CT DNA Research Articles

DNA interactions and biocidal activity of metal complexes of benzothiazole Schiff bases: synthesis, characterization and validation

Binary complexes of Cu(II), Ni(II) and Co(II) were synthesized using two novel Schiff bases $$\mathbf{L}_{1}$$ = 2-(-(benzothiazol-6-ylimino)methyl)-4-chlorophenol (BTEMCP), $$\mathbf{L}_{2}$$ = 2-(-(benzothiazol-6-ylimino)methyl)-4-nitrophenol. The Schiff bases and metal complexes were characterized by analytical and spectral methods like elemental analysis, Mass, $$^{1}\hbox {H}$$ -NMR, $$^{13}\hbox {C}$$ -NMR, UV-Vis, IR, ESR, SEM, EDX, XRD and magnetic susceptibility measurements. From the analytical data, square planar geometry has been proposed for all the metal complexes. The binding interaction between the metal complexes and DNA was investigated by means of electronic absorption, fluorescence spectroscopy and viscosity measurements. The DNA cleavage ability of the metal complexes was also evaluated by agarose gel electrophoresis method. These studies revealed that the complexes showed an intercalative mode of binding to CT DNA and also effectively cleaved the supercoiled pBR DNA. The synthesised compounds were evaluated for in vitro antibacterial activity against Gram positive and Gram negative bacteria, and found that the metal complexes showed more potent biocidal activity than the Schiff bases. SYNOPSIS Six mononuclear metal complexes of benzothiazole Schiff bases were synthesized and characterized. The DNA interaction studies showed that all the complexes bind with CT-DNA through an intercalative mode. Supercoiled pBR322 DNA was effectively cleaved by the complexes . All the metal complexes exhibited better antibacterial activity than the Schiff bases from which they were prepared.

The binding mode of pyrazolone derived copper(II), cobalt(II), nickel(II) and zinc(II) Schiff base coordination compounds with Calf-Thymus DNA

Transition metal complexes with Schiff base ligands possess remarkable biomedical activities that could be used to develop new metal-based anticancer agents. Anticancer metal complexes have attracted considerable interest since the successful application of platinum based anti-cancer drugs. Pyrazolone derivatives have been extensively studied and have shown a wide range of pharmacological activities and anticancer activities. A new class of mononuclear copper (II), cobalt(II), nickel(II) and zinc(II) complexes were synthesized using pyrazolone precursor Schiff base, obtained by the condensation of 4–aminoantipyrine with salicylaldehyde and respective metal(II) chloride. They have been characterized by elemental analysis, magnetic susceptibility, molar conductance measurements, UV-Vis., FT-IR, 1H NMR spectra and EPR studies. Their magnetic susceptibility values of the complexes at room temperature are consistent with octahedral geometry around the central metal ions. These complexes show lower conductance values, supporting their non-electrolytic nature. The data show that the complexes have composition of [ML2] type. In vitro DNA binding profiles of all complexes have been explored by absorption titrations, cyclic voltammetry technique and viscosity measurements. The corroborative results of above experiments suggest that these complexes are efficient metallo intercalators and can interact with CT-DNA. The binding constant values (Kb) clearly signify that the complexes bind to DNA through intercalation mode. The binding affinity of these complexes with CT DNA is found to be in the order, Cu(II)>Zn(II)>Co(II)>Ni(II), among them copper complex displaying a higher binding propensity as compared to other complexes.

DNA interaction, antioxidant and in vitro cytotoxic activities of some mononuclear metal(II) complexes of a bishydrazone ligand

