relatively healthy period normally lasts a number of years normally lasts a number of years before fullblownbefore fullblown

• AIDS appears. Full-blown AIDS is AIDS appears. Full-blown AIDS is characterised by a wide variety of characterised by a wide variety of diseases suchdiseases such as bacterial infections, as bacterial infections, neurological diseases and cancers. neurological diseases and cancers. Treatment is more effective whenTreatment is more effective when a a mixture of antiviral agents is usedmixture of antiviral agents is used

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14.4. Antiviral drugs14.4. Antiviral drugs

• It has been found that viruses utilise It has been found that viruses utilise a number of virus-specific enzymes a number of virus-specific enzymes during replication.during replication.

• These enzymes and the processes These enzymes and the processes they control are significantly different they control are significantly different from those of thefrom those of the host cell to make host cell to make them a useful target for medicinal them a useful target for medicinal chemists.chemists.

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•Consequently, antiviral drugsConsequently, antiviral drugs normally act by inhibiting viral normally act by inhibiting viral nucleic acid synthesis, inhibiting nucleic acid synthesis, inhibiting attachment to andattachment to and penetration of penetration of the host cell or inhibiting viral the host cell or inhibiting viral protein synthesis.protein synthesis.

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Nucleic acid synthesis Nucleic acid synthesis inhibitorsinhibitors

• Nucleic acid synthesis inhibitors usually Nucleic acid synthesis inhibitors usually act by inhibiting the polymerases or act by inhibiting the polymerases or reversereverse transcriptases required for transcriptases required for nucleic acid chain formation. However, nucleic acid chain formation. However, because they are usuallybecause they are usually analogues of analogues of the purine and pyrimidine bases found in the purine and pyrimidine bases found in the viral nucleic acids, they are oftenthe viral nucleic acids, they are often incorporated into the growing nucleic incorporated into the growing nucleic acid chain.acid chain.

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• In this case their general mode of In this case their general mode of actionaction frequently involves conversion frequently involves conversion to the corresponding 50-triphosphate to the corresponding 50-triphosphate by the host cell’sby the host cell’s cellular kinases. cellular kinases. This conversion may also involve This conversion may also involve specific viral enzymes in the initialspecific viral enzymes in the initial monophosphorylation step.monophosphorylation step.

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• These triphosphate drug derivatives are These triphosphate drug derivatives are incorporated into theincorporated into the nucleic acid chain nucleic acid chain where they terminate its formation. where they terminate its formation. Termination occurs because the drugTermination occurs because the drug residues do not have the 30-hydroxy group residues do not have the 30-hydroxy group necessary for the phosphate ester formationnecessary for the phosphate ester formation required for further growth of the nucleic acid required for further growth of the nucleic acid chain. This effectively inhibits thechain. This effectively inhibits the polymerases and ranscriptases that catalyse polymerases and ranscriptases that catalyse the growth of the nucleic acid (Fig. 10.45the growth of the nucleic acid (Fig. 10.45).).

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•This effectively inhibits the This effectively inhibits the polymerases and ranscriptases polymerases and ranscriptases that catalyse the growth of the that catalyse the growth of the nucleic acid (Fig. 10.45).nucleic acid (Fig. 10.45).

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AciclovirAciclovir• Aciclovir was the first effective Aciclovir was the first effective

antiviral drug. It is effective against a antiviral drug. It is effective against a number ofnumber of herpes viruses, notably herpes viruses, notably simplex, varicella-zoster (shingles), simplex, varicella-zoster (shingles), varicella (chickenpox) andvaricella (chickenpox) and Epstein–Epstein–Barr virus (glandular fever). It may Barr virus (glandular fever). It may be administered orally and by be administered orally and by intravenousintravenous injection as well as injection as well as topically. Orally administered doses topically. Orally administered doses have a low bioavailability.have a low bioavailability.

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• The action of aciclovir is more The action of aciclovir is more effective in virus-infected host cells effective in virus-infected host cells because the viralbecause the viral thymidine kinase is thymidine kinase is a more efficient catalyst for the a more efficient catalyst for the monophosphorylation of aciclovir monophosphorylation of aciclovir thanthan the thymidine kinases of the the thymidine kinases of the host cell.host cell.

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• This leads to an increase in the This leads to an increase in the concentration of theconcentration of the aciclovir aciclovir triphosphate, triphosphate, which has 100-fold which has 100-fold greater affinity for viral DNA greater affinity for viral DNA polymerase thanpolymerase than human DNA human DNA polymerase. polymerase.

