Effects on tumor size
Although there are no published reports on the growth of T47D-KBluc xenografts in mice, data from parental T47D xenografts show a slow, linear growth pattern rather than exponential (48). To better assay for antitumor activity of polyamide 1, we conducted similar experiments over a longer period of time. T47D-KBluc xenografted tumors were grown for 2 weeks before initiating treatment, and treatment with polyamide 1 was conducted twice per week for a total of 4 weeks. We observed no significant change in tumor size at the experimental endpoint, although we found a sustained suppression of luciferase output in the polyamide-treated arm as compared with vehicle-treated, consistent with our initial observations. The IC 50 for cytotoxicity of polyamide 1 in cell culture is 0.47 μmol/L, which we believe to be higher than the concentrations achieved within the tumor tissues in this study.
2 QSAR Lab Ltd., Trzy Lipy 3 St, 80-172 Gdańsk, Poland
* Correspondence: firstname.lastname@example.org; Tel.: +48-42-6314-600; Fax +48-42-6314-610
Abstract: The expanding applications of nanotechnology seem to be a response to many technological, environmental, and medical challenges. The unique properties of nanoparticles allow for developing new technologies and therapies. Among many investigated compounds is manganese and its oxides, which in the form of nanoparticles, could be a promising alternative for gadolinium-based contrast agents used in diagnostic imaging. Manganese, which is essential for living organisms as an enzyme cofactor, under excessive exposure—for example, due to water contamination or as an occupational haz- ard for welders—can lead to neurological disorders, including manganism—a condition similar to Par- kinson’s disease. This review attempts to summarise the available literature data on the potential appli- cations of manganese and manganese oxide nanoparticles and their biologicalactivity. Some of the pub- lished studies, both in vitro and in vivo, show negative effects of exposure to manganese, mainly on the nervous system, whereas other data suggest that it is possible to develop functionalised nanoparticles with negligible toxicity and novel promising properties.
result from a physical phenomena such as insolubility (due to aggregation) at the higher concentration. In general, β -aryl polyamides show decreased solubility relative to β - amino compounds owing to their reduced charge at physiological pH.
Other than delivery, perhaps the most important future challenge lies in developing new methods to define the concentration-dependent effects of Py-Im polyamides on gene expression in living cells. Analytical techniques such as MPE footprinting and affinity cleavage have proven essential to the design of sequence- selective DNA-binding agents, 35,36 and reveal dose-dependent binding patterns that can be used to directly guide applications in vitro. 37,38 Quantitative fluorescence analysis of polyamide uptake, as performed here, combined with recently developed high-throughput sequencing strategies for analysis of protein-DNA binding 39,40 and gene expression 39 represent promising approaches to similarly footprint polyamide-induced perturbations and binding events in vivo, and thereby define the relationship between nuclear concentration and gene regulatory effects.
Phenotypes of bone marrow subpopulations were analyzed to determine which hematopoietic cell subsets were affected and to determine if CB7993113 could protect all subsets from DMBA-induced toxicity. No significant changes were seen in the resident bone marrow T cell (CD3 + ) or natural killer cell (NK1.1 + ) populations following treatment with DMBA, CH223191, CB7993113, or combinations of DMBA with either antagonist (data not shown). In contrast, DMBA treatment significantly reduced the number of pre/pro-B cells (IgM - /B220 + /CD43 + /HSA - ), pro-B cells (IgM - /B220 + /CD43 + /HSA + ) (Hardy and Hayakawa, 2001), and neutrophils (CD11b hi /GR-1 hi ) (Sukhumavasi et al., 2007) (Figure 9C, 9D, 9E). Notably, pretreatment of mice with either antagonist significantly inhibited DMBA-induced toxicity in all three cell populations. These data confirm the ability of a prototypic AHR ligand to adversely affect bone marrow cells destined to contribute to the adaptive immune response, i.e., pre/pro- and pro-B cells, and to the innate immune response, i.e., neutrophils. Importantly, CB7993113 prevented a significant DMBA-induced loss of these three bone marrow cell subsets. These data demonstrate the achievement of physiologically relevant doses of CB7993113 in vivo, and suggest that this non-toxic antagonist could be used to block AHR activity either induced acutely by environmental ligands or chronically during a variety of pathological conditions.
