DNA Alkyltransferase By O6 Benzylguanine Analogs Biology Essay

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Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). These causal factors may occur together or in a particular sequence which further leads to carcinogenesis. There is a lot of evidence which indicate that carcinogenesis in humans involves a number of steps which alter the genome and lead to the transformation to cancerous derivatives (2). There are so many different types of cancer in a multitude of organs.

According to the American cancer society, this year, about 565,650 Americans are will die of cancer, more than 1,500 people a day. Cancer is the second most common cause of death in the US, exceeded only by heart disease. In the US, cancer accounts for 1 of every 4 deaths. The American Cancer Society estimates that in 2008 about 170,000 cancer deaths were caused by tobacco use (2).

Major treatment of cancer involves targeting various pathways that lead to Apoptosis or programmed cell death, which plays a very important role to maintain the balance of cell growth and cell disease.

The traits which are considered as 'Hallmarks of cancer' (2)(Hanahan and Weinberg)comprise of

Self-sufficiency in growth signals (oncogenes): The basic platform for cancer is set by a number of mutations which result in the generation of the oncogenes. Any normal cells can be initiated to being transformed to a progressively growing cancer cell by these growth signals (GS). Bernard Weinstein, coined the term "oncogene addiction" which describes that all the different alterations in the genome which result in the formation of the malignant tumor seem to be dependent on the activity of the oncogenes(4). It is believed that metamorphosis of any normal cell cannot occur in the absence of these growth signals.

Insensitivity to anti-growth signals :

According to Hanahan and weinberg, the anti-growth signals function by two mechanisms. Cells may quit their cycle of proliferation and enter into the quiescent (G0) state. The appearance of some extracellular signals may result in their re-emergance. The second alternative is that the cells may be impelled to get into postmitotic states renounced its ability to proliferate. Normally, the tumor suppressor genes are normal genes which tends to slow the process of cell division, repairing mistakes in DNA , and apoptosis. When tumor suppressor genes become insensitive, cells tend to proliferate without control, leading to cancer. About 30 TSG s have been discovered till today, which include p53, BRCA1, BRCA2, APC, and RB1.(American cancer society)

Limitless replicative potential

This inherent characteristic of cancerous cell is acquired by the cell for its uncontrolled replication. The unlimited replicative potential of the cancer cells is dependant on how the telomeric DNA is maintained. The activity of telomerase is highly activated in many of the tumors, moreover the telomeres also tend to be shortened in length in most of the tumors as compared to normal tissues. Thus we see that this telomere/telomerase pathway is highly studied in cancer research. The evasion of the cellular senescence may also result in the unlimited replicative potential. (3)

Sustained angiogenesis

The growth and survival of the tumor requires blood vessels. Angiogenesis is the development of new blood vessels from the existing blood vessel precursors. About 30 Years ago, Judah Folkman isolated a number of proteins and interpreted the process which governs angiogenesis. Many invivo tests suggest that angiogenesis sustains the progressive development of the tumor explants. Hanahan and folkman through their transgenic mouse model studies suggest the involvement of a 'angiogenic switch' in the early stages for the progress of the tumor. Infact they could visualize the angiogenic switch in the intial stages of breast and cervical cancer(6).

Tissue invasion and metastasis.

According to Hanahan and Weinberg, metastasis is responsible for 90% of cancers. In metastasis the primary tumor mass tend to spread to neighboring tissues, further invading the blood/lymph into other organs .Thus the cancer cells tend to establish new tumors in locations very far from the primary tumor region. This process is seen in the later stages of the disease. Another parameter involved in the metastasis are the extracellular proteases.

Evasion of apoptosis: A balance of cell division and cell death is essential to maintain homeostatis in multicellular organisms. Apoptosis is a form of programmed cell death of unwanted or damaged cells Apoptosis or programmed cell death is an effective mechanism by which damaged cells and other oncogenic mutations are removed. Since most of the current cancer therapies work by apoptosis ,the resistance to the same plays a important role in the advancement of cancer. Apoptosis can be either be blocked by the accumulation of the anti apoptotic molecules or by the improper functioning of the pro- apoptotic proteins as suggested by the apoptosis escape mechanisms by Simone Fulda(5) . A number of mechanisms are thought to be involved in the development of the resistance against apoptosis. The most common being the impairment of the proapoptotic regulator which is due to mutation of the p53 tumor suppressor gene. Thus inactivating its product, p53 protein,which is thought to be involved in more than 50% of cancers in humans .The final result is the excision of one of the important sensor coding for the DNA damage which leads to the ineffectiveness of the apoptotic pathway.(hanbarg). Any dysregulation in the process either activation or inhibition is usually associated with degenerative disorders and cancer. Thus apoptotic pathways are of immense intrest in drug discovery and development

Fig 1 :- Apoptosis by intrinsic and extrinsic pathways.

Apoptosis can be triggered by internal events or an extrinsic pathway in which the disruption of mitochondria and cytochrome C lead to downstream activation of caspases (figure 1). Alternatively, extrinsic pathways also lead to apoptosis: specific ligands bind to the death surface receptors, such as the receptors of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) super family. The immune cells mediate the extrinsic pathway to initiate intracellular signalling downstream activation of caspases.

Down the line prevention and cancer treatment are hot topics of the present day research. As a means of developing several cancer treatments researchers are manipulating DNA damage and repair mechanisms that leads to programmed cell death.