A tridentate ONO donor bishydrazone ligand, 3-(((3-oxoindolin-2-ylidene)hydrazono) methyl)quinoxalin-2(1H)-one (L) was synthesized. Using the ligand, mononuclear metal complexes of the type [M(L)(OAc)] (M=Co(II), Ni(II), Cu(II) and Zn(II), OAc=–OCOCH3) were synthesized. The newly synthesized compounds were thoroughly characterized by various analytical and spectral techniques. Tetrahedral geometry was proposed for Co(II), Ni(II) and Zn(II) complexes, while square planar geometry has been assigned to the Cu(II) complex. The in vitro antibacterial, antifungal and antioxidant studies reveal that the Cu(II) complex of the bishydrazone ligand possess higher activities. DNA binding studies suggest that the present compounds interact with CT DNA through intercalation. This was further supported by molecular docking. Gel electrophoresis studies substantiated the involvement of free radicals in the cleavage of pUC18 DNA. In vitro cytotoxicity of the compounds against MCF-7 cell line was also examined. The molecular modeling studies reveal that the compounds strongly interact with VEGFR-2 kinase receptor through hydrogen bonding, π-π, π-cation and sigma-π interactions. The bioavailability and bioactivity determination of the compounds suggest that the present compounds may be utilized as drug candidates.

Synthesis, characterization, in vitro cytotoxicity and DNA interaction study of phosphanegold(I) complexes with dithiocarbamate ligands

A new series of phosphanegold(I) dithiocarbamate complexes with general formula [Au(PR3)(S2CNR′2)], 1–5 (Where R=Methyl, ethyl, isopropyl and R′=Methyl, ethyl) were synthesized and characterized by elemental analysis, FTIR and multinuclear NMR spectroscopy. The molecular structure of one of them, [Au(PMe3)(S2CNMe2)] (1) was determined by single crystal X-ray crystallography, which revealed linear geometry around the Au(I) metal center. The in vitro cytotoxicity of all Au(I) complexes was studied against two human cancer cell lines, A549 and HepG2. All the complexes showed significantly higher potency (4 to 6-fold for A549 and 3 to 5-fold for HepG2 cell line) than the commercial chemotherapeutic drug, cisplatin. The in vitro DNA binding abilities of Au(I) complexes 1–5 were examined by employing different biophysical techniques, viz., UV–Vis titrations, fluorescence spectroscopy and circular dichroism. The UV–Vis titration of Au(I) complexes with CT DNA revealed that the complexes bound to CT DNA via electrostatic interactions, and the intrinsic binding constant (Kb) values for 1–5 were found to be 3.90×104, 4.74×104, 6.81×104, 8.53×104 and 2.48×104M−1 respectively, which suggested the highest binding propensity for complex 4. In addition, molecular docking studies of the Au(I) complexes were performed with B–DNA to visualize the preferential docking position, and the results showed the binding of complexes to the adenine–thymine residues in the minor groove of the DNA.

Synthesis and structure elucidation of new open cubane tetranuclear [CuII4] Cluster: Evaluation of the DNA/HSA interaction and pBR322 DNA cleavage pathway and cytotoxicity

New open–cubane tetranuclear copper(II) complex of formulation {[Cu4(HL)4(H2O)2]·(CH3OH)2·(H2O)5}(1) derived from Schiff base ligand 2–((E)–(1,3–dihydroxy–2–methylpropan–2–ylimino)methyl)–6–methoxyphenol (HL) was synthesized. The characterization of both ligand (HL) and tetranuclear Cu complex was carried out by analytical, spectroscopic and single crystal X–ray diffraction. The structure analyses revealed that in the tetranuclear core, the metal centers are mutually interconnected via two phenoxo oxygen and two alkoxo oxygen atoms of four H2L2− ligand to generate a distorted single–open [Cu4(μ3–O)2]6+ open cubane core with the four Cu atoms disposed in an extended “butterfly–like” arrangement. Binding of complex 1 with CT DNA was investigated by using vivid spectroscopic techniques to study the mode of the interaction of 1 towards CT DNA. The results obtained indicated that 1 avidly binds to CT DNA via electrostatic mode with an intrinsic binding constant, Kb value 4.63×104M–1. Complex 1 showed an impressive nuclease activity generating single–strand breaks and the mechanistic investigation confirms the contribution of the reactive oxygen species (ROS). Likewise, the interaction of 1 with human serum album (HSA) revealed the change in intrinsic fluorescence intensity of HSA and was supposed to be associated with the microenvironment of Trp residue. Furthermore, the interaction of complex 1 with DNA and HSA was further investigated by the molecular docking studies which corroborated well with above findings. We have also tested 1 for cytotoxicity against the PC3 (Prostate) and K562 (Leukemia) cancer cell lines. The results exhibited were moderate when compared to standard drug viz, adriamycin (ADR).