• As a result, it preferentially As a result, it preferentially competitively inhibits viral DNAcompetitively inhibits viral DNA polymerase and so prevents the virus polymerase and so prevents the virus from replicating.from replicating.

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• However, resistance has beenHowever, resistance has been reported due to changes in the viral reported due to changes in the viral mRNA responsible for the production mRNA responsible for the production of the viralof the viral thymidine kinase. thymidine kinase. Aciclovir also acts by terminating Aciclovir also acts by terminating chain formation. chain formation. The aciclovir–DNAThe aciclovir–DNA complex complex formed by the drug also formed by the drug also irreversibly inhibits DNA polymerase.irreversibly inhibits DNA polymerase.

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VidarabineVidarabine

• Vidarabine is active against herpes Vidarabine is active against herpes simplex and herpes varicella-zoster.simplex and herpes varicella-zoster.

• However, the drug does give rise to However, the drug does give rise to nausea, vomiting, tremors, dizziness nausea, vomiting, tremors, dizziness and seizures. Inand seizures. In addition it has been addition it has been reported to be mutagenic, reported to be mutagenic, teratogenic and carcinogenic in teratogenic and carcinogenic in animal studiesanimal studies..

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• Vidarabine is administered by Vidarabine is administered by intravenous infusion and topical intravenous infusion and topical application. It has a half-lifeapplication. It has a half-life of about of about one hour, the drug being rapidly one hour, the drug being rapidly deaminated to arabinofuranosyl deaminated to arabinofuranosyl hypoxanthine (ara-HX) by adenosine hypoxanthine (ara-HX) by adenosine deaminase.deaminase.

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• This enzyme is found in the serum and red This enzyme is found in the serum and red blood cells. Ara-HX, which also exhibits a blood cells. Ara-HX, which also exhibits a weak antiviral action, has a half-life of weak antiviral action, has a half-life of about 3.5 hours.about 3.5 hours.

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Zidovudine (AZT)Zidovudine (AZT)• Zidovudine was originally Zidovudine was originally

synthesised in 1964 as an analogue synthesised in 1964 as an analogue ofof thymine by J. Horwitz as a thymine by J. Horwitz as a potential antileukaemia drug. It was potential antileukaemia drug. It was found to be unsuitable for usefound to be unsuitable for use in this in this role and for 20 years was ignored, role and for 20 years was ignored, even though in 1974even though in 1974 W. Osterag et W. Osterag et al. al. reported thatreported that it was active against it was active against Friend leukaemia virusFriend leukaemia virus, a retrovirus., a retrovirus.

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•However, the identification in However, the identification in 19831983 of the retrovirus HIVas the of the retrovirus HIVas the source of AIDS resulted in the source of AIDS resulted in the virologist M. St Clair setting up avirologist M. St Clair setting up a screening programme for drugs screening programme for drugs that could attack HIVthat could attack HIV

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• Fourteen compounds were selected Fourteen compounds were selected and screened against Friend and screened against Friend leukaemia virus and a second leukaemia virus and a second retrovirus called Harvey sarcoma retrovirus called Harvey sarcoma virus. virus. This screen led to the This screen led to the discovery of zidovudine (AZT), which discovery of zidovudine (AZT), which was rapidly developed into clinical was rapidly developed into clinical use on selected patients in 1986use on selected patients in 1986..

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•AZT is converted by the action of AZT is converted by the action of cellular thymidine kinase to the cellular thymidine kinase to the 50-triphosphate. This50-triphosphate. This inhibits the inhibits the enzyme reverse transcriptase in enzyme reverse transcriptase in the retrovirus, which effectively the retrovirus, which effectively prevents itprevents it from forming the viral from forming the viral DNA necessary for viral replicationDNA necessary for viral replication..

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• The incorporation of AZT intoThe incorporation of AZT into the the nucleic acid chain also results in nucleic acid chain also results in chain termination because the chain termination because the presence of the 30-azidepresence of the 30-azide group group prevents the reaction of the chain prevents the reaction of the chain with the 50-triphosphate of the next with the 50-triphosphate of the next nucleotidenucleotide waiting to join the chain waiting to join the chain (Fig. 10.45).(Fig. 10.45).

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•AZT is also active against AZT is also active against mammalian DNAmammalian DNA polymerase and polymerase and although its affinity for this although its affinity for this enzyme is about 100-fold less enzyme is about 100-fold less this action isthis action is thought to be the thought to be the cause of some of its unwanted cause of some of its unwanted side effects.side effects.

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• Zidovudine is active against the Zidovudine is active against the retroviruses (see section 10.14.2) retroviruses (see section 10.14.2) that cause AIDSthat cause AIDS (HIV virus) and (HIV virus) and certain types of leukaemia. certain types of leukaemia.