Chemotherapy and radiation are the most important machinery for cancer treatment through DNA damage. DNA damage can also occur naturally apart from chemotherapeutic agents, but there are abundant DNA repair pathways that can detect and repair this damage to prevent the genomic integrity. While some base insertions, deletions, or point mutations may be harmless due to redundancy and conservation of the genetic code, other mutations can force cells to become malignant. However, DNA damage can also be used to treat cancer by causing enough DNA damage that leads to death of tumor cells. These DNA repair pathways have a great impact on clinical outcome and are responsible for therapeutic resistance. Therefore a revised strategy is to reverse this drug resistance by inhibiting the DNA repair pathways to enhance chemo and radiosensitivity. Several DNA repair inhibitors in combination with radiation or chemotherapy showed promising results in preclinical studies (7).


Evidence supporting the view that DNA damage lead to apoptosis is provoked by genotoxins are because of the following factors,

1. Almost all DNA repair deficient cells have hypersensitive apoptotic responses. And also respond with elevated apoptosis levels due to the killing effect of genotoxins except for the DNA Mismatch repair mutants. The following DNA repair mechanisms had shown the mutants defective in O6-methylguanine-DNA methyltransferase (MGMT), Base Excision repair (BER), Nucleotide excision repair(NER), DNA double strand break (DBS) and DNA crosslink repair.

2. Induce apoptosis by incorporating modified nucleotide precursors into DNA. The GCV ganciclovir model system demonstrates that a simple modified base present in the DNA during replication can trigger apoptosis.

Enzyme induced DSBs trigger apoptosis but not necrosis.

Fig 2:- Schematic overview of DNA repair pathways. The principal targets promising in cancer treatment are highlighted (7).


DNA, or deoxyribonucleic acid, is the fundamental building block for an individual's entire genetic makeup. They are often referred to as the "building blocks of life," since DNA encodes the genetic material which determines what an organism will develop into. In addition to maintaining the genetic blueprints for its parent organism, DNA also performs a number of other functions, which are critical to life. The DNA molecules encode for the genome, hence DNA repair refers to the processes by which the damage to the DNA molecules is identified and corrected.

The sequence of DNA can be changed because of errors during copying by the DNA polymerases involved in replication and by other agents in the environment such as certain mutagenic chemicals and also by particular types of radiation. If there is a change in the DNA sequence, whatever be the cause, and is left uncorrected, all the vegetative cells may acquire so many mutations that they stop functioning. Moreover, many mutations may also occur in the DNA of the germ cells, which will affect the formation of viable offspring. Thus, DNA sequence errors needs to be corrected in all the types of cells which play an important role for survival.(9)

The rate at which the DNA is repaired depends on number of factors, like type of cell, the age of the cell, and also the extracellular environment. A cell which cannot repair its accumulated damage enters into one of the following stages:

1. Apoptosis: A form of cell death in which a programmed sequence of events leads to the elimination of cells without releasing harmful substances into the surrounding area. Apoptosis plays a crucial role in developing and maintaining health by eliminating old cells, unnecessary cells, and unhealthy cells as described above.

2. Aging/Senescence

3. An unregulated cell division, which can result into the formation of a tumor which can be cancerous.

The importance of DNA damage and repair for the generation of cancer or carcinogenesis was recognized when it was seen that all the agents that are responsible for cancer also known as carcinogens can also result in the change in the sequence of DNA and thus are also known as mutagens. The carcinogenic chemicals play a significant role in production of tumor. The resulting damage to the DNA is beyond repair (9).

The sources of DNA Damage can be caused by either

Endogenous Factors which include some genetical disorders ,immunopathological and neurological reactions(11)

Exogenous Factors are (10)

1. Chemical agents like poisons, genotoxic, phenotoxic compounds, venoms and caustic agents.

2. Mechanical factors like truamatic injury

3. Physical Factors like ionising radiation, changes in temperature

4. Biological Agents like protozoa, microorganisms, and viruses

5. Nutritive Deficiencies due to deficiency of some basic nutrients

In most of the human tumors, endogenous mutation give rise to multiple mutations which is not a sudden event, but happens continuously with the progress of tumorigenesis. The role of different endogenous processes in the damage of the DNA has been studied extensively and occurs at a very high frequency. Based on Jackson et al invitro studies of many normal cell processes act as the sources leading to endogenous carcinogenesis.

DNA Depurination by water

Misincorporation of bases by the DNA polymerases

The cytosine deamiantion

The damage of the DNA by reactive oxygen species

Methylation of bases such as formation of 7 Methyl guanine

Exogenous factors

Types and examples of human carcinogens

Type of carcinogen


Chemical carcinogens

Nickel, cadmium, arsenic, nitrosamines, trichloro-

ethylene, arylamines, benzopyrene, aflatoxins, reactive

oxygen species

Physical carcinogens

UV irradiation (specifically UVB), ionizing radiation

Biological carcinogens

Human papilloma virus (e.g. strain 16), Epstein-Barr-

Virus, Hepatitis virus B, Helicobacter pylori,

Schistosoma mansoni

Table 1 from Advanced Biology of human cancers by Wolfgang Arthur Schulz.



Nucleotide excision repair (NER) is one of different DNA repair mechanisms in which damaged bases are removed from the genome by utilizing various biochemical steps that includes DNA damage recognition, dual incision, oligonucleotide excision, DNA re-synthesis and ligation. Over 30 different protiens are invovled in mammalian NER, The human protein substrate required for NER assemble in an ordered fashion near the base damage site.

