Topoisomerase Research Articles

DNA Topoisomerase Top3β Is Impacted by Early Life Stress in the Developing Female and Male Rat Brain

DNA Topoisomerase Top3β Is Impacted by Early Life Stress in the Developing Female and Male Rat Brain

Screening of cytotoxicity and DNA topoisomerase IIa inhibitory activity of Turkish Onosma species

Onosma L., the largest genus of Boraginaceae, is represented by 105 species in Turkey with an endemism rate of 52%. Phytochemical studies indicate that Boraginaceae plants mainly comprise naphthoquinones with a wide range of biological activities including anticancer, antiinflammatory, wound healing, and antioxidant effects. However, few taxa of the genus Onosma have been investigated in detail for their bioactivities. Considering the high rate of endemism and an inadequate number of bioactivity screening studies in literature, we aimed to evaluate the cytotoxic effects and topoisomerase inhibitory activities of some Onosma species growing in southwestern Turkey. Here, we describe a comprehensive cytotoxic activity screening study on petroleum ether, dichloromethane, and methanol extracts of the roots of 20 identified and one unidentified Onosma taxa. The MTT cell viability assay has been performed to investigate the cytotoxicity of the extracts against seven cancer cell lines (MCF-7, HeLa, Hep G2, A549, Capan-1, HCC-1937, and DU-145) and a noncancerous cell line (MRC-5), while doxorubicin was served as a positive standard. The petroleum ether extracts of O. aksoyii Aytaç&Türkmen, O. isaurica Boiss. and Heldr.,O. taurica Pallas ex Willd. var. taurica and O. alborosea Fisch. & C.A. Mey subsp. alborosea var. alborosea were determined as the most active ones based on their IC50 values. DNA topoisomerase II? inhibition assay was conducted on the petroleum ether and dichloromethane extracts of these four active species, and almost all tested extracts demonstrated strong inhibition on the enzyme at a concentration of 0.1 mg/mL. Our cytotoxicity screening results were consistent with the findings of the topoisomerase II? inhibition test. This study advocates the significant role of Onosma species in the field of anticancer drug discovery.

Synthesis, spectroscopic characterization, molecular docking and in vitro cytotoxicity investigations on 8-Amino-6-Methoxy Quinolinium Picrate: a novel breast cancer drug

Density Functional Theory (DFT) studies of the 8-Amino-6-Methoxy Quinolinium Picrate (8A6MQP) molecule have been carried out with extensive and accurate investigations of detailed vibrational and spectroscopic investigations as well as validated experimentally. The 8A6MQP sample was synthesized and characterized using FT-IR, FT-Raman, FT-NMR and UV-Vis spectroscopic techniques. Subsequently, the optimized molecular structure and harmonic resonance frequencies of the molecule were computed based on DFT/B3LYP method with a 6-311G++(d,p) basis set using the Gaussian 09 program. The experimental and calculated vibrational wavenumbers were assigned. The absorption spectrum of the molecule was computed in the liquid phase (ethanol), which exhibits n to л* electronic transition and compared with the observed UV-Vis spectrum. Frontier molecular orbital analysis shows the molecular reactivity and kinetic stability of the molecule. The Mulliken atomic charge distribution and molecular electrostatic potential surface analysis of the molecule validate the reactive site of the molecule. The natural bond orbital analysis proves the bioactivity of the molecule. Molecular docking analysis indicates that the 8A6MQP molecule inhibits the action of DNA topoisomerase 2-alpha protein, which is associated with breast cancer. In addition, the in vitro cytotoxicity analysis of the 8A6MQP molecule against human cervical cancer cell lines (ME180) and human breast cancer cell lines (MDA MB 231) were determined by MTT assay, which evidences that the title molecule exhibits higher inhibition against the breast cancer cell lines compared to that of cervical cancer cell lines. Hence, the present study paves the way for the development of novel drugs in the treatment of breast cancer. Communicated by Ramaswamy H. Sarma