While protein-level inhibitors aimed to inhibit enzyme activity or disrupt binding enjoy a large degree of commercial success, pyrrole-imidazolepolyamides (“PIPs”) and their accurate DNA base pair recognition [ 1 – 5 ] provide a promising option against a large class of so-called “undruggable” targets such as KRAS [ 6 – 8 ] which lack solvent-accessible surfaces or pockets for ligand binding. From paired heterocyclic amide building blocks of pyrrole and imidazole, PIPs can distinguish G/C and A/T (T/A) pairs via DNA minor groove binding with Im/Py and Py/Py pairs, respectively, down to the precision of a single hydrogen bond between the bases. Chemical functionalization provides versatility in regulating biological events [ 9 – 15 ] such as cell reprogramming, NF-κB-dependent gene transcription, retinal development, and the inhi- bition of specific biological targets [ 16 , 17 ]. Nevertheless, as PIPs principally bind typically well less than 20 bp, the impact of binding at genomic locations other than the intended design site remains unclear to-date. Without exploring other possible binding sites, it may be too prema- ture to conclude that unanticipated binding to non-KRAS loci has no effect, since off-target effects could also contribute to cancer cell death and mask the true efficacy of a therapeutic candidate. In spite of recent developments in next-generation sequencing, PIP binding studies, especially those involving alkylating functional groups, remain few and far between at the genome level. Hopefully with the procedure discussed in this manuscript, we can introduce a useful addition in the design pipeline to understand whether off-target binding may have a large effect on the tumor before more costly studies, e.g. animal models. Sequencing studies involving bioactive DNA-targeting polyamides conjugated with a psoralen moiety for photo- crosslinking and biotin as affinity tag [ 18 ] so far have been largely restricted to chromatinized regions in the living cell genome until the latest report with a 6-bp alkylating PIP [ 19 ], albeit in an ex vivo setting. Following our previous study of a PIP with 9-bp recognition demonstrating toxicity against KRAS G12 mutant cell lines [ 20 ], we seek to evaluate, via a new sequencing pro- cedure with the biotinylated KR12 to affinity capture nucleotides bearing binding sites for the polyamide in vivo, and computational workflow to determine whether other genomic binding sites in living cells may present an issue in the effectiveness of alkylating PIPs as therapeutic candidates. Corroborating findings here with our previous publication in which KR12 demon- strated distinguishable toxicities in KRAS G12D/G12V over wild-type cells suggested specific targeting of the mutant driver gene by the polyamide. Results here also revealed, for the first time at 9-bp precision, insights into the manner in which PIPs access the human genome in cells.
In order for DNA-binding molecules to be useful tools for gene regulation, they must be able to access DNA in biological systems. DNA in eukaryotic organisms exists in a highly condensed form as chromatin. The nucleosome core particle (NCP) represents the most basic unit of the higher order structure chromatin. The NCP consists of 147 bp of DNA wrapped twice around an octamer of histone proteins. The histone octamer contains two copies each of histones H2A, H2B, H3, and H4. One of the most interesting features of the NCP is the alignment between the major and minor-grooves of the two gyres of DNA. These aligned minor-grooves separated by a small gap between the gyres of DNA create a “super groove”, consisting of 14-16 bp of DNA that is accessible for recognition. Each of these supergrooves bring sequence elements that are 80 bp apart in the linear DNA strand into close spatial proximity. As a result, supergrooves create a recognition platform that exists solely in the context of the NCP.
ically blunted the ability of YY1 to block Tat activation (Fig.
In summary, we show that RCS-binding polyamides targeted to the HIV LTR can inhibit LSF binding to the RCS in vitro and can increase expression of the LTR. RCS-binding poly- amides selectively upregulate expression of the integrated HIV-1 promoter in three different model systems. We ob- served increasing expression in HUT78 T cells in the absence of Tat and in viral expression in the ACH2 model of persistent infection, and we observed relief of YY1-induced repression in the HeLa LTR-CAT reporter system. Pyrrole-imidazole poly- amides, which are synthetic cell-permeable small molecules, are novel and useful tools to inhibit the DNA-binding activity of transcription factors and directly modulate target gene ac- tivity in a living cell.
been synthesized to pursue higher specificity with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that they both bound to the predetermined sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method named Bind-n-Seq-based motif identification with reference sequence (Bind-n-Seq-MR). High- throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were also performed to validate the new method of analysis.
Introduction In developing an efficient sequence-specific alkylat-
ing agent, we found that the choice of alkylating moiety DNA-alkylating agents have long been of interest for and linker region are very important. We describe here a their biological properties. They constitute a major class comparative study of DNA sequence-specific alkylation of such antitumor drugs as nitrosoureas, mitomycin C, and antitumor activity of three different alkylating ImPy cisplatin, and nitrogen mustards, and they are routinely diamide conjugates 1–3 utilizing high-resolution dena- used in hospitals. Many alkylating antitumor agents are, turing gel electrophoresis and a panel of 39 human can- in fact, practically nonselective and cannot distinguish cer cell lines. To gain insight into the origin of the intense between cancer cells and normal cells; as a result, they antitumor activity of ImPyLDu86 (3) expression level, a exhibit severe toxicity to normal tissues. Recently, it has gene chip analysis was also performed.
