Strong DNA Binding Research Articles

Design, synthesis, structural inspection, and DFT calculation of some novel imine ciprofloxacin metal chelates: A novel approach for pharmaceutical applications and DNA interaction

Novel compounds with pharmacological activity were synthesized from Pd (II), Fe (III), Cr (III), Ni (II), and Cu (II) ions with 4‐{2‐(3‐carboxy‐1‐cyclopropyl‐6‐fluoro‐7‐piperazin‐1‐yl‐2,3‐dihydro‐1H‐quinolin‐4‐ylideneamino)‐phenylimino}‐1‐cyclopropyl‐6‐fluoro‐7‐piperazin‐1‐yl‐1,4‐dihydro‐quinoline‐3‐carboxylic acid (CFPD). The newly synthesized compounds have been investigated by 1H‐ and 13C‐NMR spectra, Fourier transform infrared spectra, CHN analyses, ultraviolet‐visible spectra, mass spectra, molar conductivity, and magnetic moment measurements. In addition, the pH profile of the CFPD complexes showed remarkable stability, and their stability constant was identified in solution. To find out essential characteristics for CFPD and its complexes and to investigate the molecular geometry, computational analysis occurred. Through its nitrogen and OH of carboxylate groups, the ligand interacted with the metal ions to form CFPDPd complex with a square planar geometry and CFPDCu, CFPDCr, CFPDNi, and CFPDFe complexes with octahedral geometry. The M:L ratio of 1:1 was demonstrated by the molar ratio and sequence variation techniques’ outcomes. The effect of the investigated CFPD imine ligand on bacterial community and its metal chelate was examined within vitro using a range of fungal and viral pathogens The findings showed that the effectiveness of antimicrobial went with the directive: CFPDPd complex when compared to the highly suppressor complex, fluconazol and ofloxacin as model medication. The novel ligand’s and its complexes’ in vitro cytotoxic potential against the Hep‐G2, MCF‐7, and HCT‐116 cell lines was also studied. The findings once more indicated that, when compared to vinblastine medication, the CFPDPd chelate is the most active agent. In addition, the complexes showed high reactivity in catching free radicals when their antioxidant activity was examined. By the application of viscosity, spectrum analysis, and gel electrophoreses, the interaction among metal chelates and DNA was determined. Studies on viscosity and spectrophotometric titration showed that every substance in test is a strong DNA binder. Increased hydrophobic and electrostatic interactions between aromatic rings could be the cause of this. In conclusion, these complexes have the potential to be effective bioactive agents.

Synthesis, characterization, DNA interaction, molecular docking, and α‐glucosidase inhibition studies of 3‐(pyrimidin‐2‐ylthio) groups substituted water soluble zinc (II) phthalocyanine

In this work, 3‐(pyrimidin‐2‐ylthio)phthalonitrile (n‐MP1), non‐peripherally 3‐(pyrimidin‐2‐ylthio) groups substituted Zn (II) phthalocyanine (n‐MP2), and its water soluble derivative (n‐MP3) have been firstly synthesized and characterized with spectral data. The interaction of the n‐MP3 complex with DNA was examined in vitro using UV–visible titrimetric and thermal denaturation assays and in silico by performing molecular docking studies. In addition, the antidiabetic activity of n‐MP3 was revealed spectroscopically by studying α‐glucosidase inhibition activities. The spectroscopic results indicated that n‐MP3 effectively binds to CT‐DNA with a Kb value of 2.0 × 105 M−1 and interacts with CT‐DNA via noncovalent binding mode. Besides, docking studies divulged that n‐MP3 exhibits a stronger binding tendency (BE: −10.64 kcal/mol) with DNA than the control compounds, (7.78 kcal/mol for ethidium bromide and −6.21 kcal/mol for cisplatin). Consequently, due to its strong DNA binding activity, n‐MP3 may be suitable for antimicrobial and anticancer applications after further toxicological test systems. Also, n‐MP3 complex inhibited the activity of α‐glucosidase with the IC50 value of 1.44 ± 0.08 μM, whereas IC50 value of acarbose was 237.24 ± 1.80 μM. Therefore, it can be said that n‐MP3 is a very effective α‐glucosidase inhibitor.