Novel Tetrazole-Based Antimicrobial Agents Targeting Clinical Bacteria Strains: Exploring the Inhibition of Staphylococcus aureus DNA Topoisomerase IV and Gyrase

Eleven novel imide-tetrazoles were synthesized. In the initial stage of research, in silico structure-based pharmacological prediction was conducted. All compounds were screened for antimicrobial activity using standard and clinical strains. Within the studied group, compounds 1–3 were recognized as leading structures with the most promising results in antimicrobial studies. Minimal inhibitory concentration values for compounds 1, 2, 3 were within the range of 0.8–3.2 μg/mL for standard and clinical Gram-positive and Gram-negative bacterial strains, showing in some cases higher activity than the reference Ciprofloxacin. Additionally, all three inhibited the growth of all clinical Staphylococci panels: Staphylococcus aureus (T5592; T5591) and Staphylococcus epidermidis (5253; 4243) with MIC values of 0.8 μg/mL. Selected compounds were examined in topoisomerase IV decatenation assay and DNA gyrase supercoiling assay, followed by suitable molecular docking studies to explore the possible binding modes. In summary, the presented transition from substrate imide-thioureas to imide-tetrazole derivatives resulted in significant increase of antimicrobial properties. The compounds 1–3 proposed here provide a promising basis for further exploration towards novel antimicrobial drug candidates.

A natural product, (S)-10-Hydroxycamptothecin inhibits pseudorabies virus proliferation through DNA damage dependent antiviral innate immunity

Pseudorabies virus (PRV), a member of the subfamily alphaherpesvirinae, is one of the most important pathogenes that cause acute death in infected pigs and leads to substantial economic losses in the global swine industry. Recently, China's emerging PRV mutant strains resulted in the traditionally commercial vaccines not providing complete protection. Some studies reported that PRV could infect humans and cause endophthalmitis and encephalitis under certain circumstances. It is necessary to develop alternative manners to control the virus infection. Here, by screening a library of natural products, (S)-10-Hydroxycamptothecin (10-HCPT) was revealed to inhibit PRV replication with a selective index of 270.04. And 10-HCPT inhibited PRV replication by blocking the viral genome replication but not inhibiting the viral attachment, internalization, and release. RNA interference assay showed that 10-HCPT inhibited PRV replication by targeting DNA topoisomerase 1 (TOP1). Meanwhile, 10-HCPT treatment induced DNA damage response and stimulated antiviral innate immunity. Animal challenge experiments showed that 10-HCPT effectively alleviated clinical signs and hispathology, and increased INF-β responses in lung and brain tissues of mice induced by PRV infection. The results demonstrate that 10-HCPT is a promising therapeutic agent to control PRV infection.

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Uptake of Ozenoxacin and Other Quinolones in Gram-Positive Bacteria.

The big problem of antimicrobial resistance is that it requires great efforts in the design of improved drugs which can quickly reach their target of action. Studies of antibiotic uptake and interaction with their target it is a key factor in this important challenge. We investigated the accumulation of ozenoxacin (OZN), moxifloxacin (MOX), levofloxacin (LVX), and ciprofloxacin (CIP) into the bacterial cells of 5 species, including Staphylococcus aureus (SA4-149), Staphylococcus epidermidis (SEP7602), Streptococcus pyogenes (SPY165), Streptococcus agalactiae (SAG146), and Enterococcus faecium (EF897) previously characterized.The concentration of quinolone uptake was estimated by agar disc-diffusion bioassay. Furthermore, we determined the inhibitory concentrations 50 (IC50) of OZN, MOX, LVX, and CIP against type II topoisomerases from S. aureus.The accumulation of OZN inside the bacterial cell was superior in comparison to MOX, LVX, and CIP in all tested species. The accumulation of OZN inside the bacterial cell was superior in comparison to MOX, LVX, and CIP in all tested species. The rapid penetration of OZN into the cell was reflected during the first minute of exposure with antibiotic values between 190 and 447 ng/mg (dry weight) of bacteria in all strains. Moreover, OZN showed the greatest inhibitory activity among the quinolones tested for both DNA gyrase and topoisomerase IV isolated from S. aureus with IC50 values of 10 and 0.5 mg/L, respectively. OZN intracellular concentration was significantly higher than that of MOX, LVX and CIP. All of these features may explain the higher in vitro activity of OZN compared to the other tested quinolones.

