Transcript Nucleic Acids - Farmasi Unand

Drugs that Target Nucleic Acids
Reference: Gareth Thomas
Week 15
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13. Drugs that Target Nucleic Acids
• Drugs that target DNA and RNA
either inhibit their synthesis or act
on existing nucleic acid molecules.
Those that inhibit the synthesis of
nucleic acids usually act as either
antimetabolites or enzyme
inhibitors.
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• The drugs that target existing
nucleic acid molecules can, for
convenience be broadly
classified into intercalating
agents, alkylating agents and
chain-cleaving agents.
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• However, it should be realised that
these classifications are not rigid: drugs
may act by more than one mechanism.
those drugs acting on existing DNA
usually inhibit transcription whereas
those acting on RNA normally inhibit
translation.
• In both cases the net result is the
prevention or slowing down of cell
growth and division.
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• Consequently, the discovery of new
drugs that target existing DNA and
RNA is a major consideration when
developing new drugs for the treatment
of cancer (see Appendix 4) and
bacterial and other infections due to
microorganisms.
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13.1 Antimetabolites
• Antimetabolites are compounds that
block the normal metabolic pathways
operating in cells.
• They act by either replacing an
endogenous compound in the pathway by
a compound whose incorporation into the
system results in a product that can no
longer play any further part in the
pathway, or inhibiting an enzyme in the
metabolic pathway in the cell.
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• Both these types of
intervention inhibit the
targeted metabolic pathway to
a level that hopefully has it
significant effect on the health
of the patient.
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• The structures of antimetabolites are
usually very similar to those of the
normal metabolites used by the cell.
Those used to prevent the formation of
DNA may be classified as antifolates,
pyrimidine antimetabolites and purine
antimetabolites.
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• However, because of the difficult of
classifying biologically active substances
(see section 1.6), antimetabolites that
inhibit enzyme action are also classified
as enzyme inhibitors.
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Figure 10.23. (a) The structure of folic acid. In blood, folic
acids usually have one glutamate residue. However, in the
cell they are converted to polyglutamates. (b) A Fragment
of a polyglutamate chain.
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13.1.1. Antifolates
• Folic acid (Figure 10.23) is usually
regarded as the parent of a family of
naturallv occurring compounds known as
folates.
• These folates are widely distributed in
food. They differ from folic acid in such
ways as the state of reduction of the
pteridine ring and having carbon units
attached to either or both of the N5
and N 10 atoms.
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• In the body folates are converted by a two-
step process into tetrahvdrofolates (FH4) by
the action of the enzyme dihydrofolate
reductase (DHFR). Tetrahydrofolic acid is an
essential cofactor in the biosynthesis of
purines and thymine. which are required for
DNA synthesis.
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• Folic acid antimetabolites have
structures that resemble folic acid
(Figure 10.24). They have a stronger
affinity for DHFR than folic acid and
act by inhibiting this enzyme at both
stages in the conversion of folic acid to
FH4.
• This has the effect of inhibiting the
formation of purines and thymine
required for DNA synthesis.
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Methotrexate
• This inhibits cell growth, which prevents
replication and ultimately leads to cell
death.
• Methotrexate is the only folate
antimetabolite in clinical use. It is
distributed to most body fluids but has
a low lipid solubility, which means, that
does not readilv cross the blood-brain
barrier.
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Figure 10.24. A comparison of the structures of
folic acid antimetabolites with folic acid.
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• It is transported into cells by the
folate transport system and at high
blood levels an additional second
transport mechanism comes into
operation.
• Once in the cell it is metabolised to the
polyglutamate, which is retained in the
cell for considerable periods of time.
This is probably due to the polar nature
of the polymer.
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• Methotrexate is used to treat a variety
of cancers, including head and neck
tumours, and, in low doses, rheumatoid
arthritis.
• It can cause vomiting, nausea, oral and
gastric ulceration and depression of
bone marrow, a well as other unwanted
side effects.
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13.1.2 Purine Antimetabolites
• Purine antimetaholites are exogenous
compounds, such as 6-mercaptopurine
and 6-thioguanine, with structures
based on the purine nucleus (Figure
1O.25).
• They inhibit the synthesis of DNA and
in some cases RNA by a number of
different mechanisms. For example, 6mercaptopurine is metabolised to the
ribonucleotide 6-thioguanosine-5’pltosphate.