Synthesis, characterization and DNA nuclease activity of oxo-peroxomolybdenum(VI) complexes

The synthesis and structural characterization of two oxo-peroxo molybdenum(VI) complexes, [Mo(O)(O)2(PAA)]− (1) and [Mo(O)(O)2(PAH)]− (2), with phenylacetic acid (PAA) and 2-phenylacetylhydroxamic acid (PAHH) ligands have been accomplished. The coordination geometry of the oxo-peroxo molybdenum(VI) complexes is found to be pentagonal bipyramidal where, in both cases, the ligands are coordinated in bidentate fashion through oxygen atoms. The binding affinities of 1 and 2 with calf-thymus DNA (CT DNA) are determined using absorption spectroscopic measurements. The spectroscopic as well as cyclic voltammetric (CV) studies and viscosity measurements indicate that both complexes interact with CT DNA in the groove. The intrinsic binding constants are 5.2 × 104 M−1 and 7.3 × 104 M−1 for 1 and 2, respectively, from UV–vis studies. Complexes 1 and 2 show nuclease activity with plasmid DNA in the presence of H2O2. Concentration-dependent nuclease study suggests that 2 possesses higher ability to cleave plasmid DNA compared to 1. The experimental results of the binding of 1 and 2 with DNA are further supported by molecular docking studies.

New binuclear Ni(II) metallates as potent antiproliferative agents against MCF-7 and HeLa cells

Four new binuclear nickel(II) metallates 1–4 were synthesized from the reaction between NiCl2·6H2O, 1,3-bis(diphenylphosphino)propane and [H2-(MSal-Rtsc)], where R=H, CH3, C2H5, C6H5, MSal=3-methoxysalicylaldehyde, tsc=thiosemicarbazone. The complexes were characterized by various spectral and analytical methods. Distorted square planar geometry of complexes 3 and 4 were evidenced from their crystal structures. Their binding interaction with calf-thymus DNA (CT DNA) and albumin (Bovine serum albumin) were analyzed with absorption and emission spectral titration studies. Based on the observations, the complexes 1–4 act as strong binders to CT-DNA and BSA. The antioxidant properties of the compounds were evaluated to test their free radical scavenging ability. All the complexes exhibited better antioxidant activity than the ligands and were found to be greater than that of the conventional standard vitamin C. Cell proliferation MTT assays performed for all the compounds in the human breast cancer (MCF-7) and human cervical cancer cell line (HeLa) exposed a significant growth inhibition. The complexes showed remarkably high cytotoxicity against human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) than the standard cisplatin. Among the four complexes, complex 3 was the most effective against MCF-7 (IC50 value of 4µM) and HeLa (2.33µM) compared with the other complexes 1 (8.51µM; 10.02µM), 2 (6.25µM; 2.40µM) and 4 (6.62µM; 4.33µM) respectively. Furthermore, the compounds resulted in ROS (reactive oxygen species)-mediated apoptosis, as indicated by DCFDA (2′,7′-dichlorodihydro-fluorescein diacetate) and sub-G1 DNA content was measured by fluorescent activated cell sorting (FACS) analysis.

Heteroleptic silver(I) complexes with 2,2′:6′,2″-terpyridines and naproxen: DNA interaction, EGFR/VEGFR2 kinase, growth inhibition and cell cycle arrest studies

A series of heteroleptic silver(I) complexes of the type [Ag(L1−3)(nap)] (1–3), where L1–3=4′-(4-substituted)-2,2′:6′,2″-terpyridines and nap=naproxen have been synthesized and characterized by elemental analysis and spectroscopic methods. The geometric parameters of the complexes were determined using UV–Vis and DFT calculations together with the IR spectral data. All the complexes adopted distorted tetrahedral geometry around silver(I) ion. The small HOMO-LUMO energy gap supports the bioefficacy of the complexes. DNA binding and melting experiments suggest the intercalative binding mode of the complexes to CT–DNA, which was further supported by molecular docking studies. The molecular docking studies of the heteroleptic silver(I) complexes with EGFR/VEGFR2 kinase receptors show hydrophobic, π-π, σ-π and hydrogen bonding interactions. All the complexes have been found to promote DNA cleavage through hydrolytic pathway. In vitro cytotoxicity activity of the complexes was tested against four human breast adenocarcinoma (MCF-7), cervical (HeLa), epithelioma (Hep-2) and hepatoma (HepG2) cancerous, and one normal human dermal fibroblasts (NHDF) cell lines by MTT reduction assay. The morphological study by Hoechst 33258 staining revealed that the complex 3 induces apoptosis much more effectively than the other complexes. Further, all the complexes increases the DNA synthesis in S phase with the corresponding reduction in G0–G1 and G2/M phase, which suggest the growth inhibition mechanism on HepG2 cells was DNA damage mediated S phase arrest.