The various steps involved in NER system are(13, 14),

NER operates on DNA lesions caused by UV radiation, mutagenic chemicals, chemotherapeutic drugs that form bulky DNA adducts. DNA lesions causes structural distortion in one of these strands that is recognized by a protein called XPC, which is stably bound to another protein called HHRAD23B forming a heterodimer subcomplex. Later various proteins join to felicitate specific recognition of base damage by XPA and RPA later by TFIIH, which is a subcomplex of the RNA polymerase II transcription initiation machinery. It consists of six subunits and contains two DNA helicase activities (XPB and XPD) that unwind the DNA duplex at the sites of damage. This local denaturation generates a bubble in the DNA, the ends of which comprise junctions between duplex and single stranded DNA. NER multiprotien complex is generated when ERCC1-XPF binds and forms a heterodimerix complex. XPG protein has an endonuclease activity that makes an incision 3' to the damaged DNA, followed by ERCC1-XPF heterodimeric protein that makes the 5' incision to the site of base damage.

This dual incision generates an oligonucleotide fragment 25-30 nucleotides that includes the damaged base. This fragment is excised from the genome, concomitant with restoring the potential 25-30 nucleotide gap by repair synthesis. Repair synthesis requires DNA polymerase δ or ε, and Replication protien A, RPA, PCNA to support DNA polymaerase in the reaction and RFC. The covalent integrity of the damaged strand is then restored by DNA ligase.


DNA mismatch repair pathway involves the recognition of specific mismatching base combinations that can arise during DNA replication and recombination. Some of the common examples of mismatch repair include G/T or A/C base pairing. The process involves identifying the wrongly incorporated bases by excising them and replace with the correct nucleotide. The mismatch repair process gets complicated as we go from E.coli to mammalian system.

The whole MMR process was reconstructed in the laboratories of Gou Min Li and Paul Modrich from purified recombinant constituents by including the protiens MutSα-MutLα-RPA-EXO1-PCNA-RFC-(HMGB1) system with DNA ligase I and DNA polymerase δ. In Mammalian MMR category three models have been proposed and they are, The molecular switch model, The active translocation model and The DNA bending model. The first two models are higly agreed upon and the difference between the two lies in the mode of translation of MutSα from the mismatch and their mechanisms are discussed in the following figure 3(13).

Figure 3: - "The Mismatch repair (MMR) process was recently reconstituted72,74 from either MutSα or MutSβ, MutLα, replication protein A (RPA), exonuclease-1 (EXO1), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), DNA polymerase δ (Pol δ) and DNA ligase I. The following is proposed to take place. The mismatch (red triangle)-bound MutSα (or MutSβ) recruits MutLα. The ternary complex undergoes an ATP-driven conformational switch, which releases the sliding clamp from the mismatch site. a | Clamps that diffuse upstream encounter RFC that is bound at the 5′ terminus of the strand break, and will displace it and load EXO1. The activated exonuclease commences the degradation of the strand in a 5′→3′ direction. The single-stranded gap is stabilized by RPA. When themismatch is removed, EXO1 activity is no longer stimulated by MutSα, and is actively inhibited by MutLα. Pol δ loads at the 3′ terminus of the original discontinuity, which carries a bound PCNA molecule. This complex fills the gap and DNA ligase I seals the remaining nick to complete the repair process. b | Clamps that migrate downstream encounter a PCNA molecule that is bound at the 3′ terminus of the strand break. The recruitment and the activation of EXO1 results in the degradation of the region between the original discontinuity and the mismatch, possibly through several iterative EXO1-loading events. RFC that is bound at the 5′ terminus of the discontinuity prevents degradation in the 5′→3′ direction (away from the mismatch). Once the mismatch is removed and the EXO1 activity is inhibited by bound RPA and MutLα, the gap is filled by Pol δ. DNA ligase I seals the remaining nick to complete the repair process." (13)


The double stranded breaks in the chromosome may be as a result of number of stresses such as ionising radiation, some spontaneous breaks in the chromosome during the replication of the DNA, or due to action of the endonucleases .These breaks pose a big threat to the survival of the cell. The unrepaired chromosomal breaks may segregate improperly and may be degraded resulting in aneuploidy. The two general pathways of DNA repair are

Homologous Recombination

Non Homologous End Joining (NHEJ)


The term Homologous Recombination means the DNA molecules are exchanged between homologous DNA molecules thus preserving the genomic integrity of the cell as well as ensuring the precise replication of the genome. The basic mechanism is same for the eukaryotes and prokaryotes, although the former being more complex.

The homologous recombination reaction basically involves 3 stages

Pre synaptic stage: Recombinant DNA is prepared

Synaptic Stage: Joint molecule formation between a double stranded homologous template DNA and recombinant DNA end.

Post synaptic Stage: The DNA strands are repaired and resolved. Finally the recombinant DNA is separated. One of the major factors threatening the stability of the genome may arise due to the improper DNA recombination mechanisms, which have to be controlled carefully.

Fig 4: - DNA double-strand break (DSB) repair through homologous recombination(16).

In the following model, the blue and red double-stranded DNAs represent homologous sequences.

"A DSB can be generated by DNA-damaging agents or replication of DNA containing a single-

Strand break.

The DSB is processed to a single-stranded region with a 39 overhang by a nuclease, a helicase or both.

Rad51 protein polymerizes onto the single-stranded DNA to form a nucleoprotein filament that searches for the homologous duplex DNA

After the search has been successfully completed, DNA strand exchange generates a joint molecule between the homologous damaged and undamaged duplex DNAs.

In addition to Rad51p, these steps require the coordinated action of Rad52p, Rad54p, Rad55/57p and the single-strand DNA-binding protein replication protein A (RP-A). DNA synthesis, requiring a DNA polymerase, its accessory factors and a ligase, restores the missing information.

Finally, resolution of crossed DNA strands (Holliday junctions) by a resolvase yields two intact duplex DNAs.

For simplicity, branch migration of the Holliday junctions to extend heteroduplex DNA is not indicated. Only one pair of possible recombination products is depicted(16)".