• It also inhibits cellular a-DNA It also inhibits cellular a-DNA polymerase butpolymerase but only at only at cconcentrations in excess of 100-fold oncentrations in excess of 100-fold greater than those needed to treat greater than those needed to treat the viralthe viral infection.infection.

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• The drug may be administered orally or The drug may be administered orally or by intravenous infusion. by intravenous infusion. TheThe bioavailability from oral administration bioavailability from oral administration is goodis good, the drug being distributed into , the drug being distributed into most bodymost body fluids and tissues. fluids and tissues.

• However, when used to treat AIDS it However, when used to treat AIDS it has given rise to gastrointestinalhas given rise to gastrointestinal disorders, skin rashes, insomnia, disorders, skin rashes, insomnia, anaemia, fever, headaches, depression anaemia, fever, headaches, depression and otherand other unwanted effects.unwanted effects.

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ResistanceResistance

•Resistance increases with time. Resistance increases with time. This is known to be due to This is known to be due to the the virusvirus developing mutations’developing mutations’ which result in changes in the which result in changes in the amino acid sequences in the amino acid sequences in the reversereverse transcriptase.transcriptase.

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DidanosineDidanosine

• Didanosine is used to treat some Didanosine is used to treat some AZT-resistant strains of HIV. It is also AZT-resistant strains of HIV. It is also used inused in combination with AZT to combination with AZT to treat HIV. Didanosine is administered treat HIV. Didanosine is administered orally in dosage forms thatorally in dosage forms that contain contain antacid buffers to prevent conversion antacid buffers to prevent conversion by the stomach acids to by the stomach acids to hypoxanthinehypoxanthine

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• However, in spite of the use of However, in spite of the use of buffers the bioavailability from oral buffers the bioavailability from oral administration isadministration is low. low.

• The drug can cause nausea, The drug can cause nausea, abdominal pain and peripheral abdominal pain and peripheral neuropathy, amongst otherneuropathy, amongst other symptoms. symptoms. Drug resistance occurs Drug resistance occurs after prolonged use.after prolonged use.

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• Didanosine is converted by viral and Didanosine is converted by viral and cellular kinases to the monophosphate cellular kinases to the monophosphate and then toand then to the triphosphate. In this the triphosphate. In this form it inhibits reverse transcriptase form it inhibits reverse transcriptase and in addition itsand in addition its incorporation into incorporation into the DNA chain terminates the chain the DNA chain terminates the chain because the drug has no 30-hydroxybecause the drug has no 30-hydroxy group (Fig. 10.45).group (Fig. 10.45).

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Host cell penetration inhibitorsHost cell penetration inhibitors

• The principal drugs that act in this The principal drugs that act in this manner are amantadine and manner are amantadine and rimantadine (Fig. 10.46).rimantadine (Fig. 10.46).

• Both amantadine and rimantadine Both amantadine and rimantadine are also used to treat Parkinson’s are also used to treat Parkinson’s disease. However, theirdisease. However, their mode of mode of action in this disease is different from action in this disease is different from their action as antiviral agents.their action as antiviral agents.

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Amantadine Amantadine hydrochloridehydrochloride

•Amantadine hydrochloride is Amantadine hydrochloride is effective against influenza Aeffective against influenza A virus virus but is not effective against the but is not effective against the influenza B virus. When used as a influenza B virus. When used as a prophylactic, it isprophylactic, it is believed to give believed to give up to 80 per cent protection up to 80 per cent protection against influenza A virus infectionsagainst influenza A virus infections

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prof. azaprof. aza

•The drugThe drug acts by blocking an ion acts by blocking an ion channel in the virus membrane channel in the virus membrane formed by the viral proteinM2. formed by the viral proteinM2. This isThis is believed to inhibit the believed to inhibit the disassembly of the core of the disassembly of the core of the virion and its penetration of the virion and its penetration of the host (seehost (see section 10.14.1).section 10.14.1).

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• Amantadine hydrochloride has a good Amantadine hydrochloride has a good bioavailability on oral administration, bioavailability on oral administration, beingbeing readily absorbed and distributed readily absorbed and distributed to most body fluids and tissues. to most body fluids and tissues.

• Its elimination time isIts elimination time is 12–18 hours. 12–18 hours. However, its use can result in However, its use can result in depression, dizziness, insomnia anddepression, dizziness, insomnia and gastrointestinal disturbances, amongst gastrointestinal disturbances, amongst other unwanted side effects.other unwanted side effects.