Synthesis, characterization, preliminary biological screening, and interaction studies of the bis(pyridinium)amine compound, 4,4'-[(2-aminophenyl)azanediyl]bis(1-methylpyridin-1-ium)triflate with biological macromolecules

Bis(pyridinium)amine 4,4'-[(2-aminophenyl)azanediyl]bis(1-methylpyridin-1-ium)triflate ([H2L][TfO]2) was synthesized and characterized by elemental analysis, UV–Vis, fluorescence, FT-IR, 1H, and 13C NMR spectroscopies, mass spectrometry, and a single crystal X-ray structure analysis. The potential of the title compound to interact with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was investigated with the help of UV–Vis, florescence and docking studies. UV–Vis spectroscopy indicated that the binding of [H2L][TfO]2 with CT–DNA took place via a groove binding mode. [H2L][TfO]2–BSA was characterized by moderate binding constant, electrostatic interaction and spontaneous association. [H2L][TfO]2 Displayed excellent enzyme inhibition activity with Trypsin and acetylcholine esterase. [H2L][TfO]2 was found to be an efficient antioxidant, scavenging the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in a dose dependent manner. [H2L][TfO]2 exhibited activity against six microbes selected for the study.

Selenium nanoparticles synthesized in aqueous extract of Allium sativum perturbs the structural integrity of Calf thymus DNA through intercalation and groove binding

Biomedical application of selenium nanoparticles (SeNPs) demands the eco-friendly composite for synthesis of SeNPs. The present study reports an aqueous extract of Allium sativum (AqEAS) plug-up the current need. Modern spectroscopic, microscopic and gravimetric techniques were employed to characterize the synthesized nanoparticles. Characterization studies revealed the formation of crystalline spherical shaped SeNPs. FTIR spectrum brings out the presence of different functional groups in AqEAS, which influence the SeNPs formation and stabilization. Furthermore the different aspects of the interaction between SeNPs and CT-DNA were scrutinized by various spectroscopic and cyclic voltametric studies. The results reveals the intercalation and groove binding mode of interaction of SeNPs with stacked base pair of CT-DNA. The Stern–Volmer quenching constant (KSV) were found to be 7.02×106Mˉ1 (ethidium bromide), 4.22×106 Mˉ1 (acridine orange) and 7.6×106Mˉ1 (Hoechst) indicating strong binding of SeNPs with CT–DNA. The SeNPs - CT-DNA interactions were directly visualized by atomic force microscopy. The present study unveils the cost effective, innocuous, highly stable SeNPs intricate mechanism of DNA interaction, which will be a milestone in DNA targeted chemotherapy.

Clinical applications of extended ctDNA RAS mutation determination in metastatic colorectal cancer.