Fig 5:- Model for DNA double-strand break (DSB) repair through DNA end-joining. (16)

"Upon DSB formation, the KU heterodimer binds to the DNA ends and attracts DNA-PKCS. In addition to this,chromatin structure might also be influenced.

In Saccharomyces cerevisiae, Sir2p, Sir3p and Sir4p are involved in this process, and a similar chromatin-remodelling reaction might also occur in mammals.

Subsequently, the ends are brought together and the DNA-PKCS protomers phosphorylate each

other, causing a structural change in the complex, possibly resulting in removal of DNA-PKCS.

For the later stages of the repair process, the complex containing RAD50, MRE11 and NBS1 is attracted, which might process the ends.

Finally, the DNA ligase-IV-XRCC4 complex rejoins the strands. It should be stressed that it is not known in which order the various protein complexes are attracted to the DNA end.

The two interwound DNA strands are represented as ribbons, and proteins as spheres. Higher-order chromatin structure is not indicated, although the Sir proteins act by modulating chromatin structure through interactions with histones."


Fig 6: - Schematic BER pathway and different sub-pathways in mammals(17).

"BER starts with the recognition and removal of a lesion (star) by a DNA glycosylase. Only bifunctional DNA glycosylases are able to cleave the sugar-phosphate backbone and create a 5_ phosphate (P) and a 3_ phosphate or 3_ polyunsaturated aldehyde (PUA), depending on the DNA glycosylase. After removal of the damaged base by monofunctional DNA glycosylases, strand scission is exerted by AP endonuclease, creating 3_ hydroxyl (OH) and 5_ deoxyribose-phosphate (dRP). These unconventional termini have to be restored to 3_ OH and 5_ P to allow further repair through deoxyribose-phosphatase diesterase (dRPase) activity of Pol β (5_ dRP), diesterase activity of AP endonuclease (3_PUA), phoshatase activity of polynucleotide kinase phosphatase (PNKP) (3_ P), or phosphatase activity of aprataxin (APTX) (3_ P).Repair then proceeds via short-patch or long-patch repair. During short-patch repair, Pol β incorporates one nucleotide, followed by nick ligation by the XRCC1/LigIIIα complex (predominantly) or LigI. If the 5_ lesion is refractory to cleavage by Pol β, the long-patch branch of BER is taken. Pol β and/or Pol δ/ε accomplish strand displacement by incorporating multiple nucleotides, followed by removal of the DNA flap containing the 5_ refractory moiety by Flap endonuclease and ligation of the resulting nick by LigI. Supportive BER proteins are indicated in gray."(17)


Cellular DNA is subjected to damage due to modifications by intracellular and extracellular chemicals, which can result in covalent changes and also cause 1 million molecular lesions per day per cycle, Even DNA damage is caused by the alkylating agents. These agents are widespread in nature and are used as anticancer compounds in reality. Alkylating agents are also present endogenously inside cells; for example, S-adenosylmethionine, a methyl donor for various cellular reactions, has been shown to produce methylating damage. Various lesions on heterocyclic bases like N3-methylguanine, N7-methyl guanine, O6-methyl guanine are formed by the alkylating attack on DNA. Most of these methyl or alkyl adducts are mutagenic or toxic, and cells have evolved with several proteins to detect and repair them through direct removal of the adduct. Other than the photolyase enzyme that removes the damage by photoreactivation process, that catalyzes direct reversal of the thymine dimer formed by UV light. All known direct DNA repair proteins are engaged in alkylation DNA damage repair. These are the N-terminal domain of the Escherichia coli Ada protein, the O6-alkylguanine-DNA alkyltransferase family, and the AlkB family(20).

A suitable example is alkylation adduct like O6-methyl guanine that are highly cytotoxic and are directly removed by the protein methyl guanine methyl transferase (MGMT). They do not involve breakage of phosphodiester backbone in direct reversal mechanism. This DNA repair occurs via a suicide mechanism that occurs b y transferring strochiometrically alkyl adducts from O6 atom of the guanine moiety in DNA to an internal cystine residue within the active site of MGMT without any cofactor requirements and a detailed mechanism will be described later (18).


Several methylating agents such as temozolomide, procarbazine and dacarbazine are used in cancer therapy, as they target DNA inducing about dozen DNA methylating lesions. One of them, O6-methylguanine , comprising <8% of DNA methylations, has been identified as the major lesion leading to apoptosis. O6Methylguanine has been identified as an apoptotic DNA lesion, that has unveiled the chain of events leading to apoptosis in great detail. DNA lesion O6Methylguanine alone does not trigger apoptosis directly. It requires DNA mismatch repair (MMR). MMR-deficient cells are highly resistant to O6MeG-triggered apoptosis by tolerating O6Methylguanine lesions at the expense of point mutations. MMR is provoked by the mispairing properties of O6Methylguanine-thymine mispairs. In a futile effort to cut out thymine, the MMR enzyme complex repeatedly inserts the wrong base and ends up in a faulty repair cycle resulting in secondary DNA lesions that block replication in the next cell cycle, leading to Double Stranded Breaks(8).

"Work with rodent fibroblasts showed that a hallmark of O6MeG-triggered apoptosis is the decline of Bcl-2. The Fas/CD95/Apo-1 pathway was not significantly activated, indicating that O6MeG triggers mainly the mitochondrial damage pathway in fibroblasts involving cytochrome c release from mitochondria and caspase-9/3 activation. This was independent of p53 as the cells were mutated in the gene coding for this protein (19)."