Solid-phase synthesis and biological evaluation of piperazine-based novel bacterial topoisomerase inhibitors

There is an emerging global need for new and more effective antibiotics against multi-resistant bacteria. This situation has led to massive industrial investigations on novel bacterial topoisomerase inhibitors (NBTIs) that target the vital bacterial enzymes DNA gyrase and topoisomerase IV. However, several of the NBTI compound classes have been associated with inhibition of the hERG potassium channel, an undesired cause of cardiac arrhythmia, which challenges medicinal chemistry efforts through lengthy synthetic routes. We herein present a solid-phase strategy that rapidly facilitates the chemical synthesis of a promising new class of NBTIs. A proof-of-concept library was synthesized with the ability to modulate both hERG affinity and antibacterial activity through scaffold substitutions.

Patterns of Diversity of Fusarium Fungi Contaminating Soybean Grains.

Soybean is an important, high protein source of food and feed. However, like other agricultural grains, soybean may pose a risk to human and animal health due to contamination of the grains with toxigenic Fusaria and associated mycotoxins. In this study, we investigated the diversity of Fusaria on a panel of 104 field isolates obtained from soybean grains during the growing seasons in 2017–2020. The results of species-specific PCR analyses showed that Fusarium avenaceum was the most common (n = 40) species associated with soybean grains in Poland, followed by F. equiseti (n = 22) and F. sporotrichioides (11 isolates). A set of isolates, which was not determined based on PCR analyses, was whole genome sequenced. Multiple sequence analyses using tef-1α, top1, rpb1, rpb2, tub2, pgk, cam and lsu genes showed that most of them belonged to Equiseti clade. Three cryptic species from this clade: F. clavum, F. flagelliforme and FIESC 31 (lacking Latin binomial) were found on soybean for the first time. This is the first report demonstrating the prevalence of Fusaria on soybean grains in Poland.

MYC assembles and stimulates topoisomerases 1 and 2 in a “topoisome”

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed invitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, invitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Delafloxacin, Finafloxacin, and Zabofloxacin: Novel Fluoroquinolones in the Antibiotic Pipeline.

Novel antimicrobial agents, approved for clinical use in past years, represent potential treatment options for various infections. In this review, we summarize the most important medical and microbiological features of three recently approved fluoroquinolones, namely delafloxacin, finafloxacin, and zabofloxacin. Delafloxacin possesses an anionic chemical structure, and represents broad-spectrum activity, as it targets both bacterial DNA gyrase and topoisomerase IV enzymes of gram-positive and gram-negative bacteria with equal affinity. Its molecular surface is larger than that of other fluoroquinolones, and it has enhanced antibacterial efficacy in acidic environments. Delafloxacin has been approved to treat acute bacterial skin and skin-structure infections, as well as community-acquired bacterial pneumonia. Finafloxacin has a zwitterionic chemical structure, and targets both DNA gyrase and topoisomerase IV enzymes. This enables a broad antibacterial spectrum; however, finafloxacin has so far only been approved in ear-drops to treat bacterial otitis externa. Zabofloxacin is also a broad-spectrum fluoroquinolone agent, and was first approved in South Korea to treat acute bacterial exacerbation of chronic obstructive pulmonary disease. The introduction of these novel fluoroquinolones into daily practice extends the possible indications of antibiotics into different bacterial infections, and provides treatment options in difficult-to-treat infections. However, some reports of delafloxacin resistance have already appeared, thus underlining the importance of the prudent use of antibiotics.