607 Background: Tumor tissue is currently used for RAS testing in metastatic colorectal cancer (mCRC) patients, but detection of circulating tumor (ct) DNA in plasma is being actively investigated as an alternative for detection and monitoring RAS mutations during therapy. Methods: Concordance of RAS testing in plasma using the OncoBEAM RAS CRC test and RAS testing in tissue using standard-of-care RAS detection techniques was evaluated in 114 mCRC antiEGFR-naïve patients. In discordant cases, tissue samples were re-examined by BEAMing. OncoBEAM RAS CRC assay was also used to monitor RAS mutation status in serial plasma samples from 34 patients treated with anti-VEGF +/- chemotherapy or anti-EGFR based therapy. Results: The overall agreement of the two methods was 93% (106/114 patients), with positive agreement of 96.2% (51/53) and negative agreement of 90.2% (55/61). Longitudinal monitoring of plasma samples from 13 patients with RAS mutated tumors treated with chemotherapy +/- anti-VEGF, showed a significant decrease (or disappearance) of RAS mutant allele fraction (MAF) in all cases at the time of CT-scan (8-12 weeks after onset of treatment). In 3 patients, plasma was also analyzed at week 4 showing the same dramatic decrease in MAF. RAS decline mirrored clinical and radiological response to treatment. Interestingly, RAS decline was also observed in patients with stable disease in CT-scan by RECIST1.1. In 7 of 9 patients that subsequently progressed, RAS ctDNA fraction increased accordingly. On the other hand, among 22 patients RAS wt treated with cetuximab, 6 showed emergence of RAS mutations at progression. In two cases, multiple RAS mutations were concomitantly present. Conclusions: The high overall agreement between basal plasma and tissue RAS mutation status indicates that blood-based testing with OncoBEAM RAS CRC is a viable alternative to tissue RAS testing in mCRC patients. Moreover, this study highlights the utility of OncoBEAM RAS CRC to monitor treatment of mCRC patients in two setting: (a) evaluation of response to anti-VEGF +/- chemotherapy in RAS mutant patients, as a complement to RECIST radiological criteria and (b) to monitor emergence of resistant clones during anti-EGFR treatment in RAS wt patients.

Magnetic resonance Imaging (MRI), liquid biopsies, and patient derived organoids (PDOs) as biomarkers of response to regorafenib (REG) in treatment-refractory metastatic colorectal cancer (mCRC) patients (pts).

613 Background: REG demonstrated efficacy in pre-treated mCRC pts. Lack of predictive biomarkers, potential toxicities and cost/effectiveness concerns highlight the unmet need for better patient selection. Methods: RAS mutant mCRC pts with biopsiable metastases were enrolled in this phase II trial. Tissue biopsies (6-12 cores) were obtained at baseline (BL), after 2 months if stable disease (SD) and at disease progression (PD). Dynamic contrast enhanced (DCE) MRI was acquired pre and at day 15 post-treatment. Median values of volume transfer constant (Ktrans) and enhancing fraction (EF) [K-trans*EF/100] were generated. Circulating tumour (ct)DNA was collected monthly until PD and tested for clonal RAS mutations by digital droplet PCR. PDOs derived from responders and non-responders pts were implanted orthotopically in the liver of mice and treated with REG for 5 days. Changes in tumour and fractional blood volume (fBV) were monitored by oxygen-enhanced MRI. Results: mCRC pts (n = 27) with paired MRI scans were analysed; a single target lesion per pt was chosen (25 liver and 2 pelvic metastases). Median K-trans*EF/100 product decrease was 58.2%. In the 23 analysable pts (4 received < 1 cycle of treatment due to toxicities), > 70% drop in K-trans*EF/100 (8/23) was associated with higher disease control rate (6/6 vs. 0/6, p = 0.048) measured by RECIST 1.1 at 2 months, improved progression free survival (PFS) [HR = 0.24 (0.07-0.86), p = 0.03], and 4-month PFS (58.3% VS 21.2%). Sequential tissue biopsies analysis confirmed reduction in CD31 in pts with K-trans*EF/100 drop. RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS [HR = 0.25 (0.08 - 0.83), p = 0.02] independently of K-trans*EF/100 drop. PDOs xeno-transplants treated with REG compared to controls had significant lower tumour fBV (4.5 VS 10.6, p = 0.03) and lower microvascular density measured by CD31 staining (4.3 VS 8.9, p = 0.02). Conclusions: Combining DCE MRI and ctDNA predicts depth and duration of anti-angiogenic response to REG monotherapy and may improve pt selection with potential health/economic implications. Clinical trial information: 201400357951.