Molecular and thermodynamic determinants of self-assembly and hetero-oligomerization in the enterobacterial thermo-osmo-regulatory protein H-NS.

Environmentally regulated gene expression is critical for bacterial survival under stress conditions, including extremes in temperature, osmolarity and nutrient availability. Here, we dissect the thermo- and osmo-responsory behavior of the transcriptional repressor H-NS, an archetypal nucleoid-condensing sensory protein, ubiquitous in enterobacteria that infect the mammalian gut. Through experiments and thermodynamic modeling, we show that H-NS exhibits osmolarity, temperature and concentration dependent self-association, with a highly polydisperse native ensemble dominated by monomers, dimers, tetramers and octamers. The relative population of these oligomeric states is determined by an interplay between dimerization and higher-order oligomerization, which in turn drives a competition between weak homo- versus hetero-oligomerization of protein-protein and protein-DNA complexes. A phosphomimetic mutation, Y61E, fully eliminates higher-order self-assembly and preserves only dimerization while weakening DNA binding, highlighting that oligomerization is a prerequisite for strong DNA binding. We further demonstrate the presence of long-distance thermodynamic connectivity between dimerization and oligomerization sites on H-NS which influences the binding of the co-repressor Cnu, and switches the DNA binding mode of the hetero-oligomeric H-NS:Cnu complex. Our work thus uncovers important organizational principles in H-NS including a multi-layered thermodynamic control, and provides a molecular framework broadly applicable to other thermo-osmo sensory proteins that employ similar mechanisms to regulate gene expression.

Synthesis, characterization, DNA interaction, molecular docking, and α-amylase and α-glucosidase inhibition studies of a water soluble Zn(II) phthalocyanine.

In this study, 2(3),9(10),16(17),23(24)-tetrakis-[(N-methyl-(1-benzylpiperidin-4-yl)oxy)phthalocyaninato]zinc(II) iodide (ZnPc-2) was synthesized and characterized using spectral methods (FT-IR, 1H-NMR, UV-Vis and mass spectroscopy). The interaction of ZnPc-2 with DNA was investigated by using the UV/Vis titrimetric method, thermal denaturation profile, agarose gel electrophoresis and molecular docking studies. Additionally, the antidiabetic activity of ZnPc-2 was revealed spectroscopically by studying α-amylase and α-glucosidase inhibition activities. The spectroscopic results indicated that ZnPc-2 effectively binds to calf thymus-DNA (CT-DNA) with a Kb value of 7.5 × 104 M-1 and interacts with CT-DNA via noncovalent binding mode. Gel electrophoresis results also show that ZnPc-2 binds strongly to DNA molecules and exhibits effective nuclease activity even at low concentrations. Furthermore, docking studies suggest that ZnPc-2 exhibits a stronger binding tendency with DNA than the control compounds ethidium bromide and cisplatin. Consequently, due to its strong DNA binding and nuclease activity, ZnPc-2 may be suitable for antimicrobial and anticancer applications after further toxicological tests. Additionally, antidiabetic studies showed that ZnPc-2 had both α-amylase and α-glucosidase inhibition activity. Moreover, the α-glucosidase inhibitory effect of ZnPc-2 was approximately 3500 times higher than that of the standard inhibitor, acarbose. Considering these results, it can be said that ZnPc-2 is a moderate α-amylase and a highly effective α-glucosidase inhibitor. This suggests that ZnPc-2 may have the potential to be used as a therapeutic agent for the treatment of type 2 diabetes.

Exploring the phototoxicity of GSH-resistant 2-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)quinoline-based Ir(III)-PTA complexes in MDA-MB-231 cancer cells.