Rhophilin rho GTPase binding protein 1-antisense RNA 1 (RHPN1-AS1) promotes ovarian carcinogenesis by sponging microRNA-485-5p and releasing DNA topoisomerase II alpha (TOP2A)

ABSTRACT Ovarian cancer (OC) is the most common and lethal gynecological cancer worldwide. Long non-coding RNAs (lncRNAs) and sponging microRNAs (miRNAs) serve as key regulators in the biological processes of OC. We sought to evaluate the effect of the RHPN1-AS1-miR-485-5p-DNA topoisomerase II alpha (TOP2A) axis in regulating OC progression. RHPN1-AS1, miR-485-5p, and TOP2A levels in OC tissues and cells were determined by RT-qPCR. The interaction of RHPN1-AS1/miR-485-5p/TOP2A was assessed using luciferase, RNA immunoprecipitation, and RNA pull-down assays. RHPN1-AS1 silencing allowed us to explore its biological function by measuring cell viability, proliferation, migration, invasion, and apoptosis in OC cells. In vivo experiments were performed to verify the in vitro findings. We found that the RHPN1-AS1 and TOP2A levels were significantly enhanced, whereas the miR-485-5p levels were reduced in OC tissues and cells. RHPN1-AS1 silencing attenuated cell growth, facilitated apoptosis in OC cells, and inhibited tumor growth in vivo. Notably, RHPN1-AS1 negatively regulating miR-485-5p promoted the TOP2A expression in OC cells. In conclusion, RHPN1-AS1 sponging miR-485-5p accelerated the progression of OC by elevating TOP2A expression, which makes it a promising target for the treatment of OC patients.

Utility of 1,8-diaminonaphthalene in the synthesis of some novel heterocycles as potent anti-MRSA with dual DNA gyrase and topoisomerase IV inhibition

Utility of 1,8-diaminonaphthalene in the synthesis of some novel heterocycles as potent anti-MRSA with dual DNA gyrase and topoisomerase IV inhibition

The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana.

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.

Therapeutic Efficacy of Variable Biological Effectiveness of Proton Therapy in U-CH2 and MUG-Chor1 Human Chordoma Cell Death.

Simple SummaryChordoma is a rare, slow-growing cancer of the spinal cord. Photon radiation therapy and surgery are the standard of care for chordoma. Proton radiation therapy has become an increasingly common treatment in comparison to photon radiation therapy due to the ability to reduce off-target radiation dose. However, there is still an increased risk of toxicity to the surrounding critical structures that lead to poor treatment outcomes. Moreover, the biologic effectiveness of protons to sterilize chordoma cells remains uncertain and likely varies according to the proton energy spectrum throughout the proton field. We aim to investigate the tumoricidal properties of proton radiation therapy at the middle and end of the proton radiation field and elucidate variations in the relative biological effectiveness for chordoma cells. Our study helps quantify the therapeutic value of treating chordoma near the end of the proton field, where linear energy transfer is relatively high.Background: Chordoma is a cancer of spinal cord, skull base, and sacral area. Currently, the standard of care to treat chordoma is resection followed by radiation therapy. Since, chordoma is present in the spinal cord and these are very sensitive structures and often complete removal by surgery is not possible. As a result, chordoma has a high chance of recurrence and developing resistance to radiation therapy. In addition, treatment of chordoma by conventional radiation therapy can also damage normal tissues surrounding chordoma. Thus, current therapeutic options to treat chordoma are insufficient and novel therapies are desperately needed to treat locally advanced and metastatic chordoma. (2) Methods: In the present investigation, human chordoma cell lines of sacral origin MUG-Chor1 and U-CH2 were cultured and irradiated with Proton Beam Radiation using the clinical superconducting cyclotron and pencil-beam (active) scanning at Middle and End of the Spread-Out Bragg Peak (SOBP). Proton radiation was given at the following doses: Mug-Chor1 at 0, 1, 2, 4, and 8 Gy and U-CH2 at 0, 4, 8, 12, and 16 Gy. These doses were selected based on a pilot study in our lab and attempted to produce approximate survival fractions in the range of 1, 0.9, 0.5, 0.1, and 0.01, respectively, chosen for linear quadratic model fitting of the dose response. (3) Results: In this study, we investigated relative biological effectiveness (RBE) of proton radiation at the end of Spread Out Bragg Peak assuming that the reference radiation is a proton radiation in the middle of the SOBP. We observed differences in the survival of both Human chordoma cell lines, U-CH2 and MUG-Chor1. The data showed that there was a significantly higher cell death at the end of the Bragg peak as compared to middle of the Bragg peak. Based on the linear quadratic (LQ) fit for cell survival we calculated the RBE between M-SOBP and E-SOBP at 95% CI level and it was observed that RBE was higher than 1 at E-SOBP and caused significantly higher cell killing. Proton field at E-SOBP caused complex DNA damage in comparison to M-EOBP and the genes such as DNA topoisomerase 1, GTSE1, RAD51B were downregulated in E-SOBP treated cells. Thus, we conclude that there seems to be substantial variation in RBE (1.3–1.7) at the E-SOBP compared with the M-SOBP.