In the work with human lymphocytes, "O6MeG-triggered apoptosis in stimulated lymphocytes was a late response preceded by the induction of DSBs and p53 upregulation. Furthermore, the death receptor Fas/CD95/APO-1 was enhanced in expression, whereas Bcl-2 and Bax remained unaffected. Upon inhibition of the Fas receptor, apoptosis was drastically reduced. This indicates that in lymphocytes, O6Methylguanine triggers apoptosis by activating the receptor-driven pathway rather than targeting the mitochondria. Obviously, depending on the cellular background, O6MeG is able to activate either the Fas or the Bcl-2-driven pathway, with DNA replication to be essentially involved(19). "

Figure 7:- " DNA lesions and apoptotic pathways induced by methylating agents. Primary pre-toxic DNA lesions are N7-methylguanine (N7MeG), O6-methylguanine (O6MeG) and N3-methyladenine (N3MeA). N-methylpurines block DNA replication leading to DSBs at collapsed replication forks. Likewise, O6MeG lesions processed by mismatch repair (MMR) interfere with replication, which is supposed to give rise to DSBs. Depending on the cellular background, DSBs can trigger either the death-receptor-driven or the so-called endogenous mitochondrial damage pathway of apoptosis, both ending with activation of the downstream executing caspases, such as caspase-3."(8)


In the Direct repair pathway, AlkB enzymes and AGT are the only human protein that can directly repair or reverse DNA Damage. By examining the X ray crystal structure using the visualization tools in maestro, it helps in understanding how AGT finds, captures and repairs O6 methyl guanine lesions in DNA. The whole idea of my work lies in generating a global conformer that will aid in development of novel inhibitors solely based on docking energies and inspection of their interactions in the active site of the protein ligand complexes. The topology of the crystal structure is highly conservative across prokayotic, eukaryotic and antibacterial homologs despite their low homolog sequences. Since the original development of O6-benzylguanine, structural and biochemical studies have provided a much greater understanding of the repair reaction catalyzed by alkyltransferase and the binding and reaction of this inhibitor.

The factors responsible for the recognition of damaged O6 methyl guanine and repair by direct reversal pathway are the following,

First step involves the binding of B-DNA to HTH motifs, the recognition of helix (Tyr114-Ala121), second helix (Ala127-Gly136) deep within the minor groove that accounts for 64% of the buried area. The protein structure is not altered by DNA binding as the AGT-DNA complex exhibits an r.m.s deviation of 0.8A0 from native AGT prior to binding. The DNA structural changes resulting from DNA minor groove widening by >3 A0 to perfect B-DNA and the DNA bends 15 degrees away from the protein help flip the damaged guanine nucleotide out from the major groove from the base stack. The binding affinity between the recognition helices and minor groove aids in weakening of local base pair stability. The flipping of O6-methylguanine out of the base pair stack and into the AGT active site is enhanced by the positioning of Arg 128 into the base stack through stabilizing affinity through charged hydrogen bonding with the cytosine compensates for the loss of O6-methylguanine. Arg128 intercalates via the minor groove and stabilizes the extrahelical DNA conformation. Another factor that assists nucleotide flipping is the phosphate rotation due to the close proximity of Tyr114 in AGT-DNA complex owing to both charge and steric repulsion from phosphate anion.

AGT interacts with guanine selectively over the other three bases. Because after the flipping of O6-methylguanine from the base stack, hydrogen bonds and geometric exclusions provide some selectivity to 2'-deoxyguanosine nucleotides. And also strong hydrogen bondings are formed from carbonyls of Cys145 and Val148 to exocyclic amine of guanines. Also the Tyr114 hydroxyl and Ser159 N donate hydrogen bonds to N3 and O6 of guanine respectively. The affinity for AGT towards O6-methylguanine over guanine is only about three fold, which is due to larger hydrophobic interaction derived from alkylation in the hydrophobic pocket.


Nature's strategy of dealing with the Direct reversal mechanism in the case of AGT or MGMT is to irreversibly transfer the methyl group to nucleophilic Csy 145 residue in AGT repair protein forming a alkyl cystine adduct in the active site. Here a Glu-His-Water-Cys hydrogen bond network formed to increase the reactivity of active site Cys 145. The proton transfer obtained through this network to generate a thiolate anion, explains the low pKa and high reactivity of Cys145. So the AGT protein serves as an alkyl transfer reagent rather than like an enzyme as it is degraded by ubiquitylation and proteosomal digestion(21).

Fig 8- Reaction Mechanism. A hydrogen bond network similar to the catalytic triad of serine proteases seems placed to deprotonate the active site cystine. In this mechanism, His 146 acts as water mediated general base to deprotonate Cys145, which acts as a nucleophile in the dealkylation reaction. Donation of the hydrogen bond from Tyr114 to N3 of guanine may also promote the reaction.


O6-Alkylguanine_DNA_alkyltrnasferase as can be seen from the following mechanism, is a unique DNA repair protein that acts in a single step to restore DNA with O6-alkylguanine adducts by transferring the alkyl group to an acceptor site, which in human alkyltransferase is located at Cys145. Alkyltransferase activity in tumors is an important source of resistance to therapeutic alkylating agents such as dacarbazine, temozolomide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-3-(trans-4- methylcyclohexyl)-1-nitrosourea . O6-Benzylguanine has been shown to be a potent inhibitor of human alkyltransferase. O6-Benzylguanine acts as a pseudosubstrate. After binding in the active site, it leads to the formation of S-benzylcysteine at the Cys145 acceptor site irreversibly inactivating the protein and the mechanism proceeds as described above.