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Figure 10.25. Examples of purine
antimetabolites. The purine nucleus, on which
the structures of the antimetabolites and the
endogenous compounds they replace are based,
is shown in square brackets.
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• This exogenous nucleotide inhibits
several pathways for the biosynthesis
of endogenous purine nucleotides.
• In contrast, 6-thioguanine is converted
in the cell to the ribonucleotide 6thioinosine-5’-phosphate.
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• This ribonucleotide disrupts DNA
synthesis by being incorporated into the
structure of DNA as a false nucleic
acid.
• Resistance to these two drugs arises
because of a loss of the posphorybosil
transferase required for the formation
of their ribonucleotides.
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13.1.3 Pyrimidine Antimetabolltes
• These are antimetabolites whose
structures closely to those of the
endogenous pyrimidine bases
(Figure l0.26a).
• They usually act by inhibiting one or
more of the enzymes that are
required for DNA synthesis.
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• The presence of the unreactive C5
F bond in FUdRP blocks this
methylation, which prevents the
formation of deoxythymidylic acid
(TdRP) and its subsecquent
incorporation into DNA (Figure 1 0.
261).
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• Fluorine was chosen to replace hydrogen
at the C5 position of uracil because it is
of a similar size to hydrogen (atomic
radii: F. 0.13 nm: 1-1. 0.l2nrn).
• It was thought that this similarity in
size would give a drug that ould cause
little steric disturbance to the
biosynthetic pathway as well as being
chemically inert.
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• Analogues containing larger halogen
atoms do not have any appreciable
activity.
• For example, fluorouracil is
metabolised by the same metabolic
pathway as uracil to 5-fluoro-2’deoxyuridylic acid (FUdRP).
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• FUdRP inhibits the enzyme thymidylate
synthetase, which in its normal role is
responsible for the transfer (of a
methyl group from the coenzyme
melhylenetetrahydrofolic acid (MeFI
14) to the C5 atom of deoxyuridylic acid
(UdRP).
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Figure 10.26. (a) Examples of pyrimidines that act as
antimetabolites. It should be noted that cytarabine only differs
from cytidine by the stereochemistry of the 2’ carbon. (b) The
intervention of fluorouracil in pyrimidine biosynthesis.
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Figure 10.27. Examples of topoisomerase inhibitors.
Ellipticene acts h intercalation and inhibition of
topoisomerase II enzymes. It is active against nasophar
ngeal carcinomas. Amsacrinc is used to treat oarian
carcinomas. lymphoinas and myelogenous leukaemias.
(camptotheci n is an antitumour.
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13.2. Enzyme Inhibitors
• Enzyme inhibitors may be classified for
convenience as those that inhibit the
enzymes directly responsible for the
formation of nucleic acids or the variety
of enzymes that catalyse the various
stages in the formation of the
pirimidine and purine bases required for
the formation of nucleic acids.
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13.2.1 Topoisomerases
• Topoisomerases are a group of enzymes
that are responsible for the
supercoiling, the cleavage and rejoining
of DNA.
• Their inhibition has the effect of
preventing transcription. A number of
compounds (Figure 10.27) are believed
to act by inhibiting these enzymes.
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• It is thought that some
intercalators act in this manner
although it is not clear whether the
drug binds to the topoisomerase
prior to or after the enzyme has
formed a DNA—enzyme complex.
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13.2.2 Enzyme Inhibitors for Purine and
Primidine Precursor Systems
• A wide range of compounds are active
against a number of the enzyme s stems
that are involved in the biosynthesis of
purines and pyrimidines in bacteria.
• In both of these examples the overall
effect is the inhibit ion of purine and
pyrirmidine synthesis, which results in
the inhibition of the synthesis of DNA.
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• This restricts the growth of the
bacteria and ultimately prevents it from
replicating, which gives the bodys
natural defences time to destroy the
bacteria.
• Because sulphonamides and
trimethoprim inhibit different stages in
the same metabolic pathway, they are
often used in conjunction (Figure 1
0.28).This allows the clinician to use
lower and there fore safer doses.
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• For example, sulphonamides inhibit
dihydropteroate synthetase (see
section 6.12.1), which prevents the
formation of folic acid, whereas
trimethoprim inhibits dihydrofolate
reductase, which prevents the
conversion of folic acid to
tetrahydrofolate (see section 10.13.1.1).
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• In both of these examples the overall
effect is the inhibit ion of purine and
pyrimidine synthesis, which results in
the inhibition of the synthesis of DNA.