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Rimantadine Rimantadine hydrochloridehydrochloride

•Rimantadine hydrochloride is an Rimantadine hydrochloride is an analogue of amantadineanalogue of amantadine hydrochloride. It is more hydrochloride. It is more effective against influenza A effective against influenza A virus than amantadine. Its mode virus than amantadine. Its mode ofof action is probably similar to action is probably similar to that of amantadine.that of amantadine.

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•The drug is readily absorbed whenThe drug is readily absorbed when administered orally but undergoes administered orally but undergoes extensive first-pass metabolism. extensive first-pass metabolism. However, in spite ofHowever, in spite of this, its this, its elimination half-life is double that elimination half-life is double that of amantadine. Furthermore, CNS of amantadine. Furthermore, CNS side effects areside effects are significantly significantly reduced.reduced.

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Inhibitors of viral protein Inhibitors of viral protein synthesissynthesis

• The principal compounds that act as The principal compounds that act as inhibitors of protein synthesis are the inhibitors of protein synthesis are the interferons.interferons.

• These compounds are members of a These compounds are members of a naturally occurring family of naturally occurring family of glycoprotein hormonesglycoprotein hormones (RMM 20 (RMM 20 000–160 000), which are produced 000–160 000), which are produced by nearly all types of eukaryotic cell.by nearly all types of eukaryotic cell.

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• Three general classes of interferons Three general classes of interferons are known are known to occur naturally in to occur naturally in mammals, namelymammals, namely:: the the αα-interferons -interferons produced by leucocytes, produced by leucocytes, ββ-interferons -interferons produced by fibroblasts andproduced by fibroblasts and γ-γ-interferons interferons produced by T produced by T lymphocyteslymphocytes. At least twenty . At least twenty αα-, two -, two ββ- - and two and two γγ-interferons-interferons have been have been identifiedidentified

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• Interferons form part of the human Interferons form part of the human immune system. It is believed that the immune system. It is believed that the presence ofpresence of virions, bacteria and other virions, bacteria and other antigens in the body switches on the antigens in the body switches on the mRNA that controls themRNA that controls the production production and release of interferon. This release and release of interferon. This release stimulates other cells to produce andstimulates other cells to produce and

• release more interferon.release more interferon.

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• Interferons are thought to act by Interferons are thought to act by initiating the production in theinitiating the production in the cell of proteins that protect the cell of proteins that protect the cells from viral attack. The main cells from viral attack. The main action of these proteinsaction of these proteins takes takes the form of inhibiting the the form of inhibiting the synthesis of viral mRNA and viral synthesis of viral mRNA and viral protein synthesis. protein synthesis.

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•a- Interferons also enhance a- Interferons also enhance the activity of killer T cells the activity of killer T cells associated with the immune associated with the immune system. (see section 14.5.5).system. (see section 14.5.5).

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•The main action of these proteins The main action of these proteins takes the form of takes the form of inhibiting the inhibiting the synthesis of viral mRNA and viral synthesis of viral mRNA and viral protein synthesisprotein synthesis. .

•αα- Interferons also - Interferons also enhance the enhance the activity of killer T cells activity of killer T cells associated associated with the immune system.with the immune system.

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•A number of a-interferons have A number of a-interferons have been manufactured andbeen manufactured and proven proven to be reasonably effective to be reasonably effective against a number of viruses and against a number of viruses and cancers. cancers.

• Interferons areInterferons are usually given by usually given by intravenous, intramuscular or intravenous, intramuscular or subcutaneous injection.subcutaneous injection.

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• However, theirHowever, their administration can administration can cause adverse effects, such as cause adverse effects, such as headaches, fevers and bone marrowheadaches, fevers and bone marrow depression, that are dose related.depression, that are dose related.

• The formation and release of The formation and release of interferon by viral and other interferon by viral and other pathological stimulation haspathological stimulation has resulted resulted in a search for chemical inducers of in a search for chemical inducers of endogenous interferon.endogenous interferon.

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•Administration of aAdministration of a wide range of wide range of compounds has resulted in the compounds has resulted in the induction of interferon production. induction of interferon production. However,However, no clinically useful no clinically useful compounds have been found for compounds have been found for humans’ although tilorone is humans’ although tilorone is effectiveeffective in inducing interferon in inducing interferon in in mice.mice.

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prof. azaprof. aza

10.15. 10.15. Recombinant DNA (Genetic Recombinant DNA (Genetic Engineering)Engineering)

• The body requires a constant supply of The body requires a constant supply of certain peptides and proteins if it is to certain peptides and proteins if it is to remain remain healthhealth and function normally. and function normally. Many oMany off these pepti these peptiddes and proteins es and proteins are only produced in are only produced in aa small small quantities. quantities. They will They will bbe produced only e produced only if the correct genes are present in the if the correct genes are present in the cell. cell. CCononsequentlysequently, , if a gene is missing if a gene is missing or defective an essential protein willor defective an essential protein will

• not be produced, not be produced, which can lead to a which can lead to a diseased statediseased state..