Pyrrole-Imidazole (Py-Im) polyamides are programmable, sequence specific DNA minor groove binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24hr period. Liver, kidney, and lung samples were collected postmortem.
15. Johnston HJ, Hutchison GR, Christensen FM, Aschberger K, Stone V (2010) The biological mechanisms and physicochemical characteristics responsible for driving fullerene toxicity. Toxicol Sci 114:162 – 182
16. Aschberger K, Johnston HJ, Stone V, Aitken RJ, Tran CL, Hankin SM, Peters SA, Tran CL, Christensen FM (2010) Review of fullerene toxicity and exposure appraisal of a human health risk assessment, based on open literature. Regul Toxicol Pharmacol 58:455 – 473
We report a Py-Im polyamide with activity against enzalutamide-resistant CaP in cell and animal models. Polyamide ARE-1, targeted to the sequence 5`-WGWWCW-3`, which is similar to the ARE and GRE half site, attenuates ligand induced AR and GR transcriptional activity, is more potent than enzalutamide and bicalutamide in cell culture, and is active against enzalutamide resistant xenografts. Long term treatment of LREX’ cells with ARE-1 also decreases nascent RNA synthesis. In biophysical experiments, polyamides can halt RNAP2 elongation directly upstream of a polyamide binding site (19). We hypothesize this stalling of RNAP2 promotes ubiquitination and degradation of RPB1, ultimately interfering with RNA synthesis, which may contribute to efficacy against treatment refractory CaP. Other molecules that interfere with RNA synthesis are proposed as potential drug candidates for CaP (13,20).
The selective modulation of eukaryotic gene expression by small molecules may have important implications in the field of chemical biology and human medicine. Pyrrole-imidazolepolyamides are a class of synthetic ligands that can be programmed to bind the minor groove of specific DNA sequences. 1 Antiparallel, side-by-side N-methylpyrrole (Py) and N- methylimidazole (Im) carboxamides (Im/Py) pairs distinguish G·C from C·G base pairs, N- methyl-3-hydroxypyrrole (Hp)/Py shows specificity for T·A over A·T, whereas Py/Py pairs are specific for both T·A and A·T. 2–5 By linking two strands of these heterocyclic oligomers via a - amino butyric acid (GABA) turn unit, hairpin Py-Im polyamides can be programmed to bind a large library of DNA sequences with affinities comparable to natural DNA-binding proteins. 6–8 Hairpin polyamides have been shown to localize to the nuclei of living cells, and regulate endogenous gene expression by disrupting protein / DNA interfaces. 9–17 Cyclic polyamides containing a second GABA turn unit exhibit further enhanced DNA binding properties. 18–21 We have recently demonstrated their gene regulatory effects on AR-activated gene expression in prostate cancer models. 22
Mutant KRAS is a therapeutically important target, and there are at present no drugs that successfully target mutant KRAS. Conventional drugs aim at inhibiting the KRAS protein or downstream targets. We decided to take a different approach by targeting the KRAS codon 12 mutation directly. We evaluated the sequence-specific alkylating activity of four Py–Im polyamide conjugates in targeting the KRAS codon 12 G12D and G12V mutation sequences. Two of the alkylating Py–Im polyamides displayed high reactivity for the target site; conjugate 4 displayed the highest selectivity and was selected for further evaluation. The computer-minimized structure of the G12D mutation sequence suggested that the structural motif of 4 was appropriate for efficient sequence-specific alkylation of the target sequence. High-throughput sequencing (Bind-n-Seq) revealed the binding specificity of conjugate 5 against 10 bp randomized sequences. Conjugate 5 displayed significant affinity for the KRAS codon 12 mutation sequences with 89- and 69-fold enrichment for the G12D and G12V sequences, respectively, but only fourfold enrichment for the WT sequence. The 9 base pair recognition of Py-Im polyamide 4 allows the recognition of approximately 9,121 sites in the human genome (GRCh38/hg38) containing the KRAS match sequence for preferential alkylation.  Our results strongly suggest that seco-CBI Py–Im polyamides are potential agents for the treatment of cancer based on individual genetic mutations. This approach may help minimize side effects and overcome the problem of drug resistance. Specifically targeting the KRAS codon 12 mutation sequence may be a viable therapeutic route for targeting tumors harboring mutant KRAS.
