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E5. DNA Binding Drugs - Biology

E5. DNA Binding Drugs - Biology


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Most drugs in use today bind to specific proteins and interfere with the activity of the protein. Stuart Schreiber at Harvard has been a leader in the this field.

Another way to inhibitor specific gene transcription is to bind a small single-stranded DNA to the dsDNA to form a triple helix. The single stranded DNA molecule can bind to exposed H bond donors and acceptors of bases not involved in Watson-Crick H-bonding interactions between complementary bases in the ds DNA.

Figure: Triple helix

The single strand binds to such donors that are accessible in the major grove of dsDNA.

Peptide-nucleic acid analogs, as shown below, are promising new drugs. They are less charged than nucleic acids (so can more easily cross a membrane) and are resistance to cleavage by proteases and nucleases. One could image them binding to specific nucleotide sequences and inhibiting processes like DNA replication and transcription.

Transcription factors that are generally activated by genetic events or upstream signaling pathways are key regulators of cell state. Due to the extensive protein-protein interfaces and general absence of hydrophobic pockets that might inhibit protein:protein interactions required for transcriptional regulation, it has been difficult to design drugs that bind to transcription factors and modulate their activity. Moellering et al. report a successful development of a direct-acting antagonist of an oncogenic transcription factor, NOTCH1. This antagonist consists of cell-permeable stabilized α-helical peptides, SAHMs that was "stapled" into a stable helix through addition of two unnatural alkenyl amino acids which through ring closure sterically restrained the peptide in an alpha helix. The helix mimicked one protein:protein interface region in the ternary complex of DNA:NOTCH1:MAML1 (which is a coactivator protein). The peptides antagonized on NOTCH signaling and cell proliferation in T-cell acute lymphoblastic leukemia cells (T-ALL).


DNA Binding Lab Structure Collection

DNA Binding Lab Structure Collection

The DNA Binding Lab structure collection is a stand-alone lab activity with a set of "unknown structures" that students explore to learn about DNA structure and the interactions between DNA and other molecules. This lab is appropriate for both high school and college students.

Many fundamental steps in biology begin when proteins bind to DNA. Proteins copy DNA, cut DNA, repair DNA, use DNA as a template to make RNA, wrap DNA up into structures that help it fit into the nucleus, and bind to DNA to control all of these processes. Other molecules, such as anti-tumor drugs, or antibiotics can also bind DNA. Sometimes these other molecules block activities like DNA replication and transcription. Sometimes, they cause problems and lead to diseases like cancer.

In this digital biology lab, students work with molecular structures to identify the major and minor grooves in DNA and see what it looks like when proteins or drugs are binding to these regions. The Molecule World DNA Binding Lab lets you add molecular modeling to your classroom toolkit.

A set of unknown structures provides an tool for assessment by letting each student investigate a different structure and determine if a protein or drug binds to the major groove, minor groove, or both, and capture an image to hand in as evidence to support their conclusion.

***We have replaced this app with a collection of structures that can be used in Molecule World. Go to the DNA Binding Lab structure collection to download the set of DNA structures and get the lab instructions.


Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter

It has been widely accepted that DNA can adopt other biologically relevant structures beside the Watson-Crick double helix. One recent important example is the guanine-quadruplex (G-quadruplex) structure formed by guanine tracts found in the MYC (or c-myc) promoter region, which regulates the transcription of the MYC oncogene. Stabilization of this G-quadruplex by ligands, such as the cationic porphyrin TMPyP4, decreases the transcriptional level of MYC. Here, we report the first structure of a DNA fragment containing five guanine tracts from this region. An unusual G-quadruplex fold, which was derived from NMR restraints using unambiguous model-independent resonance assignment approaches, involves a core of three stacked guanine tetrads formed by four parallel guanine tracts with all anti guanines and a snapback 3'-end syn guanine. We have determined the structure of the complex formed between this G-quadruplex and TMPyP4. This structural information, combined with details of small-molecule interaction, provides a platform for the design of anticancer drugs targeting multi-guanine-tract sequences that are found in the MYC and other oncogenic promoters, as well as in telomeres.

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Figures

NMR study of the MYC…

NMR study of the MYC promoter guanine-rich sequences. ( a ) The 600…

Structure of the Pu24I quadruplex.…

Structure of the Pu24I quadruplex. ( a ) Stereo view of eight superposed…

Interaction between the Pu24I quadruplex…

Interaction between the Pu24I quadruplex and different ligands as monitored by NMR. (…

NMR study of the Pu24I…

NMR study of the Pu24I –TMPyP4 complex. ( a ) The structure of…

Six superposed refined structures of…

Six superposed refined structures of the Pu24I quadruplex–TMPyP4 complex. ( a ) Side…


Contents

In humans, the TP53 gene is located on the short arm of chromosome 17 (17p13.1). [8] [9] [10] [11] The gene spans 20 kb, with a non-coding exon 1 and a very long first intron of 10 kb. The coding sequence contains five regions showing a high degree of conservation in vertebrates, predominantly in exons 2, 5, 6, 7 and 8, but the sequences found in invertebrates show only distant resemblance to mammalian TP53. [15] TP53 orthologs [16] have been identified in most mammals for which complete genome data are available.