•Consequently, if a gene is Consequently, if a gene is missing or defective an missing or defective an essential protein willessential protein will not be not be produced, produced, which can lead to a which can lead to a diseased state.diseased state.

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prof. azaprof. aza

•For example, cystic fibrosis is For example, cystic fibrosis is causedcaused by a defective gene. by a defective gene. This This faulty gene produces a defective faulty gene produces a defective membrane protein, membrane protein, cysticcystic fibrosis fibrosis transmembrane regulator transmembrane regulator (CFTR), which will not allow the (CFTR), which will not allow the free passage of chloridefree passage of chloride ions ions through the membranethrough the membrane

• The passage of chloride ions through The passage of chloride ions through a normal membrane intoa normal membrane into the lungs is the lungs is usually accompanied by a flow of usually accompanied by a flow of water molecules in the same water molecules in the same directiondirection. .

• InIn membranes that contain CFTR the membranes that contain CFTR the transport of water through the transport of water through the membrane into the lungsmembrane into the lungs is reduced.is reduced.

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•This viscous mucus clogs the This viscous mucus clogs the lungslungs and and makes breathing makes breathing difficultdifficult, a classic symptom of , a classic symptom of cystic fibrosis. It also provides cystic fibrosis. It also provides aa breeding ground for bacteria that breeding ground for bacteria that cause pneumonia cause pneumonia and other and other illnesses.illnesses.

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• Several thousand Several thousand hereditary diseases hereditary diseases found in humans are known to be found in humans are known to be caused by faultycaused by faulty genes. Recombinant genes. Recombinant DNA (rDNA) technology (genetic DNA (rDNA) technology (genetic engineering) offers a new way ofengineering) offers a new way of combating these hereditary diseases combating these hereditary diseases by either replacing the faulty genes or by either replacing the faulty genes or producing theproducing the missing peptides and missing peptides and proteinsproteins so that they can be given as a so that they can be given as a medicine (see section 10.15.2).medicine (see section 10.15.2).

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•The first step in any use of The first step in any use of recombinant DNA technology is to recombinant DNA technology is to isolate or copy theisolate or copy the required gene. required gene. There are three sources of the There are three sources of the genes required for cloning. The genes required for cloning. The two mosttwo most important are genomic important are genomic and copy or and copy or complementary DNA complementary DNA (cDNA) libraries.(cDNA) libraries.

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• In the first caseIn the first case the library the library consists of DNA fragments consists of DNA fragments obtained from a cell’s genome, obtained from a cell’s genome, whilst in the secondwhilst in the second case the case the library consists of DNA fragments library consists of DNA fragments synthesised by using the mRNA synthesised by using the mRNA for the proteinfor the protein of interestof interest..

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•The third is by the automated The third is by the automated synthesis of DNA, which is only synthesis of DNA, which is only feasible if thefeasible if the required base required base sequence is known. This may be sequence is known. This may be deduced from the amino acid deduced from the amino acid sequence ofsequence of the required protein the required protein if it is known.if it is known.

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•Once the gene has been obtained Once the gene has been obtained it is inserted into ait is inserted into a carrier (vector) carrier (vector) that can enter a host cell and be that can enter a host cell and be replicated, propagated and replicated, propagated and transcripted intotranscripted into mRNA by the mRNA by the cellular biochemistry of that cell. cellular biochemistry of that cell. This process is often referred to This process is often referred to as geneas gene cloning.cloning.

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• The mRNA produced by the cloned The mRNA produced by the cloned DNA is used by the cell ribosomes to DNA is used by the cell ribosomes to produce the protein encoded by the produce the protein encoded by the cloned DNA. In theory, cloned DNA. In theory, gene cloninggene cloning makes it possible to produce any makes it possible to produce any protein provided that it is possible to protein provided that it is possible to obtain a copy of theobtain a copy of the corresponding corresponding genegene. Products produced using . Products produced using recombinant DNA usually have recombinant DNA usually have recombinant,recombinant, r or rDNA in their r or rDNA in their names.names. prof. azaprof. aza

15.1. Gene cloning15.1. Gene cloning

•Bacteria are frequently used as host Bacteria are frequently used as host cells for gene cloning. This is cells for gene cloning. This is because they normally usebecause they normally use the the same genetic code as humans to same genetic code as humans to make peptides and proteins. make peptides and proteins. However, in bacteria theHowever, in bacteria the mechanism mechanism for peptide and protein formation is for peptide and protein formation is somewhat different.somewhat different.