O6-Benzylguanine binds much more weakly at the active site pocket since all of the interactions with the alkyltransferase-DNA binding domain are lost but it is held in a position that allows attack by Cys145 via the interactions with the guanine moiety described above and the interaction of the benzyl group with the side-chain of Pro140. Mutation of this Pro residue profoundly reduces the ability of O6-benzylguanine to inactivate human alkyltransferase . Despite the weak binding, the reactivity of benzyl in bimolecular displacement reactions such as that occurring in the alkyltransferase active site facilitates the inactivating reaction. However, the rate constant for the reaction with O6-benzylguanine free base is only c. 600 M-1. sec-1, which is >10,000 times less than that for the repair of O6-methylguanine in DNA and oligodeoxyribonucleotides containing O6-benzylguanine are much more potent inactivators. Such oligodeoxyribonucleotides are not ideal for clinical use and other attempts to improve the binding of low M.W. pseudosubstrates are needed.

Several benzyl analogs of guanine were sythesized by our collaborators, namely O6-[(aminomethyl)benzyl]guanines (compounds 1- 3) and three O6-(methylbenzyl)guanines (compounds 5-7) shown in Fig. 9. It was found that the nature and position of the substitution profoundly affected the ability to inactivate human alkyltransferase.

Molecular modelling studies using one of the crystal structures available for the protein and its interaction with compounds 1-3, 5-7 and benzylguanine were carried out and provide a plausible explanation for these results and show that the meta-substituent on O6-[3-(aminomethyl)benzyl]guanine (2) provides additional interactions with the active site pocket that increase the affinity for O6-benzylguanine derivatives and generates a more potent and soluble alkyltransferase inhibitor.

Fig.9:-. Substituted O6-benzylguanines.

Inactivation of purified human alkyltransferase in vitro.

In the experimental bio essay studies done by our collaborators, Compound 1, the para- substituted (aminomethyl) derivative of O6-benzylguanine was only slightly more active than O6-benzylguanine itself but the meta-derivative 2 was considerably more active with an ED50 of 0.4 nM compared to 100 nM for O6-benzylguanine when assayed in the presence of DNA and 17 nM compared to 300 nM when assayed in the absence of DNA (Table 2).

The ortho-derivative 3 was much less active than O6-benzylguanine or the other substituted O6-benzylguanines with an ED50 of 45 µM when assayed in the presence of DNA and 80 µM when assayed in the absence of DNA (Table 2). The improved activity of an aminomethyl meta- substituent is not mimicked by a simple methyl group. There was no difference between O6-benzylguanine itself, 6 and 5, which had ED50 values of 0.30, 0.25 and 0.32 µM in the absence of DNA and 0.10, 0.16 and 0.20 in the presence of DNA (Table 2). The ortho-methyl substituted compound 7 was similar to 3 in leading to a large loss of inhibitory potency (ED50 values of 90 µM and185 µM in the presence and absence of DNA) (Table 2).

Table 2. Inactivation of purified human alkyltransferase in vitro

Alkyltransferase Inhibitor ED50 (mM)*

tested No DNA + DNA

Wild type O6-benzylguanine 0.30 0.10

Wild type O6-[3-(aminomethyl)benzyl]guanine (2) 0.017 0.004

Wild type O6-[4-(aminomethyl)benzyl]guanine (1) 0.23 0.053

Wild type O6-[2-(aminomethyl)benzyl]guanine (3) 80 45

Wild type O6-[3-(methyl)benzyl]guanine (6) 0.25 0.16

Wild type O6-[4-(methyl)benzyl]guanine (5) 0.32 0.20

Wild type O6-[2-(methyl)benzyl]guanine (7) 185 90

Wild type N6-[2-(hydroxymethyl)benzyl]

-2-aminoadenine (4) >1000 >1000

S159A mutant O6-[3-(aminomethyl)benzyl]guanine (2) 0.013 0.003

S159A mutant O6-[4-(aminomethyl)benzyl]guanine (1) 0.16 0.028

Molecular docking of inhibitors to human alkyltransferase. (Excerpt taken from our publication (23))

Computational docking studies were performed using the GLIDE program (version 4.5, Schrödinger, LLC, New York, NY, 2007). The docked structures were chosen for comparison with experimentally determined ED50 values using either the Glide Score or Emodel scoring function. For enhanced docking accuracy the best docked structures using GLIDE extra precision (XP) mode were used to calculate ligand partial charges in the protein environment and then redocked with XP using Schrödinger's QPLD (Quantum Polarized Ligand Docking) method. The Maestro user interface, version 8.0, Schrödinger, LLC, New York, NY, 2007 was employed to set up the GLIDE docking studies and for visualization of the results. The AGT X-ray crystal structure chosen for our modeling studies was human alkyltransferase bound to DNA containing O6-methylguanine and Cys145 mutated to serine to prevent the alkylation reaction from taking place. In our modeling studies, Ser145 was mutated back to cysteine. And generated a global energy conformer of the protien-ligand complex using Macromodel conformational search.

To validate the docking approach, self-docking was performed with XP/QPLD using the partial native ligand: O6-methylguanine . The RMS of the docked pose when compared to the crystallographically observed position of the O6-methylguanine moiety was 0.14 Å, and thus GLIDE produced a docking mode that closely resembled the X-ray crystal structure. Thus, our hypothesis was that GLIDE would be capable of producing docking poses for the compounds studied that are similar to the position, orientation and conformation adopted by the ligands prior to nucleophilic attack by cysteine 145 and that the docking scores obtained would correlate well with the experimentally observed ED50 values for enzyme inactivation. Although there is a crystal structure available for human alkyltransferase benzylated at Cys145, this structure was not employed to model the O6-benzylguanine analogs described in this study since the GLIDE program is unsuitable for modeling covalent bonds formed between the ligand and protein.