Human TP53 gene Edit

In humans, a common polymorphism involves the substitution of an arginine for a proline at codon position 72. Many studies have investigated a genetic link between this variation and cancer susceptibility however, the results have been controversial. For instance, a meta-analysis from 2009 failed to show a link for cervical cancer. [17] A 2011 study found that the TP53 proline mutation did have a profound effect on pancreatic cancer risk among males. [18] A study of Arab women found that proline homozygosity at TP53 codon 72 is associated with a decreased risk for breast cancer. [19] One study suggested that TP53 codon 72 polymorphisms, MDM2 SNP309, and A2164G may collectively be associated with non-oropharyngeal cancer susceptibility and that MDM2 SNP309 in combination with TP53 codon 72 may accelerate the development of non-oropharyngeal cancer in women. [20] A 2011 study found that TP53 codon 72 polymorphism was associated with an increased risk of lung cancer. [21]

Meta-analyses from 2011 found no significant associations between TP53 codon 72 polymorphisms and both colorectal cancer risk [22] and endometrial cancer risk. [23] A 2011 study of a Brazilian birth cohort found an association between the non-mutant arginine TP53 and individuals without a family history of cancer. [24] Another 2011 study found that the p53 homozygous (Pro/Pro) genotype was associated with a significantly increased risk for renal cell carcinoma. [25]

  1. an acidic N-terminus transcription-activation domain (TAD), also known as activation domain 1 (AD1), which activates transcription factors. The N-terminus contains two complementary transcriptional activation domains, with a major one at residues 1–42 and a minor one at residues 55–75, specifically involved in the regulation of several pro-apoptotic genes. [26]
  2. activation domain 2 (AD2) important for apoptotic activity: residues 43–63. rich domain important for the apoptotic activity of p53 by nuclear exportation via MAPK: residues 64–92.
  3. central DNA-binding core domain (DBD). Contains one zinc atom and several arginine amino acids: residues 102–292. This region is responsible for binding the p53 co-repressor LMO3. [27] (NLS) domain, residues 316–325.
  4. homo-oligomerisation domain (OD): residues 307–355. Tetramerization is essential for the activity of p53 in vivo. involved in downregulation of DNA binding of the central domain: residues 356–393. [28]

Mutations that deactivate p53 in cancer usually occur in the DBD. Most of these mutations destroy the ability of the protein to bind to its target DNA sequences, and thus prevents transcriptional activation of these genes. As such, mutations in the DBD are recessive loss-of-function mutations. Molecules of p53 with mutations in the OD dimerise with wild-type p53, and prevent them from activating transcription. Therefore, OD mutations have a dominant negative effect on the function of p53.

Wild-type p53 is a labile protein, comprising folded and unstructured regions that function in a synergistic manner. [29]

DNA damage and repair Edit

p53 plays a role in regulation or progression through the cell cycle, apoptosis, and genomic stability by means of several mechanisms:

  • It can activate DNA repair proteins when DNA has sustained damage. Thus, it may be an important factor in aging. [30]
  • It can arrest growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition—if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle.
  • It can initiate apoptosis (i.e., programmed cell death) if DNA damage proves to be irreparable.
  • It is essential for the senescence response to short telomeres.

WAF1/CIP1 encoding for p21 and hundreds of other down-stream genes. p21 (WAF1) binds to the G1-S/CDK (CDK4/CDK6, CDK2, and CDK1) complexes (molecules important for the G1/S transition in the cell cycle) inhibiting their activity.

When p21(WAF1) is complexed with CDK2, the cell cannot continue to the next stage of cell division. A mutant p53 will no longer bind DNA in an effective way, and, as a consequence, the p21 protein will not be available to act as the "stop signal" for cell division. [31] Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this cell type, p53 activates numerous microRNAs (like miR-302a, miR-302b, miR-302c, and miR-302d) that directly inhibit the p21 expression in hESCs.

The p21 protein binds directly to cyclin-CDK complexes that drive forward the cell cycle and inhibits their kinase activity, thereby causing cell cycle arrest to allow repair to take place. p21 can also mediate growth arrest associated with differentiation and a more permanent growth arrest associated with cellular senescence. The p21 gene contains several p53 response elements that mediate direct binding of the p53 protein, resulting in transcriptional activation of the gene encoding the p21 protein.

The p53 and RB1 pathways are linked via p14ARF, raising the possibility that the pathways may regulate each other. [32]

p53 expression can be stimulated by UV light, which also causes DNA damage. In this case, p53 can initiate events leading to tanning. [33] [34]

Stem cells Edit

Levels of p53 play an important role in the maintenance of stem cells throughout development and the rest of human life.

In human embryonic stem cells (hESCs)s, p53 is maintained at low inactive levels. [35] This is because activation of p53 leads to rapid differentiation of hESCs. [36] Studies have shown that knocking out p53 delays differentiation and that adding p53 causes spontaneous differentiation, showing how p53 promotes differentiation of hESCs and plays a key role in cell cycle as a differentiation regulator. When p53 becomes stabilized and activated in hESCs, it increases p21 to establish a longer G1. This typically leads to abolition of S-phase entry, which stops the cell cycle in G1, leading to differentiation. Work in mouse embryonic stem cells has recently shown however that the expression of P53 does not necessarily lead to differentiation. [37] p53 also activates miR-34a and miR-145, which then repress the hESCs pluripotency factors, further instigating differentiation. [35]

In adult stem cells, p53 regulation is important for maintenance of stemness in adult stem cell niches. Mechanical signals such as hypoxia affect levels of p53 in these niche cells through the hypoxia inducible factors, HIF-1α and HIF-2α. While HIF-1α stabilizes p53, HIF-2α suppresses it. [38] Suppression of p53 plays important roles in cancer stem cell phenotype, induced pluripotent stem cells and other stem cell roles and behaviors, such as blastema formation. Cells with decreased levels of p53 have been shown to reprogram into stem cells with a much greater efficiency than normal cells. [39] [40] Papers suggest that the lack of cell cycle arrest and apoptosis gives more cells the chance to be reprogrammed. Decreased levels of p53 were also shown to be a crucial aspect of blastema formation in the legs of salamanders. [41] p53 regulation is very important in acting as a barrier between stem cells and a differentiated stem cell state, as well as a barrier between stem cells being functional and being cancerous. [42]

Other Edit

Apart from the cellular and molecular effects above, p53 has a tissue-level anticancer effect that works by inhibiting angiogenesis. As tumors grow they need to recruit new blood vessels to supply them, and p53 inhibits that by (i) interfering with regulators of tumor hypoxia that also affect angiogenesis, such as HIF1 and HIF2, (ii) inhibiting the production of angiogenic promoting factors, and (iii) directly increasing the production of angiogenesis inhibitors, such as arresten. [43] [44]

p53 by regulating Leukemia Inhibitory Factor has been shown to facilitate implantation in the mouse and possibly humans reproduction. [45]

p53 becomes activated in response to myriad stressors, including but not limited to DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress, [46] osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. This activation is marked by two major events. First, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Second, a conformational change forces p53 to be activated as a transcription regulator in these cells. The critical event leading to the activation of p53 is the phosphorylation of its N-terminal domain. The N-terminal transcriptional activation domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases transducing stress signals.