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• It is not restricted toIt is not restricted to the the chromosomes but can also occur chromosomes but can also occur in extranuclear particles called in extranuclear particles called plasmids. Plasmids areplasmids. Plasmids are large large circular supercoiled DNA circular supercoiled DNA molecules whose structure molecules whose structure contains at least one gene and acontains at least one gene and a start site for replication.start site for replication.

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•However, the number of genes However, the number of genes found in a plasmid is fairly found in a plasmid is fairly limited,limited, although bacteria will although bacteria will contain a number of identical contain a number of identical copies of the same plasmid.copies of the same plasmid.

• It is possible to isolate the It is possible to isolate the plasmids of bacterial cells.plasmids of bacterial cells.

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•The isolated DNA molecules can The isolated DNA molecules can bebroken open by cleaving the bebroken open by cleaving the phosphate bonds between phosphate bonds between specific pairs of bases by the specific pairs of bases by the actionaction enzymes known as enzymes known as restriction enzymes or restriction enzymes or endonucleasesendonucleases

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•Each of these enzymes, of whichEach of these enzymes, of which over 500 are known, will only over 500 are known, will only cleave the bonds between specific cleave the bonds between specific nucleosides. For example,nucleosides. For example, EcoR I EcoR I cleaves the phosphate link between cleaves the phosphate link between guanosine and adenosine whilst guanosine and adenosine whilst Xho I cuts theXho I cuts the chain between chain between cytidine and thymine nucleosides.cytidine and thymine nucleosides.

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•Cutting the strand can result in Cutting the strand can result in either blunteither blunt ends, where the ends, where the endonuclease cuts across both endonuclease cuts across both chains of the DNA at the same chains of the DNA at the same points, orpoints, or cohesive ends (sticky cohesive ends (sticky ends), where the cut is ends), where the cut is staggered from one chain to the staggered from one chain to the otherother (Fig. 10.47).(Fig. 10.47).

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•The new non-cyclic structure of The new non-cyclic structure of the plasmid is known as the plasmid is known as linearised DNA inlinearised DNA in order to order to distinguish it from the new insert distinguish it from the new insert or foreign DNA. or foreign DNA.

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• This foreign DNA must contain the This foreign DNA must contain the required gene, a second gene system required gene, a second gene system that confers resistance to a specific that confers resistance to a specific antibiotic and any other necessary antibiotic and any other necessary information. It should be remembered information. It should be remembered that a eukaryotic gene is made upthat a eukaryotic gene is made up of of exons separated by introns, which are exons separated by introns, which are sequences that have no apparent use.sequences that have no apparent use.

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• Figure 10.47 (a) Blunt and (b) cohesive Figure 10.47 (a) Blunt and (b) cohesive cuts with compatible adhesive cutscuts with compatible adhesive cuts

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• Mixing the foreign DNA and the Mixing the foreign DNA and the linearised DNA in a suitable medium linearised DNA in a suitable medium results in theresults in the formation of extended formation of extended plasmid loops when their ends come plasmid loops when their ends come into contact (Fig. 10.48). Thisinto contact (Fig. 10.48). This contact contact is converted into a permanent bond is converted into a permanent bond by the catalytic action of an enzyme by the catalytic action of an enzyme calledcalled DNA ligase.DNA ligase.

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• Figure Figure 10.48. A 10.48. A represenrepresentation of tation of the main the main steps in steps in the the insertion insertion of a gene of a gene into a into a plasmidplasmid

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•ThisThis contact is converted into a contact is converted into a permanent bond by the catalytic permanent bond by the catalytic action of an enzyme calledaction of an enzyme called DNA DNA ligase. When the chains are ligase. When the chains are cohesive the exposed single cohesive the exposed single chains of new DNA mustchains of new DNA must contain contain a complementary base sequence a complementary base sequence to the exposed ends of the to the exposed ends of the linearised DNAlinearised DNA..

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• TheThe hydrogen bonding between hydrogen bonding between these complementary base pairs these complementary base pairs tends to bind the chainstends to bind the chains together together prior to the action of the DNA ligase, prior to the action of the DNA ligase, hence the name ‘‘sticky ends’’. The hence the name ‘‘sticky ends’’. The newnew DNA of the modified plasmid is DNA of the modified plasmid is known as recombinant DNA (rDNA).known as recombinant DNA (rDNA).