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors

Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.Graphical

Abstract P047: A phase 1/2 trial of CBX-12, an alphalexTM peptide drug conjugate, in patients with advanced or metastatic refractory solid tumors

Abstract Background Tumor-targeted drug delivery technologies are urgently needed to overcome the lack of tumor selectivity, a major drawback of conventional chemotherapy. In addition, the acidic intercellular microenvironment in solid tumors traps weak acid/base chemotherapy agents, preventing necessary intracellular concentrations in tumour cells. An alphalex conjugate, which contains a low-pH insertion peptide, a linker, and a payload, is designed to overcome these limitations. Unlike antibody drug conjugates, these peptide drug conjugates target tumors in an antigen-agnostic manner. At pH ≥7.0, the peptide is unstructured. In the low-pH tumor microenvironment, the peptide forms an alpha helix, inserts directionally in the cell membrane delivering the linker and payload intracellularly where the linker is cleaved. CBX-12 consists of the pH-sensitive peptide, a self-immolating linker, and the topoisomerase 1 (TOP1) inhibitor exatecan. Methods CBX-12-101 is a first-in-human, open-label, dose-escalation, safety, pharmacokinetics (PK), and biomarker study of CBX-12 in patients with advanced or metastatic refractory solid tumors. CBX-12 is administered as a 1-hour intravenous infusion. Two dosing schedules, daily x 5 every 3 weeks (Part A) and daily x 3 every 3 weeks (Part B), are being evaluated. The starting dose in Part A is 0.25 mg/kg. Single-patient cohorts will be enrolled initially, with dose-escalations up to 100% of the prior dose, until a patient has a ≥ Grade 2 adverse event (AE) considered possibly related to CBX-12 during Cycle 1 (the DLT period), at which time 2 additional patients will be enrolled in that cohort, and a 3 + 3 design will subsequently be utilized. Further dose escalations may be no more than 50% of the prior dose. After at least 2 cohorts have been evaluated in Part A, Part B will open for accrual. Maximum tolerated doses and recommended phase 2 doses will be determined for each dosing schedule. The PK of the CBX-12 conjugate and free exatecan will be determined. The stability of the conjugate in circulation will be evaluated by the ratio of CBX-12 to free exatecan. Pre- and on-treatment tumor biopsies are required. Biomarker and pharmacodynamic evaluations including measuring intratumor exatecan levels, TOP1, gamma H2AX and schlafen-11 are planned. Antitumor activity will be assessed per RECIST v 1.1. Phase 2 expansion cohorts are planned in patients with platinum-resistant ovarian and small cell lung cancer. As of July 2021, patients are enrolling in Part A Cohort 2 at a dose of 0.50 mg/kg. Citation Format: Anthony Tolcher, Joseph Paul Eder, David Sommerhalder, Sophia Gayle, Paul Pearson, Deb Chapman, Arthur P. DeCillis, Anish Thomas, Funda Meric-Bernstam. A phase 1/2 trial of CBX-12, an alphalexTM peptide drug conjugate, in patients with advanced or metastatic refractory solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P047.

