Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

SN-38, an active metabolite of irinotecan, inhibits transcription of nuclear factor erythroid 2-related factor 2 and enhances drug sensitivity of colorectal cancer cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) significantly contributes to drug resistance of cancer cells, and Nrf2 inhibitors have been vigorously pursued. Repurposing of existing drugs, especially anticancer drugs, is a straightforward and promising strategy to find clinically available Nrf2 inhibitors and effective drug combinations. Topoisomerase inhibitors SN-38 (an active metabolite of irinotecan), topotecan, mitoxantrone, and epirubicin were found to significantly suppress Nrf2 transcriptional activity in cancer cells. SN-38, the most potent one among them, significantly inhibited the transcription of Nrf2, as indicated by decreased mRNA level and binding of RNA polymerase II to NFE2L2 gene, while no impact on Nrf2 protein or mRNA degradation was observed. SN-38 synergized with Nrf2-sensitive anticancer drugs such as mitomycin C in killing colorectal cancer cells, and irinotecan and mitomycin C synergistically inhibited the growth of SW480 xenografts in nude mice. Our study identified SN-38 and three other topoisomerase inhibitors as Nrf2 inhibitors, revealed the Nrf2-inhibitory mechanism of SN-38, and indicate that clinically feasible drug combinations could be designed based on their interactions with Nrf2 signaling.

Study of nitrogen heterocycles as DNA/HSA binder, topoisomerase inhibitors and toxicological safety.

Four new nitrogen-containing heterocyclic derivatives (acridine, quinoline, indole, pyridine) were synthesized and their biological properties were evaluated. The compounds showed affinity for DNA and HSA, with CAIC and CAAC displaying higher binding constants (Kb) of 9.54 × 104 and 1.06 × 106, respectively. The fluorescence quenching assay (Ksv) revealed suppression values ranging from 0.34 to 0.64 × 103 M-1 for ethidium bromide (EB) and 0.1 to 0.34 × 103 M-1 for acridine orange (AO). Molecular docking confirmed the competition of the derivatives with intercalation probes at the same binding site. At 10 µM concentrations, the derivatives inhibited topoisomerase IIα activity. In the antiproliferative assays, the compounds demonstrated activity against MCF-7 and T47-D tumor cells and nonhemolytic profile. Regarding toxicity, no acute effects were observed in the embryos. However, some compounds caused enzymatic and cardiac changes, particularly the CAIC, which increased SOD activity and altered heart rate compared to the control. These findings suggest potential antitumor action of the derivatives and indicate that substituting the acridine core with different cores does not interfere with their interaction and topoisomerase inhibition. Further investigations are required to assess possible toxicological effects, including reactive oxygen species generation.

Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor.

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 µM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 µM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.

Novel bacterial topoisomerase inhibitors: unique targeting activities of amide enzyme-binding motifs for tricyclic analogs.

Antimicrobial resistance has made a sizeable impact on public health and continues to threaten the effectiveness of antibacterial therapies. Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of antibacterial agents with a unique binding mode and distinct pharmacology that enables them to evade existing resistance mechanisms. The clinical development of NBTIs has been plagued by several issues, including cardiovascular safety. Herein, we report a sub-series of tricyclic NBTIs bearing an amide linkage that displays promising antibacterial activity, potent dual-target inhibition of DNA gyrase and topoisomerase IV (TopoIV), as well as improved cardiovascular safety and metabolic profiles. These amide NBTIs induced both single- and double-strand breaks in pBR322 DNA mediated by Staphylococcus aureus DNA gyrase, in contrast to prototypical NBTIs that cause only single-strand breaks. Unexpectedly, amides 1a and 1b targeted human topoisomerase IIα (TOP2α) causing both single- and double-strand breaks in pBR322 DNA, and induced DNA strand breaks in intact human leukemia K562 cells. In addition, anticancer drug-resistant K/VP.5 cells containing decreased levels of TOP2α were cross-resistant to amides 1a and 1b. Together, these results demonstrate broad spectrum antibacterial properties of selected tricyclic NBTIs, desirable safety profiles, an unusual ability to induce DNA double-stranded breaks, and activity against human TOP2α. Future work will be directed toward optimization and development of tricyclic NBTIs with potent and selective activity against bacteria. Finally, the current results may provide an additional avenue for development of selective anticancer agents.

Oligonucleotide-Recognizing Topoisomerase Inhibitors (OTIs): Precision Gene Editors for Neurodegenerative Diseases?

Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.

Ricolinostat suppresses proliferation, promotes apoptosis, and enhances the antiproliferative activity of topoisomerase inhibitors in cervical cancer cells.