The protein kinases that are known to target this transcriptional activation domain of p53 can be roughly divided into two groups. A first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. A second group of protein kinases (ATR, ATM, CHK1 and CHK2, DNA-PK, CAK, TP53RK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress. Oncogenes also stimulate p53 activation, mediated by the protein p14ARF.

In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 (also called HDM2 in humans), binds to p53, preventing its action and transports it from the nucleus to the cytosol. Mdm2 also acts as an ubiquitin ligase and covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome. However, ubiquitylation of p53 is reversible. On activation of p53, Mdm2 is also activated, setting up a feedback loop. p53 levels can show oscillations (or repeated pulses) in response to certain stresses, and these pulses can be important in determining whether the cells survive the stress, or die. [47]

MI-63 binds to MDM2, reactivating p53 in situations where p53's function has become inhibited. [48]

A ubiquitin specific protease, USP7 (or HAUSP), can cleave ubiquitin off p53, thereby protecting it from proteasome-dependent degradation via the ubiquitin ligase pathway . This is one means by which p53 is stabilized in response to oncogenic insults. USP42 has also been shown to deubiquitinate p53 and may be required for the ability of p53 to respond to stress. [49]

Recent research has shown that HAUSP is mainly localized in the nucleus, though a fraction of it can be found in the cytoplasm and mitochondria. Overexpression of HAUSP results in p53 stabilization. However, depletion of HAUSP does not result to a decrease in p53 levels but rather increases p53 levels due to the fact that HAUSP binds and deubiquitinates Mdm2. It has been shown that HAUSP is a better binding partner to Mdm2 than p53 in unstressed cells.

USP10 however has been shown to be located in the cytoplasm in unstressed cells and deubiquitinates cytoplasmic p53, reversing Mdm2 ubiquitination. Following DNA damage, USP10 translocates to the nucleus and contributes to p53 stability. Also USP10 does not interact with Mdm2. [50]

Phosphorylation of the N-terminal end of p53 by the above-mentioned protein kinases disrupts Mdm2-binding. Other proteins, such as Pin1, are then recruited to p53 and induce a conformational change in p53, which prevents Mdm2-binding even more. Phosphorylation also allows for binding of transcriptional coactivators, like p300 and PCAF, which then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes. Deacetylase enzymes, such as Sirt1 and Sirt7, can deacetylate p53, leading to an inhibition of apoptosis. [51] Some oncogenes can also stimulate the transcription of proteins that bind to MDM2 and inhibit its activity.

If the TP53 gene is damaged, tumor suppression is severely compromised. People who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disorder known as Li-Fraumeni syndrome.

The TP53 gene can also be modified by mutagens (chemicals, radiation, or viruses), increasing the likelihood for uncontrolled cell division. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. [52] Loss of p53 creates genomic instability that most often results in an aneuploidy phenotype. [53]

Increasing the amount of p53 may seem a solution for treatment of tumors or prevention of their spreading. This, however, is not a usable method of treatment, since it can cause premature aging. [54] Restoring endogenous normal p53 function holds some promise. Research has shown that this restoration can lead to regression of certain cancer cells without damaging other cells in the process. The ways by which tumor regression occurs depends mainly on the tumor type. For example, restoration of endogenous p53 function in lymphomas may induce apoptosis, while cell growth may be reduced to normal levels. Thus, pharmacological reactivation of p53 presents itself as a viable cancer treatment option. [55] [56] The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of head and neck squamous cell carcinoma. It delivers a functional copy of the p53 gene using an engineered adenovirus. [57]

Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such example, human papillomavirus (HPV), encodes a protein, E6, which binds to the p53 protein and inactivates it. This mechanism, in synergy with the inactivation of the cell cycle regulator pRb by the HPV protein E7, allows for repeated cell division manifested clinically as warts. Certain HPV types, in particular types 16 and 18, can also lead to progression from a benign wart to low or high-grade cervical dysplasia, which are reversible forms of precancerous lesions. Persistent infection of the cervix over the years can cause irreversible changes leading to carcinoma in situ and eventually invasive cervical cancer. This results from the effects of HPV genes, particularly those encoding E6 and E7, which are the two viral oncoproteins that are preferentially retained and expressed in cervical cancers by integration of the viral DNA into the host genome. [58]

The p53 protein is continually produced and degraded in cells of healthy people, resulting in damped oscillation. The degradation of the p53 protein is associated with binding of MDM2. In a negative feedback loop, MDM2 itself is induced by the p53 protein. Mutant p53 proteins often fail to induce MDM2, causing p53 to accumulate at very high levels. Moreover, the mutant p53 protein itself can inhibit normal p53 protein levels. In some cases, single missense mutations in p53 have been shown to disrupt p53 stability and function. [59]

Suppression of p53 in human breast cancer cells is shown to lead to increased CXCR5 chemokine receptor gene expression and activated cell migration in response to chemokine CXCL13. [60]

One study found that p53 and Myc proteins were key to the survival of Chronic Myeloid Leukaemia (CML) cells. Targeting p53 and Myc proteins with drugs gave positive results on mice with CML. [61] [62]

Most p53 mutations are detected by DNA sequencing. However, it is known that single missense mutations can have a large spectrum from rather mild to very severe functional affects. [59]