Selected Articles from This Issue

Dysregulation of the protein arginine methyltransferase 5 (PRMT5) is associated with lymphomas, lung cancers, and breast cancers. In this First Disclosure, Brehmer and colleagues outline JNJ-64619178, an inhibitor of PRMT5 with potent anti-proliferative activity. JNJ-64619178 occupies the SAM and substrate pockets of PRMT5, trapping it in a catalytically inactive state. PMRT5 inhibition by JNJ-64619178 increased alternatively spliced events and resulted in the killing of primary AML samples ex vivo. Taken together, the results support the continued clinical study of JNJ-64619178 (NCT03573310).The approval of Trastuzumab deruxtecan demonstrates the potency of the DNA topoisomerase I inhibitor DXd in antibody-drug conjugates (ADCs). DXd-ADCs exhibit a short systemic half-life and ability to optimize the drug-antibody ratio. In this First Disclosure, Okajima and colleagues outline the design of another DXd-containing ADC targeting Trophoblast cell surface antigen 2 (TROP2). Datopotamab deruxtecan is designed to have a shorter half-life than sacituzumab govitecan, a TROP2-directed ADC approved by the FDA for TNBC. Dato-DXd showed potent anti-tumor activity and was absent of severe gastrointestinal toxicity. The results support the first-in-human phase 1 study for patients with advanced solid tumors (NCT03401385).Chromosome region maintenance 1 is overexpressed in ovarian cancer and its inhibition has been shown to achieve a 30% disease control rate in recurrent disease. Here, Handley and colleagues seek to enhance the effects of selinexor through synergistic drug combinations. Their screen found the PARP inhibitor olaparib as the most likely candidate. Selinexor decreased the expression of DNA damage repair proteins, generating an increased susceptibility to PARP inhibitors. The combination reduced tumor nodules and weight in vivo in a mouse model. Their results outline an actionable combination for advanced ovarian cancer patients.The advent of KRAS inhibitors, as well as the disagreement between retrospective studies in immunotherapy and KEYNOTE-189, demonstrate the need to characterize the KRAS mutational landscape of non-small cell lung cancer (NSCLC). Therefore, Judd and colleagues sequenced 592 genes from 17,095 NSCLC specimens to assess co-occuring genomic alterations, tumor mutational burden (TMB), and PD-L1 expression. They demonstrate the rate of co-mutation depends on the KRAS mutation type. Mutated KRAS was significantly increased in PD-L1 expression compared to wild type, with KRAS G12C the most likely to be PD-L1 positive. These studies inform future therapeutic intervention for KRAS-mutated NSCLC.

Abstract LBA033: YM155 induces DNA damage and cell death in anaplastic thyroid cancer cells by inhibiting DNA topoisomerase IIα at the ATP binding site