Effective treatment for enzalutamide-resistant prostate cancer is an unmet need. The LREX’
prostate cancer model is resistant to the antiandrogen enzalutamide via activation of an alternative nuclear hormone receptor (NHR), glucocorticoid receptor (GR), which has similar DNA binding specificity to the androgen receptor (AR). Small molecules that target DNA to interfere with protein-DNA interactions may retain activity against enzalutamide- resistant prostate cancers where ligand binding domain antagonists are ineffective. A pyrrole-imidazole (Py-Im) polyamide designed to bind the consensus androgen response element half-site has antitumor activity against hormone sensitive prostate cancer. In enzalutamide-resistant LREX’ cells this polyamide interferes with both androgen receptor and glucocorticoid receptor driven gene expression while enzalutamide interferes with only that of androgen receptor. Genomic analyses indicate immediate interference with the androgen receptor transcriptional pathway. Long-term treatment with the polyamide demonstrates a global decrease in RNA levels, consistent with inhibition of transcription.
Pyrrole-Imidazolepolyamides (PAs) are a family of minor groove binding ligands which have proven ability to organize non-natural functionality along DNA nanostructures with high affinity and selectivity 34,35,37 . Despite being considerably smaller in molecular weight relative to ODNs, PAs can organize small molecule fluorophores 36 through to larger molecular weight structures such as proteins 38,39 , DNA nanorings 40–42 and metal nanoparti- cles 43 . We recently reported the versatility of PAs to construct a DNA-based photonic wire 36 . Using this SUBJECT AREAS:
Exogenous chemical and biological methods to control directly expression of selected endogenous genes could have broad implications for human medicine. Towards this goal, a number of technological approaches are currently being investigated. Polydactyl zinc finger proteins are a programmable class of DNA binding proteins capable of sequence- specific binding (1, 2). These designed proteins have been used to inhibit expression of target genes (3), and transcriptional activator domain-zinc finger conjugates have been used to activate expression of target genes (4). The RNA-interference pathway can be used to regulate gene expression at the post-transcriptional level (5). siRNA and shRNA molecules enlist cellular machinery to degrade selected mRNA targets (6, 7). RNAi technology has been highly effective in achieving potent and specific knock-down of target mRNAs and is now widely used to probe target gene function (8). However, bioavailability and delivery of zinc finger proteins and siRNA to targets in humans could be an obstacle to their therapeutic application and continues to receive considerable attention (8). Cell- permeable small molecules that modulate protein-protein or protein-DNA interactions offer another approach to the control of endogenous gene regulation. Screening small molecule and natural product libraries for a desired effect can identify candidate molecules with high likelihoods of possessing drug-like bioavailability; drawbacks include the need to screen anew for each target protein-protein or protein-DNA interaction. Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) are a class of programmable DNA-binding small molecules previously used to disrupt protein-DNA interactions in a sequence-specific manner in cell culture (9, 10).
Keywords: Imidazole, Heterocyclic Ring, Biologicalactivity.
During the past decade, the concept of Imidazoline (I) receptors has been developed and gained consensus. Different rank order of affinity of ligands indicates the existence of at least two major classes of Imidazoline receptors I 1 and I 2 . 1 Findings from different laboratories have been shown that they are widely distributed in different tissues and species and may participate in the regulation of various physiological functions. Therefore a more definite knowledge of the structure and function of this receptor system could help to the search for therapeutic agents useful for treating efficaciously a variety of disorders such as hypertension, diabetes mellitus, gastric ulcer, endogenous depression and stroke. Some Imidazole containing compounds like clonidine, guanfacine and newly
The previous chapter detailed the evidence for polyamide-induced replication stress in an asynchronous population of DU145 human prostate carcinoma cells (1). This low-level stress was sufficient to activate ATR but not the downstream effector kinase Chk1. In order to investigate the generality of these effects more cell lines should be tested, as each cancer cell line harbors unique genetic alterations that can affect major signaling pathways. Another method for testing the generality of polyamide-induced replication stress is to test for polyamide effects in vitro using cell-free extracts that are capable of undergoing cell cycle progression including DNA replication and DNA damage response signaling. Cell extracts are beneficial because they can be easily synchronized and arrested, thus allowing the polyamide to be dosed before induction of the cell cycle in a controlled manner. Cell extracts also allow for direct comparison of different sequences of polyamides and accurate measurements of effective concentrations, given that there is no variability due to uptake across cell membranes.