The large spectrum of cancer phenotypes due to mutations in the TP53 gene is also supported by the fact that different isoforms of p53 proteins have different cellular mechanisms for prevention against cancer. Mutations in TP53 can give rise to different isoforms, preventing their overall functionality in different cellular mechanisms and thereby extending the cancer phenotype from mild to severe. Recents studies show that p53 isoforms are differentially expressed in different human tissues, and the loss-of-function or gain-of-function mutations within the isoforms can cause tissue-specific cancer or provides cancer stem cell potential in different tissues. [12] [63] [64] [65] TP53 mutation also hits energy metabolism and increases glycolysis in breast cancer cells. [ citation needed ]

The dynamics of p53 proteins, along with its antagonist Mdm2, indicate that the levels of p53, in units of concentration, oscillate as a function of time. This "damped" oscillation is both clinically documented [66] and mathematically modelled. [67] [68] Mathematical models also indicate that the p53 concentration oscillates much faster once teratogens, such as double-stranded breaks (DSB) or UV radiation, are introduced to the system. This supports and models the current understanding of p53 dynamics, where DNA damage induces p53 activation (see p53 regulation for more information). Current models can also be useful for modelling the mutations in p53 isoforms and their effects on p53 oscillation, thereby promoting de novo tissue-specific pharmacological drug discovery.

p53 was identified in 1979 by Lionel Crawford, David P. Lane, Arnold Levine, and Lloyd Old, working at Imperial Cancer Research Fund (UK) Princeton University/UMDNJ (Cancer Institute of New Jersey), and Memorial Sloan-Kettering Cancer Center, respectively. It had been hypothesized to exist before as the target of the SV40 virus, a strain that induced development of tumors. The TP53 gene from the mouse was first cloned by Peter Chumakov of The Academy of Sciences of the USSR in 1982, [69] and independently in 1983 by Moshe Oren in collaboration with David Givol (Weizmann Institute of Science). [70] [71] The human TP53 gene was cloned in 1984 [8] and the full length clone in 1985. [72]

It was initially presumed to be an oncogene due to the use of mutated cDNA following purification of tumor cell mRNA. Its role as a tumor suppressor gene was revealed in 1989 by Bert Vogelstein at the Johns Hopkins School of Medicine and Arnold Levine at Princeton University. [73] [74]

Warren Maltzman, of the Waksman Institute of Rutgers University first demonstrated that TP53 was responsive to DNA damage in the form of ultraviolet radiation. [75] In a series of publications in 1991–92, Michael Kastan of Johns Hopkins University, reported that TP53 was a critical part of a signal transduction pathway that helped cells respond to DNA damage. [76]

In 1993, p53 was voted molecule of the year by Science magazine. [77]

As with 95% of human genes, TP53 encodes more than one protein. Several isoforms were discovered in 2005, and so far 12 human p53 isoforms have been identified (p53α, p53β, p53γ, ∆40p53α, ∆40p53β, ∆40p53γ, ∆133p53α, ∆133p53β, ∆133p53γ, ∆160p53α, ∆160p53β, ∆160p53γ). Furthermore, p53 isoforms are expressed in a tissue dependent manner and p53α is never expressed alone. [12]

The full length p53 isoform proteins can be subdivided into different protein domains. Starting from the N-terminus, there are first the amino-terminal transactivation domains (TAD 1, TAD 2), which are needed to induce a subset of p53 target genes. This domain is followed by the proline rich domain (PXXP), whereby the motif PXXP is repeated (P is a proline and X can be any amino acid). It is required among others for p53 mediated apoptosis. [78] Some isoforms lack the proline rich domain, such as Δ133p53β,γ and Δ160p53α,β,γ hence some isoforms of p53 are not mediating apoptosis, emphasizing the diversifying roles of the TP53 gene. [63] Afterwards there is the DNA binding domain (DBD), which enables the proteins to sequence specific binding. The carboxyl terminal domain completes the protein. It includes the nuclear localization signal (NLS), the nuclear export signal (NES) and the oligomerisation domain (OD). The NLS and NES are responsible for the subcellular regulation of p53. Through the OD, p53 can form a tetramer and then bind to DNA. Among the isoforms, some domains can be missing, but all of them share most of the highly conserved DNA-binding domain.

The isoforms are formed by different mechanisms. The beta and the gamma isoforms are generated by multiple splicing of intron 9, which leads to a different C-terminus. Furthermore, the usage of an internal promoter in intron 4 causes the ∆133 and ∆160 isoforms, which lack the TAD domain and a part of the DBD. Moreover, alternative initiation of translation at codon 40 or 160 bear the ∆40p53 and ∆160p53 isoforms. [12]

Due to the isoformic nature of p53 proteins, there have been several sources of evidence showing that mutations within the TP53 gene giving rise to mutated isoforms are causative agents of various cancer phenotypes, from mild to severe, due to single mutation in the TP53 gene (refer to section Experimental analysis of p53 mutations for more details).


Platinum-intercalator conjugates: from DNA-targeted cisplatin derivatives to adenine binding complexes as potential modulators of gene regulation

Nuclear DNA is the cellular target for many cancer treatments, and DNA-directed chemotherapies continue to play an important role in drug discovery in the postgenomic era. The majority of DNA-targeted anticancer agents bind through covalent interactions, non-covalent intercalation or groove binding, or hybrid binding modes. The sequence and regiospecificity of these interactions and the resulting structural alterations within the biopolymer play an important role in the mechanism of action of these drugs. DNA-binding proteins and/or DNA-processing enzymes, which also interact with DNA in a sequence- and groove-specific manner, are mediators of the cytotoxic effect produced by these agents. Thus one major goal in the design of new clinical agents of this type is to produce new types of adducts on DNA, which may lead to unprecedented cell kill mechanisms. Platinum-intercalator conjugates are such a class of hybrid agents acting through a dual DNA binding mode. The platinum center (usually a cis-diaminedichloroPt(II) unit) dominates the DNA adduct profiles in the majority of these species-the result of the metal's tendency to form cross-links in runs of consecutive guanine bases in the major groove of DNA. This paradigm has been broken recently for the first time with the design of cytotoxic platinum-acridinylthiourea conjugates, a class of adenine-affinic minor-groove directed agents. This review summarizes major advancements in the chemistry and biology of platinum-intercalators from 1984 to 2004, with emphasis being placed on the interplay between chemical structure, mechanism of DNA binding, and biological properties.