In addition to O6-methylguanine, compounds 1-3, 5-7 and O6-benzylguanine were docked to human alkyltransferase using the GLIDE XP. Each pose of these compounds was redocked with XP using the QPLD method. The coefficient of determination (i.e., the square of the correlation coefficient, R2) was calculated between QPLD Glide Scores vs. log(ED50) values, determined in the presence of DNA, as part of a linear regression analysis. An acceptable R2 value of 0.86 between log(ED50) values and XP/QPLD Glide Scores was obtained (Fig. 10).

Emodel scores were also used for correlation studies. It had been previously observed by Bytheway and Cohran that Emodel scores correlated better with log(ED50) values than Glide Scores in their particular study. In our study, the coefficient of determination obtained between E-Model scores (obtained from XP/QPLD calculations) vs. log(ED50) values via a linear regression analysis gave an exceptional R2 of 0.96, shown in Fig. 11. It is noteworthy that Emodel is a composite scoring function, derived from a combination of the Glide Score itself coupled with Coulombic energy, van der Waals energy, and ligand strain energy terms, and is used by GLIDE to select the best docking pose for each individual inhibitor regardless of the scoring function subsequently used and to rank order the inhibitors. Infact much effort was put to generate this global conformer of the AGT protein ligand complex that gave astonishing correlation results. Prior to this global conformer generation, studies were pursued with native PDB 1T38 structure and prepared the protein ligand complex using protein preparation wizard of Maestro as described earlier.

It is noteworthy that the coefficient of determination for the initial SP and XP dockings (without QPLD) were 0.41 and 0.34 between the experimental Log(ED50) values and the Glide scores for the orginal protien complex with SER 145 residue(data not shown). And coefficient of determination for the initial XP redockings with QPLD were 0.55 for XP and 0.462 for Emodel studies(data not shown).

Later on to see the impact on the binding free energies, Solvation factors are also taken into consideration, we present here the MM/GBSA calculations that were carried out based on the quantum mechanics (QM)-polarized ligand docking (QPLD)-derived partial charges(22). The binding free energies are calculated incorporating protein flexibility of 4A at the active site and also calculating the binding free energies with and with out ligand strain. A correlation was calculated between Dgbind Vs Log(ED50 ) via a linear regression analysis and the coefficient of determination is 0.29 with ligand strain correlation coefficient is 0.28 without ligand strain. So usage of Macromodel conformatinal studies helped in obtaining an excellent coefficient of determination 0.96.

Figure 10 :- Plot of log(ED50) values versus QPLD_Glide_scores. Numbering corresponds to compounds listed in Table 3. The ED50 values determined in the presence of DNA from Table 2 were used.

Figure 11:- Plot of log(ED50) values versus E-Model scores. Numbering corresponds to compounds listed in Table3. The ED50 values determined in the presence of DNA from Table 2 were used.

From examination of the QPLD based docking modes, one can conclude that the binding affinities correlate quite well with the number of hydrogen bonds and good van der Waals contacts formed between the inhibitor and the alkyltransferase binding site. There is insufficient space to adequately accommodate the ortho substituted benzylguanines. Thus, due to fewer favorable van der Waals contacts and due to their fewer hydrogen bonds, compounds 3 and 7 are the least potent of the inhibitors we tested in the present study. On the other hand, compounds 1 and 2 exhibit four and three hydrogen bonds respectively and both form good van der Waals contacts with the receptor. All the values of these parameters are summarized in Table 3.

Table 3. Docking parameters derived from docking compounds 1-3, 5-7, O6-benzylguanine and O6-methylguanine to human alkyltransferase.

Compound Inhibitor E-model Glide H-bonds Good Bad

Number Score Score vdw vdw

Contacts Contacts

2 O6-[3-(aminomethyl)benzyl]guanine -88.1 -9.42 4 297 8

1 O6-[4-(aminomethyl)benzyl]guanine -82.0 -9.46 3 312 8

5 O6-[4-(methyl)benzyl]guanine -81.5 -9.54 3 306 7

O6-benzylguanine -78.2 -8.97 3 287 4

6 O6-[3-(methyl)benzyl]guanine -77.5 -9.30 3 289 8

O6-methylguanine -56.6 -7.16 4 177 3

3 O6-[2-(aminomethyl)benzyl]guanine -52.2 -6.75 2 260 5

7 O6-[2-(methyl)benzyl]guanine -52.0 -6.12 1 180 1

* vdw Van der Waals

Fig. 12 shows the key hydrogen bonding interactions of compound 2 with human alkyltransferase. Interactions with residues Tyr114, Cys145, Ser159 and Asn137 are seen. (All except for the interaction with Tyr114 involve interaction with protein backbone rather than the amino acid sidechains.)

Fig. 12. The key hydrogen bonding interactions of compound 2 (O6-[3-(aminomethyl)benzyl]guanine) with the protein residues represented in stick model. The inhibitor is represented in green for carbon, blue for nitrogen and red for oxygen. The rest of the protein as pale green ribbon cartoon.

Fig. 13 shows an overlay of all of the inhibitor poses in the active site with O6-methylguanine.. It is clearly apparent that compounds 3 and 7 are not oriented in the catalytic site of human alkyltransferase in same way as the other inhibitors.

Fig. 13. Overlay of the position of all of the docked compounds in the human alkyltransferase active site with the native ligand O6-methylguanine and parent inhibitor O6-benzylguanine. All of the potent inhibitor poses (1, 2, 5, 6 and O6-benzylguanine) in green are oriented in the same way as the native ligand O6-methylguanine. The ineffective inhibitors O6-[2-(aminomethyl)benzyl]guanine (3) in red and O6-[2-(methyl)benzyl]guanine (7) in yellow are not oriented in the same way as O6-methylguanine. The rest of the color code is the same as Fig 12.