Abstract Introduction: Anaplastic thyroid cancer (ATC) is among the most aggressive of all human cancers with a median survival of 4.3 months. Currently, there is no effective treatment for most ATC patients - surgery, radiation, and chemotherapy all fail to significantly prolong ATC patient survival. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing anaplastic thyroid cancer cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials (not including ATC), and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. A number of additional mechanisms have been proposed for YM155. The purpose of this study was to investigate the mechanisms underlying YM155-mediated ATC cell death. Methods: ATC cell line THJ16T was used as the model for this study. AlamarBlue was used to measure cell viability. Immunofluorescent detection of phosphorylated histone H2AX (γ-H2AX, p-Ser139) foci was used as a surrogate marker for double-strand DNA breaks. siRNA was used to knockdown topoisomerase 2α (Top2α), and Lipofectamine-3000 was used to overexpress Top2α plasmids. In-vivo complex of enzyme assay was used to measure the association between Top2α and DNA. Modeling software Molecular Operating Environment and the crystal structure of human Top2α bound to AMP-PNP (Protein Data Bank # 1ZXM) were used to estimate binding affinities for ATP and YM155 in the ATP binding site of Top2α. Site-directed mutagenesis was used to introduce mutation Ser148 → Ala into a recombinant human Top2α. Results: Previously, we presented gene expression data from ATC patients showing significant overexpression of Top2α compared to benign thyroid samples. ATC cell lines that overexpress Top2α are more sensitive to YM155 and exhibit larger increases in DNA damage with YM155 treatment. In this study, ATC cells grown to be resistant to YM155 show decreased expression of Top2α, and overexpression of Top2α re-sensitizes resistant cells to YM155. Molecular modeling shows favorable binding for YM155 in the Top2α ATP binding site and identifies key amino acids for YM155-Top2α interaction. An engineered Top2α mutant abrogates the effect of YM155 as measured by cell viability and DNA damage assays, confirming the contribution of Top2α to YM155 mechanism of action. Conclusions: Our results strongly suggest that Top2α is an important target mediating YM155’s anticancer activity in ATC. We propose that Top2α inhibition by YM155 leads to DNA damage and eventual cell death. Given YM155's potent anti-proliferative effects in ATC cells with sparing of benign thyroid cells, along with a plausible mechanism of action, we suggest further evaluation and optimization of YM155 for future clinical application in ATC. In conclusion, our study lays the foundation for targeting the enzymatic activity of Top2α as a novel therapeutic strategy for the treatment of ATC, with YM155 serving as the predicate drug. Citation Format: Ryan Mackay, Paul Weinberger, Elahe Mahdavian, Qinqin Xu. YM155 induces DNA damage and cell death in anaplastic thyroid cancer cells by inhibiting DNA topoisomerase IIα at the ATP binding site [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA033.

Design and synthesis of ciprofloxacin-sulfonamide hybrids to manipulate ciprofloxacin pharmacological qualities: Potency and side effects

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections. Although being potent, susceptibility to CNS side effects limits their use. It was observed that improvements in absorption, activity and side effects were achieved via modifications at the N atom of the C7 of the side chain. To meet the increasing demand for development of new antibacterial agents, nineteen novel ciprofloxacin-sulfonamide hybrid molecules were designed, synthesized and characterized by IR, 1H NMR and 13C NMR as potential antibacterial agents with dual DNA gyrase/topoisomerase IV inhibitory activity. Most of the synthesized compounds showed significant antibacterial activity that was revealed by testing their inhibitory activity against DNA gyrase, DNA topoisomerase IV as well as their minimum inhibitory concentration against Staphylococcus aureus. Six ciprofloxacin-sulfonamide hybrids (3f, 5d, 7a, 7d, 7e and 9b) showed potent inhibitory activity against DNA topoisomerase IV, compared to ciprofloxacin (IC50: 0.55 μM), with IC50 range: 0.23–0.44 μM. DNA gyrase was also efficiently inhibited by five ciprofloxacin-sulfonamide hybrids (3f, 5d, 5e, 7a and 7d) with IC50 range: 0.43–1.1 μM (IC50 of ciprofloxacin: 0.83 μM). Compounds 3a and 3b showed a marked improvement in the antibacterial activity over ciprofloxacin against both Gram-positive and Gram-negative pathogens, namely, Staphylococcus aureus Newman and Escherichia coli ATCC8739, with MIC = 0.324 and 0.422 μM, respectively, that is 4.2-fold and 3.2-fold lower than ciprofloxacin (MIC = 1.359 μM) against the Gram-positive Staphylococcus aureus, and MIC = 0.025 and 0.013 μM, respectively, that is 10.2-fold and 19.6-fold lower than ciprofloxacin (MIC = 0.255 μM) against the Gram-negative Escherichia coli ATCC8739. Also, the most active compounds showed lower CNS and convulsive side effects compared to ciprofloxacin with a concomitant decrease in GABA expression.