Methods

Generation of mutants according to a specific mutation ratio

We used a genetic algorithm (GA) algorithm as the mutation strategy for offspring generation. In the first generation, 10 3 gene sequences were randomly generated each sequence produced 10 4 offspring with a mutation rate of 10 −4 . As the structural modeling and docking processes are computationally very expensive, the size of the population and the frequency of mutations were reduced to a computationally manageable level. In the GA algorithm, population size was controlled by deleting the lowest-ranking individuals, allowing only the top 5% of individuals containing mutations. If the eligible sequence number exceeded 1000, then the top 1000 mutant sequences were selected. The original sequences with the highest scores were used to fill the population if the eligible sequence number was less than 1000. The DNA sequence of each individual was translated into a protein sequence, which was then subjected to protein structure modeling and molecular docking. The final evaluation score of a mutant was calculated, according to Eq. (1). The genetic evolution was considered complete when the top-ranked mutant yielded a lower binding affinity to the drug than to ATP.

Side chain packing calculations

We used the program Scap (http://honig.c2b2.columbia.edu/scap/) to model the structures of the protein mutants 40,41 . A Perl script was used to call the Scap module, with the aim to convert the mutated residues into the corresponding variation in protein structure. Scap is used to build side chain conformations using its coordinate rotamer libraries. As we do not want the mutation to change the protein structure significantly, we chose an AMBER force field with a heavy atom model and a mixed side chain rotamer library. The other parameters were set to the default values, which allowed a relatively stable mutant protein conformation. We used the Scap program to generate structures of thousands of residue mutations.

Protein-drug docking

We used the AutoDock Vina program (http://vina.scripps.edu/index.html, version 1.1.2) to build the drug-mutant and ATP-mutant structures and calculate the binding scores 69 . Default AutoDock Vina parameters were used. The superposition module in Schrödinger 70 was used to calculate the RMSD of ATP in the crystal structure and in the mutants.

ABL structures

During the simulation, we used different ABL crystal structures for different compounds. The structure of ABL complexed with ATP was derived from the ATP-peptide conjugate complex (PDB code: 2G1T) with the protein in an inactive conformation 71 . The structure used for imatinib was 2HYY with the complexed protein in an inactive conformation 72 . The structure used for nilotinib was 3CS9 with the complexed protein in an inactive conformation 73 . For dasatinib, we used 2GQG with the complexed protein in an active conformation 48 . Ponatinib was designed for the T315I mutant. It avoids steric hindrance with the side chain of isoleucine. 3IK3 (with a T315 mutation) complexed with ponatinib was also used. The mutant protein 3IK3 adopts an inactive conformation 62 . The wide-type protein structure in this case was obtained by mutating I315 back to T315.

EGFR structures

The structure of EGFR tyrosine kinase domain in complex with ATP was derived from the thiophosphoric acid O-[(adenosyl-phospho)phosphor]-S-acetamidyl-diester complex (PDB code: 2GS6) with the protein in an active conformation 74 . The structure used for gefitinib was 4I22 with the complexed protein in an active conformation 75 .

Compounds and substrates

Imatinib (MW493.6), nilotinib (MW529.52), dasatinib (MW488.01), and ponatinib (MW532.56) were purchased from Selleck (https://www.selleck.cn/). The substrate peptide was obtained from GL Biochem (Shanghai) Ltd. and had a sequence of Lys-Lys-Gly-Glu-Ala-Ile-Tyr-Ala-Ala-Pro-Phe-Ala-NH2 (Directory peptide Cat # 86721).

Expression and purification of ABL and mutants

The kinase domains of human c-ABL (residues 222–500, NM_005157.5) were subcloned into the NdeI and XhoI restriction sites of the pET-28a vector 76 . The plasmids were transformed into Escherichia coli BL21 (DE3) cells, plated on LB agar containing kanamycin (50 μg mL −1 ), and grown overnight at 37 °C. The next day, the colonies from the plates were resuspended in expression media (LB agar containing kanamycin, 50 μg mL −1 ). Cultures were grown to an OD600 of 1.2 at 37 °C and cooled for 1 h with shaking at 16 °C prior to induction for 22 h at 16 °C with 0.1 mM IPTG. Cells were harvested by centrifugation at 7000 × g at 4 °C for 15 min and stored at −80 °C. The bacterial pellet was resuspended in Buffer A [50 mM Tris (pH 8.0), 500 mM NaCl, 20 mM imidazole] for immediate purification using nickel ion affinity chromatography. Elution of the protein from the column was achieved using a Buffer B gradient [50 mM Tris, 500 mM NaCl, 500 mM imidazole (pH 8.0)], which increased from 0% to 100% over 15 min. The eluted protein was concentrated to 2 mL and subjected to gel filtration using an S200 column 77,78 . The buffer used for the gel filtration was Buffer C [50 mM Tris, 500 mM NaCl (pH 8.0)]. The protein was eluted at 55 min. The purity of the protein was verified by SDS-PAGE.

For the mutants, primers were designed based on the predicted mutations and synthesized by GENEWIZ. Mutations were made using the Fast Site-Directed Mutagenesis kit from TIANGEN, according to the manufacturer’s instructions, and verified by DNA sequencing. The mutant proteins were expressed and purified following the expression and purification of the wt protein.

In vitro kinase inhibition assay

To assess the ability of the drug to inhibit the wt and mutant kinases, we used the ADP-Glo Kinase Assay kit from Promega 79 , which measures the amount of ADP produced in the reaction. The inhibition rates of the drugs at different concentrations were calculated by comparing the amount of ADP produced with and without the drug. Data were analyzed using the Hill1 model in the OriginLab2018 software package.