Metal complexes play a crucial role in photo-activated chemotherapy (PACT), which has recently been used to treat specific disorders. Triple-negative breast cancer has an enormously high rate of relapse due to the existence and survival of cancer stem cells (CSCs) characterized by increased amounts of glutathione (GSH). Hence, designing a phototoxic molecule is an enticing area of research to combat triple-negative breast cancer (TNBC) via GSH depletion and DNA photocleavage. Herein, we focus on the application of PTA and non-PTA Ir(III) complexes for phototoxicity in the absence and presence of GSH against MDA-MB-231 TNBC cells. Between these two complexes, [Cp*IrIII(DD)PTA]·2Cl (DDIRP) exhibited better phototoxicity (IC50 ∼ 2.80 ± 0.52 μM) compared to the non-PTA complex [Cp*IrIII(DD)Cl]·Cl (DDIR) against TNBC cells because of the high GSH resistance power of the complex DDIRP. The significant potency of the complex DDIRP under photo irradiation in both normoxia and hypoxia conditions can be attributed to selective transportation, high cellular permeability and uptake towards the nucleus, GSH depletion by GSH-GSSG conversion, the ability of strong DNA binding including intercalation, and oxidative stress. The strong affinity to serum albumin, which serves as a carrier protein, aids in the transport of the complex to its target site while preventing glutathione (GSH) deactivation. Consequently, the complex DDIRP was developed as a suitable phototoxic complex in selective cancer therapy, ruling over the usual chemotherapeutic drug cisplatin and the PDT drug Photofrin. The ability of ROS generation under hypoxic conditions delivers this complex as a hypoxia-efficient selective metallodrug for the treatment of TNBC.

An Outlook of the Structure Activity Relationship (SAR) of Naphthalimide Derivatives as Anticancer Agents.

The efficacy of drugs against cancer in clinical settings may be limited due to pharmacokinetic issues, side effects and the emergence of drug resistance. However, a class of anticancer drugs known as naphthalimides have proven to be very effective. These derivatives have demonstrated to be effective in treating different types of cancers and exhibit strong DNA binding affinity. The anticancer properties of the naphthalimide derivatives allow them to target a number of cancer cell lines. Researchers have investigated the anticancer activity of numerous naphthalimide derivatives, such as heterocyclic fused, non-fused substituted, metal-substituted and carboxamide derivatives. Surprisingly, some derivatives demonstrate greater activity than the reference norms, such as cisplatin, amonafide, mitonafide and others and are selective against many cell lines. The primary objective of this research is to comprehend the effects of various substitution patterns on the structure-activity relationship (SAR) of these derivatives and the instances in which they enhance or reduce this biological activity.

Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

Synthesis, DNA binding and biological evaluation of benzimidazole Schiff base ligands and their metal(ii) complexes.

Two novel benzimidazole ligands (E)-2-((4-(1H-benzo[d]imidazole-2-yl)phenylimino)methyl)-6-bromo-4-chlorophenol (L1) and (E)-1-((4-(1H-benzo[d]imidazole-2-yl)phenylimino)methyl)naphthalene-2-ol (L2) with their corresponding Cu(ii), Ni(ii), Pd(ii) and Zn(ii) complexes were designed and synthesized. The compounds were characterized by elemental, IR, and NMR (1H & 13C) spectral analyses. Molecular masses were determined by ESI-mass spectrometry, and the structure of ligand L1 was confirmed by single crystal X-ray diffraction analysis. Molecular docking was carried out for the theoretical investigation of DNA binding interactions. The results obtained were verified experimentally by UV/Visible absorption spectroscopy in conjunction with DNA thermal denaturation studies. It was observed that ligands (L1 and L2) and complexes (1-8) were moderate to strong DNA binders, as evident from the binding constants (K b). The value was found to be highest for complex 2 (3.27 × 105 M-1) and lowest for 5 (6.40 × 103 M-1). A cell line study revealed that breast cancer cells were less viable to the synthesized compounds compared to that of standard drugs, cisplatin and doxorubicin, at the same concentration. The compounds were also screened for in vitro antibacterial activity for which complex 2 showed a promising broad-spectrum effect against all tested strains of bacteria, almost in the proximity of the reference drug kanamycin, while the rest of the compounds displayed activity against selected strains.

Hook, Line, and Sinker! Spectroscopic Studies of Bi-Modular Mono- and Bis-1,8-naphthalimide-Ru(bpy)3-conjugates as DNA "Light Switches".