Mixed ligand ruthenium arene complexes containing N-ferrocenyl amino acids: Biomolecular interactions and cytotoxicity against MCF7 cell line

Synthesis, characterization, DNA and protein binding as well as anticancer activity of the organometallic complexes [Ru(η6-p-cym)(L)Cl] (where, p-cym = p-cymene MeC6H4Pri, L = N-ferrocenyl amino acid conjugates) RAFcA 1–4 have been described. The complexes 1–4 exhibited strong DNA/BSA binding and anticancer activity against breast cancer MCF7 cell line. Their binding with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) have been examined by absorption and emission spectral studies. Strong interactions between complexes and CT-DNA have been affirmed by absorption spectral and EB displacement studies, while interaction with BSA via static quenching was explored by fluorescence titration. Cytotoxicity, apoptosis and in vitro anticancer activity of 1–4 toward MCF7 cell line have been investigated by MTT assay. The IC50 values (37.1 μM - 86.6 μM) were found to be distinctly lower than those of NAMI-A and RAPTA complexes for MCF7 cell line which was followed by gene expression studies to confirm apoptosis as the mode of cell death.

Synthesis, characterization, structure, DNA binding aspects and molecular docking study of a novel Schiff base ligand and its bis(μ-chloro) bridged Cu(II) dimer

A novel Schiff base ligand, 2-(benzothiazol-2-yl-hydrazono)-1,2-diphenyl-ethanone (LH), has been synthesized out of the 1:1 condensation of benzil and 2-hydrazino benzothiazol in methanol. Reaction of equimolar proportion of LH and pre-activated CuCl2·2H2O in methanol generates bis(μ-chloro)bis(2-(benzothiazol-2-yl-hydrazono)-1,2-diphenyl-ethanoate)-dicopper(II), [Cu2(μ-Cl)2(L)2] (1) in copious yield. C, H and N microanalyses, ESI-MS, FT-IR, UV–Vis spectral studies have been undertaken to characterize LH and 1. The X-ray crystal structure of 1 has been determined. LH undergoes keto-enol tautomerism. The tautomeric aspects of LH have been corroborated through DFT calculations. LH binds to copper(II) in its enolate form to give rise the symmetric bis(μ-chloro) bridged copper(II) dimer. Both LH and 1 show the efficacy to bind CT DNA. The binding constants and the thermodynamic binding parameters have also been determined. A favourable major groove binding efficacy has been exposed for both the ligand and complex through molecular docking.

MA08.01 A Highly Sensitive Next-Generation Sequencing Platform for Detection of NSCLC EGFR T790M Mutation in Urine and Plasma

Non-invasive genotyping of NSCLC patients by circulating tumor (ct)DNA is a promising alternative to tissue biopsies. However, ctDNA EGFR analysis remains challenging in patients with intrathoracic disease, with a reported 26-57% T790M mutation detection rate in plasma (Karlovich et al., Clin Cancer Res 2016; Wakelee et al., ASCO 2016). We investigated whether a mutation enrichment NGS could improve mutation detection in plasma and urine from TIGER-X, a phase 1/2 study of rociletinib in patients with EGFR mutation-positive advanced NSCLC. The therascreen (Qiagen) or cobas (Roche) EGFR test was used for EGFR T790M analysis in tumor biopsies. Urine and plasma were analyzed by trovera mutation enrichment NGS assay (Trovagene). Of 174 matched tissue, plasma and urine specimens, 145 (83.3%) were T790M+ by central tissue testing, 142 (81.6%) were T790M+ by plasma, and 139 (79.9%) were T790M+ by urine. Urine and plasma combined identified 165 cases (94.8%) as T790M+. Of 25 cases positive by ctDNA but negative/inadequate by tissue, 16 were double-positive in plasma and urine, unlikely to be false positive (Figure 1). T790M detection rate was higher for extrathoracic (n=119) vs intrathoracic (n=55) disease in plasma (87.4% vs 69.1%, p=0.006) but not urine (81.5% vs 76.4%, p=0.42). Combination of urine and plasma identified T790M in 92.7% of intrathoracic and 95.8% of extrathoracic cases (p=0.47). In T790M+ patients, objective response rate was similar whether T790M mutation was identified by tissue, plasma or urine: 37.4%, 33.1% and 36.6%, respectively. 4 of 9 patients T790M+ by urine but negative by tissue responded, and 2 of 8 patients T790M+ by plasma but negative by tissue responded. Mutation enrichment NGS testing by urine and plasma combined identified 94.8% of T790M+ cases. Combination of urine and plasma may be considered before tissue testing in EGFR TKI resistant NSCLC, including patients without extrathoracic metastases.