In vitro enzyme activity assay

ITC experiments were carried out using an ITC200 instrument (Microcal Inc.). ITC has been demonstrated to directly measure the kinetics and thermodynamic parameters (k cat, K M, ΔH) of enzymatic reactions 80 . A one-step method was used to measure the enzymatic parameters. The BCR-ABL concentration was in the nanomolar range, with the ATP/substrate concentration at least three orders of magnitude higher than the enzyme concentration and above the K M [BCR-ABL (10 nM) and substrate (1 mM) in the cell, and ATP (1 mM) in the syringe].

The thermal change (Q) is proportional to the reaction enthalpy (ΔH) and the number of moles of product (n), whereas the moles of product equal the total volume (V) multiplied by the concentration [P]:

According to Eq. (3), the ΔH of the reaction can be obtained by integrating the curve of the Method 1 experiment


Result and Discussion

Experimental studies shows that the ligands (Table 2) used in this study are good antileishmanials ( 4-15 ), but docking of these compounds revealed a great variation in their binding energy. Initially, totally five poses were saved from which best pose with lowest energy was chosen for each compound. Although the predicted free energy of binding is a useful descriptor of ligand–receptor complementarity, the choice of the ‘best’ docking model was ultimately dictated by various parameters of ADME/T study. Our results show that several compounds were not following the given range limit of few ADME/T properties. These include PISA (1/4 component, i.e. carbon and attached hydrogens of the SASA range, 0–400 Å 2 ), QlogPC16 (predicted log of hexadecane/gas partition coefficient range, 4–18), QlogPo/w (predicted log of octanol/water partition coefficient range, −2 to 6), QlogS (Predicted log of aqueous solubility range, −6 to 0.5 m ). QPPMDCK (predicted apparent MDCK cell permeability range, poor < 25 nm/seconds and great > 500 nm/seconds), QPlogHERG (predicted IC50 value of blockage of HERG K+ channel range, <−5) were also found to be violated by 2, 5-bis-(4-amidinophenyl)thiophene (5c) and berenil, respectively (not shown in Table 4).

S. No Compound id G score SASA FOSA FISA PISA WPSA Vol QPpolrz QPlogPC16 QPlogPoct QPlogPw QPlogPo/w QPlogS QPlogHERG PHOA Rule of five Toxicity
1 1a −5.987491 560.887 190.08 127.05 243.8 0 945.45 32.727 9.978 17.222 11.989 2.019 −4.088 −5.436 88.721 0 NO
2 1b −4.727568 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NO
3 1c −4.637121 738.471 0 98.531 639.9 0 1291.9 49.089 16.002 22.977 14.476 4.779 6.764 −8.195 100 0 NO
4 2a −5.621212 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A NO
5 2b −6.474291 551.198 374.14 3.851 173.2 0 1002.4 34.331 8.879 13.409 6.734 3.049 −2.499 −5.046 100 0 NO
6 2c −5.619455 734.312 512.31 176.53 45.47 0 1196 39.575 12.012 23.791 15.36 1.983 −5.99 −5.923 67.157 1 NO
7 2d −5.509143 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A High
8 3 −10.93399 564.425 0 266.67 297.8 0 934.31 28.302 12.334 23.996 19.586 −0.02 −2.279 −5.883 40.131 1 NO
9 4a −11.09014 767.378 426.04 121.51 149.4 70.47 1374.1 45.769 13.979 23.249 12.698 4.021 6.408 −5.821 100 0 NO
10 4b −12.05836 573.216 0 220.97 352.2 0 972.25 32.267 12.575 22.054 15.909 1.681 −3.356 −5.995 57.842 1 NO
11 5a −10.94681 851.379 0 125.74 624.8 100.9 1506.4 54.963 19.511 29.664 16.42 6.064 8.293 −8.614 86.713 2 NO
12 5b −11.71767 889.017 152.68 88.545 571.1 76.73 1632.1 58.194 19.809 29.374 16.427 6.575 8.222 −8.235 96.017 2 NO
13 5c −11.4314 429.265 218.29 210.98 0 0 692.77 21.571 6.541 35.538 6.102 1.414 −3.354 −3.44 57.975 1 NO
14 5d −11.41968 911.9 152.18 102.79 605.6 51.37 1647.7 59.149 20.27 29.734 16.805 6.506 8.509 −8.63 93.195 2 NO
15 5e −10.88554 603.176 0 225.4 349.5 28.32 1020.4 34.168 13.239 22.196 15.51 2.19 −4.031 −6.23 60.07 1 NO
16 6 −9.29 537.669 484.39 53.272 0 0 966.55 31.243 8.127 14.109 6.819 2.467 −2.056 −4.93 82.28 0 NO
  • SASA, total solvent accessible surface area (range, 300–1000 Å 2 ) FOSA, hydrophobic component of the SASA (range, 0–750 Å 2 ) FISA, hydrophilic component of the SASA (range, 7–330 Å 2 ) PISA, 1/4 component of the SASA (range, 0–400 Å 2 ) WPSA, weakly component of SASA (range, 0–150 Å 2 ) Volume, total solvent accessible area (range: 500–2000 Å 3 ) QPpolrz, Predicted polarizability (range, 13–70 Å 3 ) QPlogPC16, rredicted log of hexadecane/gas partition coefficient (range, 4–18) QPlogPoct, predicted log of octanol/gas partition coefficient (range, 8–43) QPlogPw, predicted log of water/gas partition coefficient (range, 5–48) QPlogPo/w predicted log of octanol/water partition coefficient (range, −2 to 6) QplogS, predicted log of aqueous solubility (range, −6 to 0.5 m ) QPlogHERG, predicted IC50 value for blockage of HERG K+ channels (range, concern below −5) PHOS, per cent human oral absorption GScore, Glide Score.
  • The values in bold are out of range from the category of drug.

Hydrophobic drugs with high partition coefficients are preferentially distributed to hydrophobic compartments such as lipid bilayers of cells while hydrophilic drugs (low partition coefficients) preferentially are found in hydrophilic compartments such as blood serum. As adequate solubility and permeability is a prerequisite for drug absorption from the gastrointestinal tract, it plays a significant role for the resulting bioavailability of orally administered drugs. By violating these properties, a compound may be associated with side-effects, high or poor solubility (QlogS) that can result in poor absorption and distribution in the body.