Bi-chromophoric ruthenium polypyridyl complexes comprising one or two nitro-1,8-naphthalimide groups are shown to be effective DNA binders with off–on light switching properties. The binding to DNA was investigated using a combination of studies such as UV–visible absorption and emission titrations, thermal denaturation, and circular dichroism spectroscopy. The DNA affinity was shown to be sensitive to both the linker length and the number of naphthalimides (one vs two) contained in these systems and binding constants ranging from 106 to 107 M–1 for salmon testes DNA. The strong DNA binding is attributed to the combination of naphthalimide intercalation and the electrostatic interaction of the ruthenium complex. Large emission enhancements from the metal to ligand charge transfer (MLCT) emission arising from the metal complex were observed upon DNA binding, which was attributed to the interruption of intramolecular electron transfer quenching processes. Moving the nitro substitution from the 4-position to the 3-position is found to result in modification of the DNA binding and the resulting optical properties. The off–on light switch phenomena reported demonstrate the potential of these complexes to act as DNA probes.

Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers.

Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine’s influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compositions. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug–membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug’s Raman signature was observed only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug–membrane interactions, making it a powerful tool for in vitro drug screening.

Nucleus-targeting imaging and enhanced cytotoxicity based on naphthalimide derivatives

Organelles possess critical biological effects in cellular processes. However, the relationship between organelle targeting and antitumour activity is a challenging issue. In this paper, a number of amide/acylhydrazine modified naphthalimide derivatives were designed and synthesized. Interestingly, amide modified naphthalimide derivatives NI-A-NH and NI-C-NH with (R)-piperdine and (S)-pyrrolidine functionalization exhibited enhanced cytotoxicity compared with acylhydrazine modified derivatives NI-A-2NH and NI-C-2NH. However, acylhydrazine modified derivatives NI-B-2NH and NI-D-2NH with (S)-piperdine and achiral piperdine conjugates possessed better cytotoxicity than NI-B-NH and NI-D-NH with amide modifications. Fluorescence imaging, DNA binding interactions and cell cycle analyses were further completed to clarify that the nucleus-targeting effects showed enhanced cytotoxic activity, strong DNA binding and the blocking of cells in S phase. These results provide a preliminary theoretical basis for the further design of organelle-targeting antitumour drugs.

2′-Thiophenecarboxaldehyde derived thiosemicarbazone metal complexes of copper(II), palladium(II) and zinc(II) ions: Synthesis, spectroscopic characterization, anticancer activity and DNA binding studies

Heterocyclic 5′-methyl-2-thiophenecarboxaldehyde-N(4)-un/substituted thiosemicarbazones (1–3) and their metal complexes with copper(II), palladium(II) and zinc(II) ions (4–11) were synthesized and characterized by using different spectroscopic techniques like FT-IR, 1H, 13C NMR, UV–Visible and electron paramagnetic spectroscopy, etc. The crystal structures of the synthesized thiophene thiosemicarbazone ligands 1, 2 and 3 were determined unambiguously by single-crystal X-ray diffraction analysis. The EPR spectra of the copper(II) complexes, 4, 6 and 9 have shown rhombic, normal axial and inverse axial symmetry respectively. Furthermore, the DNA binding study was performed on calf-thymus DNA by absorbance, fluorescence and viscosity measurement methods. Notably, the copper(II) and palladium(II) complexes have strong DNA binding interactions compared to the zinc(II) complexes. The DNA binding constants measured for these complexes were found to be in the range of 1.83 × 104–1.45 × 105 M−1. Further, the newly synthesized complexes were also tested for their in vitro anticancer activity against three types of human cancer cell lines (COLO-205, MCF-7 and HepG-2) and one normal cell line (HEK-293). Importantly, the zinc(II) complex (8) exhibited potent anticancer activity against HepG-2 with an IC50 value of 11.65 ± 1.753 µM compared to the standard drug cis-platin. The copper(II) complex, 9 against COLO- 205 with an IC50 value of 23.08 ± 1.354 µM, complex 4 against MCF-7 and HepG-2 with IC50 values of 39.35 ± 1.825 µM and 35.26 ± 0.354 µM, respectively, and complex 6 against HepG-2 with an IC50 value of 38.30 ± 1.385 µM have displayed moderate activity compared to the reference drug cis-platin.

Multiple phenyl ring appended Re-based complexes for strong visible light absorption and DNA binding.