Kinetic and mechanistic study on the reactions of ruthenium(ii) chlorophenyl terpyridine complexes with nucleobases, oligonucleotides and DNA.

In this study, we investigated the ability of Ru(ii) polypyridyl complexes to act as DNA binders. The substitution reactions of three Ru(ii) chlorophenyl terpyridine complexes, i.e. [Ru(Cl-Ph-tpy)(en)Cl]Cl (1), [Ru(Cl-Ph-tpy)(dach)Cl]Cl (2) and [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) (Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2''-terpyridine, en = 1,2-diaminoethane, dach = 1,2-diaminocyclohexane, bpy = 2,2'-bipyridine), with a mononucleotide guanosine-5'-monophosphate (5'-GMP) and oligonucleotides such as fully complementary 15-mer and 22-mer duplexes with a centrally located GG-binding site for DNA, and fully complementary 13-mer duplexes with a centrally located GG-binding site for RNA were studied quantitatively by UV-Vis spectroscopy. Duplex RNA reacts faster with complexes 1-3 than duplex DNA, while shorter duplex DNA (15mer GG) reacts faster compared with 22mer GG duplex DNA. The measured enthalpies and entropies of activation (ΔH≠ > 0, ΔS≠ < 0) support an associative mechanism for the substitution process. 1H NMR spectroscopy studies performed on complex 3 demonstrated that after the hydrolysis of the Cl ligand, it is capable to interact with guanine derivatives (i.e., 9-methylguanine (9MeG) and 5'-GMP) through N7, forming monofunctional adducts. The molecular structure of the cationic compound [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) was determined in the solid state by X-ray crystallography. The interactions of 1-3 with calf thymus (CT) and herring testes (HT) DNA were examined by stopped-flow spectroscopy, in which HT DNA was sensibly more reactive than CT DNA. The reactivity towards the formation of Ru-DNA adducts was also revealed by a gel mobility shift assay, showing that complexes 1 and 2 have a stronger DNA unwinding ability compared to complex 3. Overall, the complexes with bidentate aliphatic diamines proved to be superior to those with bpy in terms of capability to bind to the here studied biomolecules.

Ruthenium(ii) p-cymene complexes of a benzimidazole-based ligand capable of VEGFR2 inhibition: hydrolysis, reactivity and cytotoxicity studies.

The design of Ru or other metal-based anticancer agents may achieve better and faster optimization if the ligands used are also designed to have standalone functions. In this scenario, even after dissociation from the metal complex under adverse conditions, the ligand would have anti-cancer properties. In our work, we have generated a bispyrazole-containing benzimidazole ligand with potency against vascular endothelial growth factor receptor 2 (VEGFR2), which is known to have roles in vasculogenesis/angiogenesis. This ligand was used to obtain ternary Ru(ii) p-cymene complexes with the formulations [(η6-p-cymene)Ru(HL)(Cl)](Cl) (1), [(η6-p-cymene)Ru(HL)(Br)](Br) (2) and [(η6-p-cymene)Ru(HL)(I)](I) (3). 1H NMR data supports that hydrolysis of the complex is governed by halide substitution, and the extent of hydrolysis followed the trend 3 > 1 > 2. All the complexes have low affinity towards DNA bases (average Kb ∼ 103 M-1 for CT DNA); however, all the complexes are cytotoxic in nature, with IC50 values less than 15 μM. The presence of excess glutathione (GSH) liberates HL from the complexes in solution. The ability of the Ru complex to impair mitochondrial function and reduce the cellular GSH pool is thought to be the reason that it retains activity in the presence of GSH despite the ability of GSH to degrade the complexes. The chloride analogue 1 shows the best in vitro cytotoxicity against a prostate cancer cell line (LNCaP), with an IC50 of 6.4 μM. The complexes show anti-proliferative activity by the mitochondria-mediated intrinsic apoptotic pathway. Docking studies showed that HL has high affinity towards vascular endothelial growth factor receptor 2 (VEGFR2). The complexes show anti-metastatic activity (in vitro) at almost non-toxic dosages, and the effect is sustained even 48 h after removal of the complexes from the culture media.