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• However, the randomHowever, the random nature of the nature of the techniques used to form the modified techniques used to form the modified plasmids means that some of theplasmids means that some of the linearised DNA reforms the plasmid linearised DNA reforms the plasmid without incorporating the foreign without incorporating the foreign DNA, that is, aDNA, that is, a mixture of both types mixture of both types of plasmid is formed.of plasmid is formed.

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•The modified plasmids are The modified plasmids are separated from theseparated from the unmodified unmodified plasmids when they are plasmids when they are reinserted into a bacterial cell.reinserted into a bacterial cell.

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• The new plasmids are reinserted into The new plasmids are reinserted into the bacteria by a process known as the bacteria by a process known as transformation.transformation.

• Bacteria are mixed with the new Bacteria are mixed with the new plasmids in a medium containing plasmids in a medium containing calcium chloride. Thiscalcium chloride. This medium makes medium makes the bacterial membrane permeable the bacterial membrane permeable to the plasmidto the plasmid..

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•However, not all bacteriaHowever, not all bacteria will will take up the modified plasmids. take up the modified plasmids. Such bacteria can easily be Such bacteria can easily be destroyed by specific antibioticdestroyed by specific antibiotic action since they do not contain action since they do not contain plasmids with the appropriate plasmids with the appropriate protecting gene.protecting gene.

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•This makesThis makes isolation of the isolation of the bacteria with the modified bacteria with the modified plasmids relatively simple. plasmids relatively simple.

•These modified bacteriaThese modified bacteria are are allowed to replicate and, in doing allowed to replicate and, in doing so, produce many copies of the so, produce many copies of the modified plasmid.modified plasmid.

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•UnderUnder favourable conditions one favourable conditions one modified bacterial cell can produce modified bacterial cell can produce over 200 copies of the newover 200 copies of the new plasmid. The gene in these plasmid. The gene in these modified plasmids will use the modified plasmids will use the bacteria’s internal machinery tobacteria’s internal machinery to automatically produce the automatically produce the appropriate peptide or protein.appropriate peptide or protein.

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•Since many bacteria replicate at Since many bacteria replicate at aa very rapid rate this technique very rapid rate this technique offers a relatively quick way of offers a relatively quick way of producing large quantities ofproducing large quantities of essential naturally occurring essential naturally occurring compounds that cannot be compounds that cannot be produced by other means.produced by other means.

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•Plasmids are not the only vectors Plasmids are not the only vectors that can be used to transport that can be used to transport DNA into a bacterial hostDNA into a bacterial host cell. cell.

•Foreign DNA can also be inserted Foreign DNA can also be inserted into bacteriophages and cosmids into bacteriophages and cosmids by similarby similar techniques.techniques.

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• Bacteriophages (phage) are viruses Bacteriophages (phage) are viruses that specifically infect bacteria whilst that specifically infect bacteria whilst aa cosmid is a hybrid between a cosmid is a hybrid between a phage and a plasmid that has been phage and a plasmid that has been especially synthesised forespecially synthesised for use in use in gene cloning. Plasmids can be used gene cloning. Plasmids can be used to insert fragments containing up to to insert fragments containing up to 10 kilobasepairs10 kilobasepairs (kbp), phages up to (kbp), phages up to 20 kbp and cosmids 50 or more kbp.20 kbp and cosmids 50 or more kbp.

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• It is not always necessary to use a It is not always necessary to use a vector to place the recombinant vector to place the recombinant DNA in a cell. If theDNA in a cell. If the cell is large cell is large enough, the recombinant DNA enough, the recombinant DNA may be placed in the cell by using may be placed in the cell by using aa micropipette whose overall tip micropipette whose overall tip diameter is less than 1 mm.diameter is less than 1 mm.

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•Only a small amount of theOnly a small amount of the recombinant DNA inserted in this recombinant DNA inserted in this fashion is taken up by the cell’s fashion is taken up by the cell’s chromosomes. However,chromosomes. However, this this small fraction will increase to a small fraction will increase to a significant level as the cell significant level as the cell replicates (Fig. 10.48).replicates (Fig. 10.48).

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• Host cells for all methods of cloning are Host cells for all methods of cloning are usually either bacterial or mammalian usually either bacterial or mammalian in origin. For example, bacterial cells in origin. For example, bacterial cells often used are E. coli and eukaryotic often used are E. coli and eukaryotic yeast while mammalian cell lines yeast while mammalian cell lines include Chinese hamster ovary (CHO), include Chinese hamster ovary (CHO), baby hamster kidney (BHK) and African baby hamster kidney (BHK) and African green monkey kidney (VERO).green monkey kidney (VERO).