A series of facial-Re(CO)3-based acyclic complexes (1-3) possessing N∩O bis-chelating ligands and a triphenylphosphine oxide donor was synthesized and characterized. The most interesting structural feature of these complexes is that both the coordinated ligands are functionalized with external phenyl rings. Single crystal X-ray diffraction analysis confirms that each molecule demonstrates a variety of intermolecular supramolecular interactions via the external phenyl rings. All the complexes displayed strong absorption (ε = 14 000-40 000 M-1 cm-1) over wide visible region (400-600 nm). Due to the interesting structural and photophysical properties of the complexes, a satisfactory DNA binding ability was observed. The binding of the complexes with DNA grooves was confirmed by a significant increase in the absorption intensities of the complexes in the visible region, absorption enhancement of DNA and circular dichroism.

1,2,4]Triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4′,3′-c]quinazoline derived DNA intercalators: Design, synthesis, in silico ADMET profile, molecular docking and anti-proliferative evaluation studies

In view of their DNA intercalation activities as anticancer agents, novel fifteen [1,2,4]triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4′,3′-c]quinazoline derivatives have been designed, synthesized and evaluated against HepG2 and HCT-116. The molecular design was performed to investigate the binding mode of the proposed compounds with DNA active site. The data obtained from biological testing highly correlated with that obtained from molecular modeling studies. HCT-116 was found to be more sensitive cell lines to the influence of the new derivatives. In particular, compounds 16, 18, 11 and 5 were found to be the most potent derivatives with IC50 = 3.61, 6.72, 7.16 and 5.18 µM respectively against HepG2 cell line. Also, compounds 16, 18, 11 and 5 displayed IC50 = 2.85, 3.82, 4.97 and 6.40 µM respectively against HCT-116 cell line. These derivatives displayed higher activities than doxorubicin, (IC50 = 7.94 and 8.07 µM respectively) against the two HepG2 and HCT-116 cell lines. The most active anti-proliferative derivatives 5, 6, 10, 11, 13, 16, 18, 19 and 20 were further evaluated for their DNA-binding affinity which revealed the ability of these compounds to intercalate DNA. The tested compounds displayed very strong to moderate DNA-binding affinities. Compounds 16 and 18 potently intercalate DNA at IC50 values of 26.03 and 28.37 µM respectively which were lower than IC50 of Doxorubicin (IC50 = 31.27). This finding indicated that these derivatives exhibited higher DNA binding activities than Doxorubicin. Also, compounds 11 and 5 displayed very strong DNA binding at IC50 = 30.84 and 33.56 µM respectively, which were nearly equipotent to that of doxorubicin. Moreover, most of our derivatives exhibited good ADMET profile.

Synthesis and antimicrobial evaluation of a pyrazoline-pyridine silver(I) complex: DNA-interaction and anti-biofilm activity.

The emergence of resistant bacterial strains mainly due to misuse of antibiotics has seriously affected our ability to treat bacterial illness, and the development of new classes of potent antimicrobial agents is desperately needed. In this study, we report the efficient synthesis of a new pyrazoline-pyridine containing ligand L1 which acts as an NN-donor for the formation of a novel silver (I) complex 2. The free ligand did not show antibacterial activity. High potency was exhibited by the complex against three Gram-negative bacteria, namely Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumanii with the minimum inhibitory concentration (MIC) ranging between 4 and 16μg/mL (4.2-16.7μM), and excellent activity against the fungi Candida albicans and Cryptococcus neoformans (MIC ≤ 0.25μg/mL = 0.26μM). Moreover, no hemolytic activity within the tested concentration range was observed. In addition to the planktonic growth inhibition, the biofilm formation of both Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was significantly reduced by the complex at MIC concentrations in a dose-dependent manner for Pseudomonas aeruginosa, whereas a biphasic response was obtained for MRSA showing that the sub-MIC doses enhanced biofilm formation before its reduction at higher concentration. Finally, complex 2 exhibited strong DNA binding with a large drop in DNA viscosity indicating the absence of classical intercalation and suggesting the participation of the silver ion in DNA binding which may be related to its antibacterial activity. Taken together, the current results reveal that the pyrazoline-pyridine silver complexes are of high interest as novel antibacterial agents, justifying further in vitro and in vivo investigation.

Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

Modeling of the PTF1‐J and PTF1‐L Heterotrimeric Transcription Factor Complexes

Students in Biomedical Research Studies at Thomas Jefferson High School in Dallas, Texas participate in an ongoing research program in collaboration with the University of Texas Southwestern Medical Center to study the structure and function of a transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a heterodimer of E12/E47 and Ptf1a (basic helix‐loop‐helix transcription factors), and either Rbpj or Rbpjl (Rbpj is also present in Notch signaling). The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells of the exocrine pancreas. The Thomas Jefferson students using 3D printing technology has generated models for the PTF1‐J and PTF1‐L transcription factor complexes. The models provide the orientation of the Ptf1a/E12 heterodimer relative to the Rbpj and Rbpjl proteins and characterizes interactions formed between Ptf1a (tryptophan 298) and a hydrophobic pocket present in the beta‐trefoil domain of Rbpj or Rbpjl. Determination of the structures for the PTF1‐J and PTF1‐L complexes will help elucidate the structural determinants and the mechanism for the strong cooperative DNA binding of these complexes.Support or Funding InformationDallas Junior League: Grant for Innovative TeachingTexas Medical Association Award for Excellence in Science Teaching

Molecular mechanisms of Evening Complex activity in Arabidopsis

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

Fighting Against Promoter DNA Hyper-Methylation: Protective Histone Modification Profiles of Stress-Resistant Intestinal Stem Cells.

Aberrant DNA methylation in stem cells is a hallmark of aging and tumor development. Recently, we have suggested that promoter DNA hyper-methylation originates in DNA repair and that even successful DNA repair might confer this kind of epigenetic long-term change. Here, we ask for interrelations between promoter DNA methylation and histone modification changes observed in the intestine weeks after irradiation and/or following Msh2 loss. We focus on H3K4me3 recruitment to the promoter of H3K27me3 target genes. By RNA- and histone ChIP-sequencing, we demonstrate that this recruitment occurs without changes of the average gene transcription and does not involve H3K9me3. Applying a mathematical model of epigenetic regulation of transcription, we show that the recruitment can be explained by stronger DNA binding of H3K4me3 and H3K27me3 histone methyl-transferases as a consequence of lower DNA methylation. This scenario implicates stable transcription despite of H3K4me3 recruitment, in agreement with our RNA-seq data. Following several kinds of stress, including moderate irradiation, stress-sensitive intestinal stem cell (ISCs) are known to become replaced by more resistant populations. Our simulation results suggest that the stress-resistant ISCs are largely protected against promoter hyper-methylation of H3K27me3 target genes.

Synthesis, structural characterization, in vitro DNA binding, and antitumor activity properties of Ru(II) compounds containing 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline

The octahedral Ru(II) complexes containing the 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline ligand of type [Ru(N-N)2(L)]2+, where N-N = phen (1,10-phenanthroline) (1), bpy (2,2'-bipyridine) (2), and dmb (4,4'-dimethyl-2,2'-bipyridine) (3); L(dmpip) = (2(2,6-dimethoxypyridine-3-yl)1Himidazo(4,5-f)[1, 10]phenanthroline), have been synthesized and characterized by UV–visible absorption, molar conductivity, elemental analysis, mass, IR, and NMR spectroscopic techniques. The physicochemical properties of the Ru(II) complexes were determined by UV–Vis absorption spectroscopy. The DNA binding studies have been explored by UV–visible absorption, fluorescence titrations, and viscosity measurements. The supercoiled pBR322 DNA cleavage efficiency of Ru(II) complexes 1–3 was investigated. The antimicrobial activity of Ru(II) complexes was done against Gram-positive and Gram-negative microorganisms. The in vitro anticancer activities of all the complexes were investigated by cell viability assay, apoptosis, cellular uptake, mitochondrial membrane potential detection, and semi-quantitative PCR on HeLa cells. The result indicates that the synthesized Ru(II) complexes probably interact with DNA through an intercalation mode of binding with complex 1 having slightly stronger DNA binding affinity and anticancer activity than 2 and 3.