This restricts the growth of the
bacteria and ultimately prevents it from
replicating, which gives the bodys
natural defences time to destroy the
bacteria.
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• Because sulphonamides and
trimethoprim inhibit different stages in
the same metabolic pathway, they are
often used in conjunction (Figure 1
0.28).This allows the clinician to use
lower and therefore safer doses.
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Figure 10.28. Sequential blocking using
sulphamethoxazole and Trimethoprim.
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13.3. Intercalating Agents
• Intercalating agents are compounds
that insert themselves between the
bases of the DNA helix (Figure 10.29).
This insertion causes the DNA helix to
partially unwind at the site of the
intercalated molecule.
• This inhibits transcription, which blocks
the replication process of the cell
containing the DNA.
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• However, it is not known how the partial
unwinding presents transcription but
some workers think that it inhibits
topoisomerases (see section 10.12.2.1).
Inhibition of cell replication can lead to
cell death, which reduces the size of a
tumour, the number of ‘free’ cancer
cells or the degree of infection, all of
which will contribute to improving the
health of the patient.
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Figure 10.29. A schematic representation of the
distortion of the DNA helix by intercalating
agents. The horizontal lines represent the
hydrogen-bonded bases. The rings of these
bases and intercalating agent are edge
on to the reader.
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• The insertion of an intercalation agent
appears to occur via either the minor or
major grooves of DNA.
• Compounds that act as intercalating
agents must have structures that
contain a flat fused aromatic or
heteroarornatic ring section that can
fit between the flat structures of the
bases of the DNA.
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• It is believed that these aromatic
structures are held in place by
hydrogen bonds, van der Waals’
forces and charge-transfer bonds
(see section 5.2).
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Figure 10.30. Examples of intercalating agents.
Trade name.
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• Drugs whose mode of action includes
intercalation are the antimalarials
quinine and chloroquine, the anticancer
agents mitoxantrone and doxorubicin,
and the antibiotic proflavine (Figure
10.30).
• In each of these compounds it is the
flat aromatic ring system that is
responsible for the intercalation.
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• However, other groups in the structures
may also contribute to the binding of a
drug to the DNA.
• For example, the amino group of the
sugar residue of doxorubicin forms an
ionic bond with the negatively charged
oxygens of the phosphate groups of the
DNA chain, which effectively locks the
drug into place.
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• A number of other drugs appear to have
groups that act in a similar manner.
• Some intercalating agents exhibit a
preference to certain combinations of
bases in DNA.
• For example, mitoxantrone appears to
prefer to intercalate with cytosine—
guanosine-rich sequences. This type of
behaviour does open out the possibility
of selective action in some cases.
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13.4 Alkylating Agents
• Alkylating agents are believed to bond
to the nucleic acid chains in either the
major or minor grooves.
• In DNA the alkylating agent frequently
forms either intrastrand or interstrand
crosslinks.
• Intrastrand cross-linking agents form a
bridge between two parts of the same
chain (Figure 10.31). This has the effect
of distorting the strand, which inhibits
transcription.
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Figure 10.31. A schematic representation of
the intrastrand cross-linking.
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• lnterstrand cross—links are formed
between the two separate chains of the
DNA,which has the effect of blocking
them together (Figure 10.32). This also
inhibits transcription.
• In RNA only intrastrand cross-links are
possible. However, irrespective of
whether or not it forms a bridge. the
bonding of an alkylating agent to a
nucleic acid inhibits replication of that
nucleic acid
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• In the case of bacteria this prevents an
increase in the size of the infection and
so buys the time for its immune system
to destroy the existing bacteria.
However, in the case of cancer it may
lead to cell death and a beneficial
reduction in tumour size.
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Figure 10.32. (a) The general structure of nitrogen mustards (h) The
proposee mechanism for tormimu’ interstrand
cross-links by the action of aliphatic nitrogen mustards.
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• The nucleophilic nature of the nucleic
acids means that alkylating agents are
usually electrophiles or give rise to
electrophiles.
• For example, it is believed that a weakly
electrophilic β-carbon atom of an
aliphatic nitrogen mustard alkylating
agent, such as mechlorethamine
(Mustine),
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• is converted to the more highly
electrophilic aziridine ion by an internal
nucleophilic substitution of a b-chlorine
atom.
• This is thought to be followed by the
nucleophilic attack of the N7 of a
guanine residue on this ion by what
appears to bean SN2 type of
mechanism.
