Nucleic acids

Structure of DNA As with protein DNA has a primary , secondary and tertiary structure

The primary structure of DNA

The primary structure of DNA is the way in which the DNA building blocks are linked together. Whereas protein have over 20building blocks from which to choose , DNA has only four the nucleosides deoxyadenosine , deoxyguanosine , deoxycytidine and deoxythymidine.(Fig7.1)

Each nucleoside is constructed from two components a deoxyribose sugar and a base . The sugar is the same in all four nucleoside and only the base is different . The four possible bases are two bicyclic purines and only the base is different . The four possible bases are two bicyclic purines ( adenine and guanine), and two smaller pyrimidine structures ( cytosine and thymine) (Fig 7.2 )

 Fig 7.1

 Fig 7.2

 The nucleoside building blocks are joined together through phosphate groups which link the 5-hydroxy group of one nucleoside unit to the 3`- hydrogen group of the next (Fig7.3)

 With only four types of building blocks available , the primary structure of DNA is far less varied than the primary structure of protein . As a result , It was long thought that DNA only had a minor role to play in cell biochemistry , since it was hard to see how such an apparently simple molecule could have anything to do with the mysteries of the genetic code . The solution to this mystery lies in the secondary structure of DNA.

The secondary structure of DNA

Watson and Crick solved the secondary structure of DNA by building a model that fitted all the known experimental results. The structure consists of two DNA chains arranged together in a double helix of constant diameter ( Fig 7.4).The double helix can be seen to have a major groove and minor groove which are of some important to the action of several antibacterial agents.

The structure relies crucially on the pairing up of nucleic acid bases between the two chains . Adenine pairs only with thymine via two hydrogen bonds, whereas guanine pairs only with cytosine via there hydrogen bonds. Thus a bicyclic purine base is always linked with a smaller monocyclic pyrimidine base to allow the constant diameter of the double helix. The double helix is further stabilized by the fact that the base pairs are stacked one on top of each other , allowing hydrophilic interactions between the heterocyclic rings .The polar sugar-phosphate backbone is placed to the out side of the structure and therefore can form favourable polar interactions with water .

The fact that adenine always binds to thymine , and cytosine always bind to guanine means that the chain are complementary with each other . In other words , one chain can be visualized as a negative image of its partner . It is now possible to see how replication is feasible . If the double helix unravels , then a new chain can be constructed on each of the original chain (Fig7.5) . In other words , each of the original chain can act as a template for the construction of a new and identical double helix.

The tertiary structure of DNA The tertiary structure of DNA is often neglected or ignored , but it is important to the action of quinoline group of antibacterial agent. The double helix is able to coil into a 3D shape and this is known as supercoiling . During replication , the double strand of DNA must unravel but, because of the tertiary supercoiling this leads to a high level of strain which has to be relived by the temporary cutting , then repair ,of the DNA chain . These procedures are enzyme-catalyzed and these are the enzymes which are inhibited by the quinolone antibacterical agents.

Drug acting on DNA In general we can classify the drugs which act on DNA into three groups: 1.intercalating cytostatic agents 2.alkylating agents 3.chain cutters

Intercalating agents :
Intercalating drugs are compounds which are capable of slipping between the layers of nucleic acids bases and their pairs and disrupting the shape of the double helix . This disruption prevents replication and transcription . One example is the antibacterial agent proflavine.

7.6

Drugs which work in this way must be flat in order to fit between the base pairs , and must therefore be aromatic or heteroaromatic in nature. Some drugs prefer to approach the helix via the major groove , whereas others prefer access via the minor groove . Several antibiotics , such as the antitumour agents actinomycin D and adriamycin (Fig7.7) operate by intercalating DNA .

Acitomycin D contains pentapeptide , but the important feature is the flat trycyclic , heteroaromatic structure which is able to slide into the double helix . It appears to favour in interaction with guanine-cytosine base pairs and in particular ,between two adjacent guanin bases on alternate strands of helix . Actinomycin D is further held in position by hydrogen bond interaction between the nucleic acid bases of DNA and the cyclic pentapeptides positioned on the out side of the helix .