Molecular Docking parameters employed: - The computational modeling studies relied upon the GLIDE (Grid-based Ligand Docking from Energetics) program (Glide, version 4.5, Schrödinger, LLC New York, NY 2007) for the docking simulations. These simulations were performed using the X-ray crystal structure of the human O6-alkylguanine-DNA alkyltransferase bound to a DNA oligonucleotide and containing O6-methylguanine determined at 3.2 Å resolution (PDB ID:1T38) [The protein databank: Berman, H.M., J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, and P.E. Bourne, The Protein Data Bank. Nucleic Acids Res, 2000, 28, 235-42.]. For our studies, DNA was removed (except for O6‑methylguanine , see Fig. 14. Solvent molecules in the protein crystal structure were deleted, except for a water molecule in the active site (WAT 1), and the protein was then prepared for the docking studies by processing it using Schrodinger's protein preparation facility. This procedure minimizes the protein to 0.30 Å RMSD using the OPLS-2001 force field.

Later the protein/ligand complex was taken and pursued Macromodel Mixed torsional /Low-mode sampling conformational search (MacroModel, version 9.5, Schrödinger, LLC, New York, NY, 2007) to obtain the global energy conformer of the protein/ligand complex. This procedure employed OPLS 2001 force field, with Distance-dependent electrostatic treatment with 2.0 units of dilectric constant. 5A0 around the native ligand and water molecule were allowed to minimize. Later ASN 157 and ARG 135 residues along with the rest of the protein residues were frozen for the conformational search. The ligand was allowed to translate and rotate in the active site and generated 892 conformers. The lowest energy conformer that retained the native ligand pose in the X-ray crystal structure of PDB 1T38 was carried for further studies.O6-Methylguanine, O6-benzylguanine and the O6-benzylguanine derivatives were prepared using Schrodinger's LigPrep facility.

Fig. 14. Prepared protein of the human alkyltransferase without DNA. O6-methylguanine and WAT1 also shown.

The initial docking studies were done with GLIDE ( version 4.5, Schrödinger, LLC, New York, NY, 2007) operating in either SP or XP mode . Maestro, version 8.0, Schrödinger Suite 2007, LLC New York, NY 2007) was employed as the graphical user interface and for generation of the graphics used in the figures. The best docked structures were chosen using the Glide_gscore function (Glide Score). For enhanced docking accuracy the best docked structures from XP were used to calculate the ligand partial charges and then redocked using Schrödinger's QPLD (Quantum Polarized Ligand Docking) method.To validate the docking approach, self docking was performed using the partial native ligand: O6-methylguanine using XP/QPLD. The RMS of the docked pose when compared to the crystallographically observed position was 0.14 Å, and thus GLIDE produced a docking mode that closely resembled the X-ray crystal structure.

CONCLUSIONS: (Excerpt taken from our publication 23)

The addition of a meta aminomethyl- group to O6-benzylguanine forming compound 2 results in an approximately 20-fold improvement in the ability to inactivate purified human alkyltransferase. This improvement is seen in assays conducted with or without added DNA. It is noteworthy that the molecular modeling studies showed a similar trend and an R2 of 0.90(data not shown) for a linear regression analysis of log(ED50), determined in the absence of DNA, vs. XP/QPLD Glide Scores and an R2 of 0.98(data not shown) for log(ED50), determined in the absence of DNA, vs. Emodel scores were obtained. As previously noted, inactivation of alkyltransferase by O6-benzylguanine is enhanced 3-4 -fold by the presence of DNA, which stimulates the rate of alkyl transfer . In contrast, some O6-benzylguanine derivatives with bulky adducts are much less potent inhibitors in the presence of DNA since they cannot be accommodated in the active site when DNA is bound there . The inactivation of alkyltransferase by compounds 1 and 2 was increased by the presence of DNA indicating that these compounds, like O6-benzylguanine, do not compete with DNA for access to the active site. However compound 1, the para aminomethyl- derivative did not show any improvement in inhibitory potency over O6-benzylguanine itself. These results are very well explained by the molecular modelling studies, which indicate the formation of an additional hydrogen bond when compound 2 is bound in the active site. The aminomethyl- group from 2 but not 1 is able to interact with Asn137. This also explains why a simple methyl substitution (compound 6) was ineffective since it cannot form this bond. The modelling studies also show clearly why the ortho- substituted O6-benzylguanine derivatives (3 and 7) are much less effective (> 200-fold) than O6-benzylguanine since, due to steric clashes, they cannot be positioned in the same way as the parent compound.

Our results not only demonstrate that 2 may be a valuable alkyltransferase inhibitor but they also show clearly the value of molecular modelling for the design of improved alkyltransferase inhibitors. The remarkable R2 values obtained using XP/QPLD with E-Model scoring for ranking the compounds tested here shows that the structure of human alkyltransferase bound to DNA containing O6-methylguanine and Cys145 mutated to serine can clearly be used as a starting structure for this process. The mutation of Ser145 back to cysteine, as is appropriate for the wild type enzyme, and refinement of the structure obtained with MacroModel was essential in order to obtain the R2 values we report. The model we have generated should be highly useful in the design of more potent inhibitors by performing additional modeling studies on O6-benzylguanine analogs and by virtually screening databases of commercially available compounds to identify potential new lead compounds for further elaboration. So far obtained more than 100 lead hits that can be probable drug molecules by the virtual screening docking studies by Glide SP and XP docking in the global energy conformer, carried using NCI Diversity set obtained from NIH.

My future research involves finding Lead molecules to inhibit AGT protien. So presently pursuing virtual screening docking studies by docking large libraries of Zinc Leadlike compounds ( around 1.8 million compounds) in the Global energy conformer of AGT 1T38 obtained through macro model studies.