The interactions and other scores resulted from docking studies for each compound are summarized in Table 5. The results are briefly described in the following section.

Ligand Receptor Interaction
LIG.NH (37) DT4.A.O2 (148) Hydrogen bond
LIG DA6.A Hydrophobic
LIG DT7.A Hydrophobic
LIG DT18.B Hydrophobic
LIG DA19.B Hydrophobic

Acridine and its derivatives

Acridine family includes a wide range of tricyclic molecules with various biologic properties. Considered as potential antiparasitic agents since the 1990s, numerous acridine derivatives have been synthesized and successfully assessed for their antimalarial, trypanocidal or antileishmanial properties ( 19-21 ). According to a previous study, N-[6-(acetylamino)-3-acridinyl] acetamide (compound 1c) and N-[6-(benzoylamino)-3-acridinyl] (compound 1b) benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite ( 12 ). But our docking score suggests that only acridine (compound 1a GScore, −5.987491) was the best with no violation of any ADME/T descriptor (Table 4). Compound 1c was found to violate few ADME/T properties when analyzed by QikProp.

Berberin and its derivatives:

Berberine, a quaternary alkaloid and a minor groove binder ( 23 ), has demonstrated experimental and clinical efficacies against both visceral ( 4, 6, 7 ) and cutaneous ( 4 ) leishmaniases. Despite the apparent potential of this compound as an antileishmanial drug, only Putzer ( 22 ) has described the antileishmanial activity of derivatives of berberine and failed to present any biologic or chemical data to substantiate his claims. Further, in support of this study, Vennerstrom et al. ( 9 ) suggested that tetrahydroberberine (2c) was proven less toxic and more potent against Leishmania donovani than berberine (2a), N-methyl tetrahydro berberinium iodide (2b), berberine chloride (2d). In contrast to these results, we have found major variation in the data when applied in silico automated docking calculations. Our results show that N-methyl tetrahydro berberinium iodide (compound 2b) was having the best GScore (−6.474291) among all with no violence of ADME/T parameters. When studied using ToxTree software, berberine chloride (compound 2d) was analyzed as highly toxic among the all compounds (Table 4).

Berenil

Berenil (diminazene aceturate), a minor groove binder in AT-rich domain ( 24 ), has been proven a good antileishmanial previously ( 5, 8 ). Our in silico docking calculation with a GScore of −10.93398 and ADME/T study with no violence of any parameters is in agreement with previous studies (Table 4).

Pentamidine and its derivatives

Pentamidine (compound 5a) is one of the few antileishmanial drugs currently available. It belongs to the diamidine class of drugs, which has been suggested to exert antiparasitic activity by binding to DNA, interfering with polyamine metabolism and disrupting of mitochondrial membrane potential ( 25 ). Typically, these dicationic molecules bind to DNA by selectively interacting with AT-rich regions of the minor groove. The complementarity of the curvature of the dicationic molecules with that of the minor groove of DNA has been considered an important determinant in the interaction of such groove binders with DNA ( 26, 27 ). Pentamidine is already in clinical use but limited by toxicity, administration by injection, and development of resistance ( 10 ). In addition to pentamidine, many of diamidine molecules –N-[2-(methylsulfanyl)-4-<5-[3-(methylsulfanyl)-4-(pyridine-2-imidamido) phenyl] fura N-2-Yl> phenyl] pyridine-2-carboximidamide (5b), 2 5-bis-(4-amidinophenyl)thiophene (5c), N-(3-chloro-4-<5-[2-chloro-4-(pyridine-2-imidamido) phenyl] fura N-2-yl> phenyl) pyridine-2-carboximidamide (5d), 2 4-bis-(4-amidinophenyl)furan (5e) – exhibited promising activity against Leishmania. The antileishmanial activity of several such dicationic molecules was reported earlier ( 10, 11, 13, 28 ).

Docking studies of these compounds including pentamidine reveal that all these compounds were scored a good GScore, but only compounds 5c and 5e satisfied the ADME/T parameters’ range and other three compounds viz. 5a, 5b, 5d seemed to violate few filters.

Diindolyl methane

(3,3′)-Diindolylmethane (DIM, compound 6) is a natural compound, product of acid condensation of indole-3-carbinol (I3C), found in most cruciferous vegetables of the genus Brassica. DIM inhibits an unusual bisubunit topoisomerase I from L. donovani and has been found to interact both with free enzyme and with DNA. A study has been carried out to verify the interaction between DIM-DNA ( 15 ). Their data and our result of its in silico interaction with DNA (GScore, −9.29) and ADME/T study showed that the compound can be a future drug molecule.

Duocarmycine and its derivative

The duocarmycins bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position ( 29 ). Seco-hydroxy-aza-CBI-TMI, analog of duocarmycin (4a), was shown to inhibit the growth of the protozoan parasites L. donovani, Leishmania mexicana, in culture ( 14 ). Seco-hydroxy-aza-CBI-TMI (4b) was also shown (Figures 1 and 2) to score a good binding energy (GScore, −12.058362), and the study of ADME/T parameters with no violence of any parameters is in agreement with the previous studies (Table 4).

Illustration of binding pocket of seco-hydroxy-aza-CBI-TMI in DNA: (A) and (B) Ligand is shown as stick and DNA residues as wire. Possible hydrogen bond is shown as yellow thick line with the atoms and distance (2.844 Å).

(A) Ligand is shown in active site pocket. Active site residues are shown as surface. (B) 2D view of the complex (generated by Poseview v1.0.0 BioSolveIT GmbH, Sankt Augustin, Germany). Hydrogen bond is shown as dashed line, and residues taking part in hydrophobic interaction are in green color. Hydrophobic interactions are shown as green line.