Adriamycin has a tetracyclic system where three of the to fit into the double helix . The amino group attached to the sugar is important in helping to lock the antibiotic into place since it can ionize and form an ionic bond with the negatively charged phosphate group on the DNA backbone. The highly effective antimalarial agent chloroquine a drug developed from quinine can attack the malarial parasite by blocking DNA transcription as part of its action . Once again a flat heteroaromatic structure is present which can intercalate DNA.

Aminoacridine agents such as proflavine are tropical antibacterial agents which were used in the second world war to treat surface wounds . The best agents are completely ionized at pH 7 and they interact with DNA in the same way as adriamycin . The flat tricyclic ring intercalates between the DNA base pairs and interacts by van der waals forces , while the amino cations from ionic bonds with the negatively charged phosphate groups on the sugar phosphate backbone .

Alkylating agents :
Alkylating agents are highly electrophilic compound which will react with nucleophiles to form covalent bonds . There are several nucleophils group in DNA and in particular the 7-nitrogen of guanine . Drugs with two alkylating groups could therefore react with a guanine on each chain and cross-link the strands such that they cannot unravel during replication and transcription .

Alternatively , the drug could link two guanine groups on the same chain such that the drug us attached like a limpet to the side of the DNA helix . Such an attachment would mask that portion of DNA and block access to the necessary enzyme required for DNA function . Miscoding due to alkylated guanine unit is also possible . The guanine base usually exists as the keto tautomer and base pairs with cytosine . Once alkylated , however , guanine prefers the enol tautomer and is more likely to base pair with thymine. Such miscoding ultimately leads to an alteration in the amino acid sequence of proteins , which in turn leads to disruption of protein structure and function .

Since alkylating agents are very reactive , they will react with any good nucleophile and so are not very selective in their action. They will alkylate protein and others macromolecules as well as DNA . Neverthless , alkylating drugs have been useful cells and so disruption of DNA function will affect these cells more drastically than normal cells. The nitrogen mustered compound mechlorethamine was the first alkylating agent to be used .The nitrogen atom is able to displace a chloride ion intramolecularly to form the highly electrophilic aziridine ion. Alkylation of DNA take place. Since the process can be repeated , cross-linking between chains will occurs.

The side reaction mentioned above can be reduced by lowering the reactivity of the alkylating agent . For example , putting an aromatic ring on the nitrogen atom instead of a methyl group has such an effect . The lone pair of the nitrogen is pulled into the ring and is less available to displace the chloride ion. As a result the intermediate aziridine ion is less easily formed and only strong nucleophiles such as guanine will now react with it .

Another approach which has been used to direct these alkylating agents more specially to DNA has been to attach a nucleic acid binding block onto the molecule . For example , uracil mustard contains one of the nucleic acid bases. This drug has been used successfully in the treatment of chronic lymphatic leukaemia and has a certain selectivity for the tumour cells over normal cells . This is because tumour cells generally divide faster than normal cells . As a result , nucleic acid synthesis is faster and tumour are hungrier for nucleic acid binding blocks . The tumour cells therefore take more than their share of the binding blocks. The tumour cells therefore take more than their share of binding blocks . Unfortunately , this approach has not so far succeeded in achieving the high levels of selectivity desired for effective eradication of tumour cells.

Cisplatin is a very useful antitumour agent for the treatment of testicular and ovarian tumours. Its discovery was fortuitous in the extreme , arising from research carried out to investigate the effect of an electric current on bacterial growth . During these experiment , it was discovered that bacterial cell division was inhibited .Further research led to the discovery that an electrolysis product from the platinum electrodes was responsible for the inhibitor and the agent was eventually identified as cis diammonia dichloroplatinum(II), known as cisplatin .

Drugs acting by chain cutting: Bleomycin is a large glycoprotein which appears to be able to cut the strands of DNA and then prevent the enzyme DNA ligase from repairing the damage. It seems to act by abstracting hydrogen atoms from DNA . The resultant radicals react with oxygen to form peroxy species which then fragment . The drug is useful against certain type of skin cancer .