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• In all cases small-scale cultures In all cases small-scale cultures of the host cell plus vectorof the host cell plus vector are are grown to find the culture grown to find the culture containing the host with the containing the host with the required gene that gives the bestrequired gene that gives the best yield of the desired protein.yield of the desired protein.

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•Once this culture has been Once this culture has been determined the process is scaled determined the process is scaled up via a suitable pilot plant to up via a suitable pilot plant to production level (see section production level (see section 16.6). The mammalian cell line 16.6). The mammalian cell line cultures normally give poorer cultures normally give poorer yields of the desired protein.yields of the desired protein.

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prof. azaprof. aza

15.2.2 Manufacture of Pharmaceuticals15.2.2 Manufacture of Pharmaceuticals

• The bodyThe body produces peptides and produces peptides and proteproteinsins, often in extremely small , often in extremely small quantitiesquantities, which , which are essential for its are essential for its well being. The absence of the well being. The absence of the necessary’ genes means that the body necessary’ genes means that the body does not produce these essential does not produce these essential compounds, resulting in a deficiency compounds, resulting in a deficiency disease that is usually’ fatal. disease that is usually’ fatal.

• Treatment by supplying the patient Treatment by supplying the patient with sufficient amounts of twith sufficient amounts of thehe missing missing compounds is normally successful. compounds is normally successful.

•However, extraction from other However, extraction from other natural sources is usually’ natural sources is usually’ difficult and yields are often low. difficult and yields are often low. For exampleFor example,, it takes half a it takes half a million sheep brains to produce million sheep brains to produce 5mg of somatostatin5mg of somatostatin a growth a growth horhormmone that inhibits secretioone that inhibits secretionn of the pituitary growof the pituitary growthth hormone hormone. .

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•FurtFurthhermore, unless termore, unless thehe source source of the required product is of the required product is donated blood there is a limit to donated blood there is a limit to the number of cadavers available the number of cadavers available for the extraction of compoufor the extraction of compounnds ds suitable for use in husuitable for use in hummans. ans.

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• Moreover, there is also the danger Moreover, there is also the danger that compounds obtained from that compounds obtained from human sources human sources maymay bbe contaminated e contaminated by’ viruses such as HIVby’ viruses such as HIV,, hepatitis hepatitis, , Creutzfeld–Jakob disease (mad cowCreutzfeld–Jakob disease (mad cow disease)disease) and others tand others thhat are difficult at are difficult to detect. Animal sources have been to detect. Animal sources have been used but only a few human protein used but only a few human protein deficiency disorders deficiency disorders cancan bbe treated e treated withwith animal proteins. animal proteins.

prof. azaprof. aza

prof. azaprof. aza

• Gene cloGene clonniinng is used to obtain human g is used to obtain human recombirecombinnant proteins. Howeverant proteins. However,, some some proteins will also need post—proteins will also need post—translational motranslational modification dification such as such as glycosylation and/or tglycosylation and/or the modificationhe modification of aof ammino acid sequences. These ino acid sequences. These modifications may require formodifications may require formming ing different section, of the peptide chain different section, of the peptide chain in tin thhe culture e culture mmedium and cedium and chemihemically’ cally’ combining these sectiocombining these sections in vitrons in vitro. .

• The genes required for these The genes required for these processes are synthesised usiprocesses are synthesised usinng the g the required prequired peeptide aptide ass a blueprint. For a blueprint. For example, human recombinant example, human recombinant insulinsulineine may he produced in t may he produced in thihis s manner (Figure 10.12). The genes for manner (Figure 10.12). The genes for the A and B chaithe A and B chainns of insulin were s of insulin were syntsynthhesised separately. esised separately.

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• TThhey were cloned separately, using ey were cloned separately, using suitable plasmids. into two different suitable plasmids. into two different bacterial strains. One of these strains bacterial strains. One of these strains is is usedused to produce t to produce thehe A chain A chain whilst whilst the the othersothers is used to produce the B is used to produce the B strain. strain. TheThe chains are isolated and chains are isolated and attached to each other byattached to each other by in vitro in vitro didisulphide bond formation. sulphide bond formation.

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•This last step is inefficient and This last step is inefficient and human recombinanthuman recombinant insulin is insulin is now made by forming now made by forming recombinant proinsulin by gene recombinant proinsulin by gene cloning. The proinsulin iscloning. The proinsulin is converted to recombinant insulin converted to recombinant insulin by proteolytic cleavageby proteolytic cleavage

prof. azaprof. aza

prof. azaprof. aza

Figure 10.42. An outline of the synthesis of recombinant human insulin.