P3.02b-024 Dynamics of EGFR Mutational Load in Urine and Plasma Correlates with Treatment Response in Advanced NSCLC: Topic: EGFR Biomarkers

NSCLC is a heterogeneous and dynamic disease where testing for key mutations is essential. With the emergence of clonal resistance, obtaining serial biopsies to assist in the real-time treatment decision-making has proven challenging. Molecular assessments of circulating tumor (ct)DNA has been previously shown feasible utilizing blood specimens. Here we additionally investigated the utility of serial urine ctDNA analysis in NSCLC. This is a prospective observational study of patients with non-squamous, tissue-confirmed EGFR, KRAS or ALK mutant NSCLC preparing to receive a systemic treatment regimen. Urine and blood specimens were collected at baseline, on treatment and at progression for ctDNA analyses. The primary endpoints were correlation between ctDNA and tumor-based molecular results, and measurable change in ctDNA with response by RECIST v1.1. Blood and urine samples were sent to Trovagene for DNA extraction and mutation enrichment NGS. Of the 34 patients enrolled thus far, interim blinded analysis of EGFR activating mutations (L858R, exon 19 deletions) was conducted in 20 patients with EGFR-positive tumors. Of 20 patients, 17 (85%) had detectable concordant EGFR mutation in pre-treatment urine and/or plasma. Of 11 patients with matched serial ctDNA samples, detectable EGFR mutation signal was observed in pre-treatment urine and/or plasma of 9 patients. These 9 patients received first to sixth line treatment with single EGFR TKI (n=3), combination TKIs (n=3), chemotherapy (n=1), immune checkpoint inhibitors alone (n=1) or in combination with TKI (n=1). In 9 of 9 patients, changes in ctDNA levels from baseline to cycle 2 day 1 on therapy correlated with best response to treatment: a 100% decrease in urine and plasma EGFR mutation levels was observed in 6 of 6 patients with partial response (PR, n=3) or stable disease (SD, n=3), while less than a 90% decrease or an increase in urine and plasma EGFR levels was observed in patients with progressive disease (PD, n=3). Mutation enrichment NGS testing by urine and plasma ctDNA correctly identified EGFR activating mutations in 85% of patients. Monitoring EGFR levels in urine/plasma enabled accurate assessment of response in advance of radiographic evaluation and regardless of therapy type in 100% of patients, with cut-point of a 90% decrease in EGFR load discriminating between patients with disease control (PR or SD) and patients with progressive disease. With continued enrollment, our study aims to further investigate clinical utility of urine and plasma ctDNA for early detection of resistance and discontinuation of inefficient therapy.

Copper(II), cobalt(II) and nickel(II) complexes of lapachol: synthesis, DNA interaction, and cytotoxicity

Three novel copper(II), cobalt(II), and nickel(II) complexes of lapachol (Lap) containing 110-phenanthroline (phen) ligand, [M(Lap)2(phen)] (M=Cu(II), 1, Co(II), 2, and Ni(II), 3), have been synthesized and characterized using, elemental analysis and spectroscopic studies. Their interactions with calf thymus DNA (CT DNA) were investigated using viscosity, thermal denaturation, circular dichorism, fluorescence quenching, and electronic absorption spectroscopy. The DNA cleavage abilities of 1–3 have been studied, where cleavage activity of copper complex 1 is more than the complexes 2 and 3. The in vitro cytotoxic potential of the complexes 1–3 against human cervical carcinoma (HeLa), human liver hepatocellular carcinoma (HepG-2), and human colorectal adenocarcinoma (HT-29) cells indicated their promising antitumor activity with quite low IC50 values in the range of .15–2.41 μM, which are lower than those of cisplatin.

21 - Evaluation of emergent circulating tumor (ct) DNA RAS mutations in patients with metastatic colorectal cancer (mCRC) treated with panitumumab (pmab) monotherapy from the ASPECCT study

21 - Evaluation of emergent circulating tumor (ct) DNA RAS mutations in patients with metastatic colorectal cancer (mCRC) treated with panitumumab (pmab) monotherapy from the ASPECCT study