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Ribonuleic acid : The primary structure of RNA is the same as DNA, with two exception . Ribose is the sugar component rather than deoxyribose , while uracil replaces thymine as one of the bases . Base pairing between nucleic acid bases can occur in RNA , with adenine pairing to uracil and cytosine pairing to guanine . However , the pairing is between bases within the same chain, and it does not a double helix, but it does have regions of helical secondary structure .

Since the secondary structure is not uniform along the length of the RNA chain more variety is allowed in RNA tertiary structure . Three types of RNA molecules have been identified with different cell function . The three type of RNA are messenger RNA (mRNA) , transfer RNA (tRNA) and ribose RNA (rRNA) Messenger RNA is responsible for relaying the code for one particular protein from the DNA genetic bank to the protein production site . The segment of DNA required is copied by a process called transcription . The DNA double helix unravels and the stretch which is exposed act as template on which the mRNA can be built. Once complete, the mRNA leaves to seek out rRNA , while the DNA reforms the double helix.

Ribosomal RNA can be looked upon as the production site for protein synthesis . It binds to one end of the mRNA molecule , then travels along it to the other end, reading the code and constructing the protein molecule one amino acid at a time as it moves along . There are two segment to the rRNA ,known as the 50s and 30s subunits.

Transfer RNA is the crucial adaptor unit which link the triplet code on mRNA to a specific amino acid . Therefore , there has to be a different tRNA for each amino acid for each amino acids . All the tRNA are clover-leaf in shape with two different binding regions at opposite ends of the molecule . One binding region is for a specific amino acid where the amino acid is covalently linked to the terminal adenosyl residue. The other is a set of three nucleic acid bases which will base pair with a complementary triplet on the mRNA molecule.

As rRNA travels along mRNA , it revels the triplet codes on mRNA . AS a triplet code is revealed , a tRNA with the complementary GTA triplet will bind to it and bring the specific amino acid coded by that triplet . The growing peptide chain will then be grafted on to that amino acid . The rRNA will shift along thee chain to reveal the next triplet and so the process continues until the whole strand is read . The new protein is then released from rRNA , which is now available to start the process.

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Drug acting on RNA :

Several antibiotic agents are capable of acting on RNA molecule and interfering with transcription and translation. A great deal of research has also been carried out into the possibility of using oligonucleotides to block the coded message carried by mRNA . This is a approach known as antisense therapy and has interesting potential for the future . The rationale is as follows( Assuming that the primary sequence of an mRNA molecule is known , an oligonucleotide can be synthesized such that its nucleic acid bases are complementary to a specific stretch of the mRNA molecule.

Since the oligo nucleotide has a complementary base sequence it is called an antisense undergo nucleotide. When mixed with mRNA, the antisense oligonucleotide recognizes its complementary section m the mRNA interacts with it and forms a duplex structure such that the bases pair up by hydrogen bonding This section can now hinder the enzymes involved in the translation process and block protein synthesis. There are several advantages to this approach. First of all, it can be highly specific. Statistically, an oligonucleotide of 17 nucleotides should be specific for a single mRNA molecule and result in the inhibition of a single protein. Secondly, since one mRNA leads to several copies of the same protein, inhibiting mRNA should be more efficient than inhibiting the resulting protein. Both these factors should result in fewer side-effects than conventional protein inhibition.

However, there are several difficulties involved in designing suitable antisense drugs. mRNA is a large molecule with a secondary and, tertiary structure. Care has to be taken to choose a section which is exposed. There are also problems related to the pharmacokinetics of nucleotides with respect to their stability and their ability to enter cells (see Chapter 10). Nevertheless, antisense oligonucleotides provide the possibility of promising antiviral and anticancer agents for the future since they should be capable of preventing the biosynthesis of rogue proteins and have fewer side-effects than currently used drugs. The first antisense oligonucleotide to be approved for clinical trials in 1992 was for the treatment of genital warts caused by the papilloma virus.