Experimental Section

Cytotoxicity Assay

HeLa cells were seeded on 96-well tissue culture plates at 1.0 × 10 4 cells/well. After incubation in a humidified atmosphere containing 5% CO2 for 24 h at 37ଌ in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, the cells were washed with Leibovitz’s L-15 medium and treated with TAT, E3-TAT, and E5-TAT in L-15. After 1 h incubation, the cells were washed with fresh L-15 medium and incubated at 37ଌ for 3 hours. The number of live cells present in each well was then determined using the Vybrant MTT Cell Proliferation Assay Kit (Molecular probes, Carlsbad, CA). Alternatively, SYTOX® Blue, a nucleic acid fluorescent stain that only penetrates cells with compromised plasma membranes, was used to measure cell-viability by fluorescence microscopy imaging.

Live Cell Assay for the Transduction of Fluorescent Imaging agents

HeLa, COS-7 and COLO 316 cells were seeded on 8-well chamber glass slide at 3.0 × 10 4 cells/well in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and incubated for 24 h at 37ଌ in a humidified atmosphere containing 5% CO2. Cells were then incubated with L-15 medium (no FBS added) and placed on an inverted epifluorescence microscope (Model IX81, Olympus, Center Valley, PA) equipped with a heating stage maintained at 37ଌ. The microscope is configured with a spinning disk unit to perform both confocal and wide-field fluorescence microscopy. Images were collected using a Rolera-MGI Plus back-illuminated EMCCD camera (Qimaging, Surrey, BC, Canada). Images were acquired using phase contrast and three standard fluorescence filter sets: CFP (Ex = 436넠 nm / Em= 480녀 nm), Texas Red (Ex = 560녀 nm / Em= 630녵 nm), and FITC (Ex = 482넵 nm / Em= 536녀 nm). Cells were co-treated with of TAT or E3/E5-TAT (2 µM) in L-15 medium containing fluorescent imaging agents at 10 µM for the fluorescent proteins EGFP, mCherry, and tdTomato (obtained as previously described) and 2.5 mg/mL for the dextrans 10 and 70 kDa anionic Dextran-fluorescein, 10 and 70 kDa neutral Dextran-tetramethylrhodamine, and 70 kDa neutral Dextran-Texas Red.[10] After incubation at 4 or 37ଌ for 15 or 60 minutes, the cells were washed with PBS 5 times and the medium was replaced with fresh L-15. The integrity of the plasma membrane of the cells was determined by addition of the cell-impermeable DNA stain SYTOX® Blue. Cells with a blue fluorescent nucleus, detected with the CFP filter set, were considered compromised or dead. The cells that were not stained by SYTOX® Blue were further confirmed to be alive by detection of active transport of intracellular endocytic vesicles. To inhibit cell surface binding of the delivery peptides, heparin sodium salt (1 mg/mL) was added during the co-incubation step. To inhibit acidification of the endolysosomal organelles, cells were pretreated with bafilomycin (200 nM) for 20 min. Cells were then co-incubated with the fluorescent macromolecules and the delivery peptides in L-15 supplemented with bafilomycin (200 nM). The fluorescence intensities within cells were measured using the SlideBook 4.2 software (Olympus, Center Valley, PA). The mean intensity within cells imaged with the FITC or Texas Red filters was measured as the total intensity of the cell divided by its area. The same protocol was used for cells with either punctate or diffuse distributions. Mean fluorescence intensities were measured over a population of 3000 cells and experiments were reproduced 3 times.

Live Cell Assay for the delivery of PAD

Cells grown on 96-well plates were treated with PAD (20 µM) and TAT (5 µM) or E5-TAT (3, 5, or 7 µM) in L-15. For the inhibition of macropinocytosis, cells were first pretreated with 50 µM of amiloride (Sigma, MO) for 30 minutes, and then with the peptides while keeping amiloride present. After 1 h incubation, the cells were washed with fresh L-15 medium again and incubated at 37ଌ for additional 3 hours. The number of live cells present in each well was then determined using the Vybrant MTT Cell Proliferation Assay Kit (Molecular probes, Carlsbad, CA). After 1 h incubation the cells were washed with L-15. SYTOX® Blue (5 µM) was added and cells were incubated for 15 min at 37ଌ. Cells were then placed on the microscope and images were acquired in the blue fluorescence channel for detection of SYTOX® Blue and red fluorescence channel for detection of TMR.


Structures in the collection

Type Description Download structure
An MW Collection

To view the collection, you will need to download and install the iPad version of Molecule World. Once Molecule World is installed on your iPad, return to this page and download the entire DNA Binding Lab collection at once by clicking the mwc file.

A double-stranded DNA molecule. Hydrogens are shown because this structure was solved by NMR.

A double-stranded DNA molecule. Hydrogens are not seen because this structure was solved by X-ray diffraction.


DNA Intercalation by Quinacrine and Methylene Blue: A Comparative Binding and Thermodynamic Characterization Study

There is compelling evidence that cellular DNA is the target of many anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA-binding agents, quinacrine (QNA) and methylene blue (MB), with calf thymus (CT) DNA. The study revealed noncooperative binding phenomena for both the drugs to DNA with an affinity one order higher for QNA compared to MB as observed from diverse techniques, but both bindings obeyed neighbor exclusion principle. The data of the salt dependence of QNA and MB from the plot of log K versus log [Na + ] revealed a slope of 1.06 and 0.93 consistent with the values predicted by theories for the binding of monovalent cations, and have been analyzed for contributions from polyelectrolytic and nonpolyelectrolytic forces. The binding of both drugs was further characterized by strong stabilization of DNA against thermal strand separation in both optical melting and differential scanning calorimetry studies. The binding data analyzed from the thermal denaturation and from isothermal titration calorimetry (ITC) were in close proximity to those obtained from spectral titration data. ITC results revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. The heat capacity changes obtained from temperature dependence of enthalpy indicated −146 and −78 cal/(mol·K), respectively, for the binding of QNA and MB to CT DNA. Circular dichroism study further characterized the structural changes on DNA upon intercalation of these molecules. Molecular aspects of interaction of these molecules to DNA are discussed.



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