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• Since these drugs have two
hydrocarbon chains with b-chlorogroups,
each of these chlorogroups is believed
to react with a guanine residue in a
different chain of the DNA strand to
form a cross-link between the two
nucleic acid chains (Fig. 10.38).
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Figure 10.33. (a) The structure of chlorambucil and (b) a
proposed mode of action for some aromatic nitrogen
mustards.
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• The electrophilic nature of alkylating
agents means that they can also react
with a wide variety of other nucleophilic
biomacromolecules.
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• This accounts for many of the unwanted
toxic effects that are frequently
observed with the use of these drugs.
In the case of the nitrogen mustards.
attempts to reduce these side effects
have centred on reducing their
reactivity by discouraging the formation
of the aiziridine ion before the drug
reaches its site of action.
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• The approach adopted has been to
reduce the nucleophilic character of the
nitrogen atom by attaching it to an
electron-withdrawing aromatic ring.
This produced analogues that would only
react with strong nucleophiles and
resulted in the development of
chlorambucil.
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• This drug is one of the least toxic
nitrogen mustards, being active against
malignant lymphomas, carcinomas of the
breast and ovary and lymphocytic
leucaemia.
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• It has been suggested that because of
the reduction in the nucleophilicity of
the nitrogen atom these aromatic
nitrogen mustards do not form an
aziridine ion.
• Instead they react by direct
substitution of the 13-chlorine atoms by
guanine, which is a strong nucleophile,
by an S.I type of mechanism (Figure
10.33).
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Figure 10.34. Cyclophosphamide and the formation
of phosphoramide mustard, the active of the drugs
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• Further attempts to reduce the toxicity
of nitrogen mustards were based on
making the drug more selective. Those
approaches have yielded useful drugs.
The first was based on the fact that
the rapid synthesis of proteins that
occurs in tumour cells requires a large
supply of amino acid raw material from
outside the cell.
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• Consequently, it was thought that the
presence of an amino acid residue in the
structure of a nitrogen mustard might
lead to an increased uptake of that
compound.
• This approach resulted in the synthesis
of the phenylalanine mustard meiphalan
(Table 10.6)
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• The L-form of this drug is more active
than the D-form and so it has been
suggested that the L-form may be
transported into the cell by means of a
L -phenylalanine active transport
system.
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• The second approach was based on the
fact that some tumours were thought to
contain a high concentration of
phosphoramidases.
• This resulted in the synthesis of
nitrogen mustard analogues mechanism,
whose structures contained phosphorus
functional groups that could be
attacked by this enzyme.
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• It lead to the development of the
cyclophosphamide (Figure 10.34). which
has a wide spectrum of activity.
• However, the action of this prodrug has
now been shown to be due to
phosphoramide mustard formed by
oxidation by microsomal enzymes in the
liver rather than hydrolysis by tumour
phosphoramidases.
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• The acrolein produced in this proces believed to be the source of myelosuppression and haemorrhagic cystitis
associated with the use of
cyclophosphamide.
• However, coadministration of the drug
with sodium 2-mercaptoethane
sulphonate (MESNA) can relieve some
of these symptoms.
• MESNA forms a water-soluble adduct
with the acrolein, which is then
excreted in the urine.
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• Some alkylating agents act by
decomposing to produce an electrophile
that bonds to a nucleophilic group of a
base in the nucleic acid.
• For example. temozolomide (Table 10.6)
enters the major groove of DNA where
it reacts with water to from nitrogen.
carbon dioxide, an aminomidazole and a
methyl carbonium ion (CH3).
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• This methyl carbonium ion then
methylates the strongly nucleophilic N7
of the guanine bases in the major groo
e.
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• A range of different classes of
compound can act as nucleic acid
alkylating agents (Table 10.6).
• Within these classes a number of
compounds have been found to be useful
drugs.
• In many, cases their effectiveness is
improved by the use of combinations of
drugs.
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• Their modes of action are usually
not fully understood but a large
amount of information is available
concerning their structure-action
relationships.
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Table 10.6. Some examples of the classes and compounds of
anticancer agents that act by aik alkylation of nucleic acids. it is
emphasised that this table only lists some of the classes of alkylating
compound that are active against cancers.
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Figure 10.35. Development routes for antisense drugs. Examples of:
(a) a section of the backbone of a deoxy ribonu
CWICIId cleic chain; (b) backbone modifications; (c) sugar residue
modifications; and (d) base modifications,
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