Drugs related to nucleic acids and nucleic acid J building blocks

There are several important drugs which are related structurally to nucleic acids or to the building blocks for nucleic acids. Antisense oligonucleotides (see Section 7.4) and uradil mustard (see Section 7.2.2) are two such examples. Another important group of drugs which falls into this category are the various nucleoside-like structures used as antiviral agents. The vast majority of these are not active themselves but are phospho rylated by cellular enzymes to form an active nucleotide. For example, AIF (zidovudine or Retrovir) (Fig 724) was the first drug to be approved for use in the treatment of AIDS It is an analogue of thymidine and is phosphorylated to form a triphosphate.

This compound inhibits a viral enzyme called reverse transcriptase which is required for the synthesis of viral DNA. Furthermore, the triphosphate is attached to the growing DNA chain. Since the sugar unit has an azide substituents instead of the required hydroxyl group, the nucleic acid chain cannot be extended any further . Other antiviral agents such as acyclovir (Zovirax) and famciclovir (Famvir) have an incomplete sugar ring which serves as the chain terminator. Like AZT, these structures are phosphorylated to an active triphosphate form. Both structures are used in the treatment of Herpes simplex infections and shingles.

Molecular biology and genetic engineering

Over the last few years, rapid advances in molecular biology and genetic engineering have had important repercussions for pharmacology and medicinal chemistry. A detailed account of this field is not possible in an introductory text of this sort. Suffice it to say that it is possible to clone specific genes and to include these genes into the DNA of fast growing cells such that the proteins encoded by these genes are expressed in the modified cell. Since the cells are fast growing, this leads to a significant quantity of the desired protein, which permits its isolation, purification, and structural determination. Before these techniques became available, it was extremely difficult to isolate and purify many proteins from their parent cells because of the small quantities present. Even if one was successful, the low yields inherent in I the process made analysis of the proteins structure and mechanism of action very difficult. Advances in molecular biology and recombinant DNA techniques have changed all that. The following are some of the applications of genetic engineering to the medical field.

Harvesting important hormones and growth factors

The genes for important hormones or growth factors, such as insulin and human growth factor, have been included in fast growing unicellular organisms. This allows the harvesting of these proteins in sufficient quantity that they can be marketed and administered to patients who are deficient in these hormones.

Identification of new protein drug targets

Nowadays, it is relatively easy to isolate and identif a range of signalling proteins, enzymes, and receptors by cloning techniques. This has led to the identification of a growing number of isozymes and receptor subtypes which offer potential drug targets for the future. The human genome project involves the mapping of human DNA and has led to the discovery of new proteins previously unsuspected. These too may offer potential drug targets.

Study of the molecular mechanism of target proteins.

Genetic engineering allows the controlled mutation of proteins such that specific amino acids are altered. This allows researchers to identify which amino acids are important to enzyme activity or to receptor binding This in turn leads to a greater understanding of how enzymes and receptors operate at the molecular level.

Somatic gene therapy

Somatic gene therapy involves the use of a carrier virus to smuggle a healthy gene into cells in the body where the corresponding gene is defective. Once the virus has infected the cell, the healthy gene is inserted into the host DNA where it undergoes transcription and translation. This approach has great therapeutic potential for cancers, MDS, and genetic abnormalities such as cystic fibrosis. However, the approach is still confined to research labs and there is still a long way to go before it is used clinically. There are several problems still to be tackled such as how to target the viruses specifically to the defective cells, how to insert the gene into DNA in a controlled manner, how to regulate gene expression once it is in the DNA, and how to avoid immune responses to the carrier virus.

Summary In this chapter we have concentrated on drugs which are structurally related to the nucleic acid building blocks or which act on transcription, translation, and replication by acting directly on DNA and RNA. There are other drugs (e.g. nalidixic acid) which affect these processes but, since these drugs work by inhibiting enzymes rather than by a direct interaction with DNA or RNA, they have not been mentioned here.

THE END