Ricolinostat has been found to exhibit anticancer effects alone and in combination with various chemotherapeutic drugs in several cancer types. However, to the best of our knowledge, the efficacy of ricolinostat in cervical cancer is still not investigated. Therefore, in this study, we evaluated the effect of ricolinostat in cervical cancer alone and in combination with topoisomerase inhibitors. The effect of ricolinostat on cervical cancer cells was assessed using MTT, cell-cycle arrest, Annexin V/PI staining assay, reactive oxygen species (ROS) measurement, and western blot analysis. The antiproliferative effect of ricolinostat in combination with topoisomerase inhibitors was assessed using the MTT assay and synergism was computed using "CompuSyn" software. We found that ricolinostat inhibited proliferation, and induced G2/M phase arrest and apoptosis in cervical cancer cells. We further found that ricolinostat treatment resulted in increased ROS production, decreased Bcl-xL expression, and induced p21 expression. We also investigated the effect of ricolinostat in combination with topotecan and etoposide in cervical cancer cells. Ricolinostat was found to significantly enhance the antiproliferative activity of both, topotecan and etoposide, in cervical cancer cells in a concentration-dependent manner. In conclusion, our study showed that ricolinostat suppressed proliferation by inducing G2/M phase arrest and promoted apoptosis in cervical cancer cells, indicating that ricolinostat may be a promising antitumor agent in cervical cancer. Also, ricolinostat and topotecan/etoposide combination are synergistic in cervical cancer cells.

Discovery of dual inhibitors of topoisomerase I and Cyclooxygenase-2 for colon cancer therapy.

Novel tolfenamic acid derivatives based on the structure of I-1 were designed and synthesized to improve its poor target inhibition and solubility. Among them, W10 was identified as a potent dual-target inhibitor of Topo I (IC50 = 0.90 ± 0.17 µM) and COX-2 (IC50 = 2.31 ± 0.07 µM) with improved water solubility (32.33 µg/mL). Moreover, W10 also exhibited fairly potent anti-proliferative and pro-apoptosis activity via the mitochondrial pathway, as well as suppressed aberrant NF-κB/IκB activation in colon cancer cells in vitro. Additionally, W10 possessed favorable pharmacokinetic properties and excellent antitumor effects in vivo. In general, our study has demonstrated the potency of a novel Topo I/COX-2 dual inhibitor, which can potentially be developed into a chemotherapeutic candidate for colon cancer.

Inhibitors of ABCB1 and ABCG2 overcame resistance to topoisomerase inhibitors in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance rapidly emerges. The aim, therefore, is to overcome resistance to topoisomerase I (irinotecan) or II (etoposide) inhibitors in SCLCs. METHODS: To identify key factors in the chemoresistance of SCLCs, we established four cell lines resistant to etoposide or an active metabolite of irinotecan, SN-38, from SCLC cell lines and evaluated RNA profiles using parental and newly established cell lines. RESULTS: We found that the drug efflux protein, ATP-binding cassette sub-family B member 1 (ABCB1), was associated with resistance to etoposide, and ATP-binding cassette sub-family G member 2 (ABCG2) was associated with resistance to SN-38 by RNA sequencing. The inhibition of ABCB1 or ABCG2 in each resistant cell line induced synergistic apoptotic activity and promoted drug sensitivity in resistant SCLC cells. The ABC transporter inhibitors, elacridar and tariquidar, restored sensitivity to etoposide or SN-38 in in vitro and in vivo studies, and promoted apoptotic activity and G2-M arrest in resistant SCLC cells. CONCLUSIONS: ABC transporter inhibitors may be a promising therapeutic strategy for the purpose of overcoming resistance to topoisomerase inhibitors in patients with SCLC.

Design, Synthesis and Cytotoxicity of Thiazole-Based Stilbene Analogs as Novel DNA Topoisomerase IB Inhibitors.

A series of new thiazole-based stilbene analogs were designed, synthesized and evaluated for DNA topoisomerase IB (Top1) inhibitory activity. Top1-mediated relaxation assays showed that the synthesized compounds possessed variable Top1 inhibitory activity. Among them, (E)-2-(3-methylstyryl)-4-(4-fluorophenyl)thiazole (8) acted as a potent Top1 inhibitor with high Top1 inhibition of ++++ which is comparable to that of CPT. A possible binding mode of compound 8 with Top1-DNA complex was further provided by molecular docking. An MTT assay against human breast cancer (MCF-7) and human colon cancer (HCT116) cell lines revealed that the majority of these compounds showed high cytotoxicity, with IC50 values at micromolar concentrations. Compounds 8 and (E)-2-(4-tert-butylstyryl)-4-(4-fluorophenyl)thiazole (11) exhibited the most potent cytotoxicity with IC50 values of 0.78 and 0.62 µM against MCF-7 and HCT116, respectively. Moreover, the preliminary structure-activity relationships of thiazole-based stilbene analogs was also discussed.

Identification of new halogen-containing 2,4-diphenyl indenopyridin-5-one derivative as a boosting agent for the anticancer responses of clinically available topoisomerase inhibitors.

Based on previous reports on the significance of halogen moieties and the indenopyridin-5-one skeleton, we designed and synthesized a novel series of halogen (F-, Cl-, Br-, CF3- and OCF3-)-containing 2,4-diphenyl indenopyridin-5-ones and their corresponding -5-ols. Unlike indenopyridin-5-ols, most of the prepared indenopyridin-5-ones with Cl-, Br-, and CF3- groups at the 2-phenyl ring conferred a strong dual topoisomerase I/IIα inhibitory effect. Among the series, para-bromophenyl substituted compound 9 exhibited the most potent topoisomerase inhibition and antiproliferative effects, which showed dependency upon the topoisomerase gene expression level of diverse cancer cells. In particular, as a DNA minor groove-binding non-intercalative topoisomerase I/IIα catalytic inhibitor, compound 9 synergistically promoted the anticancer efficacy of clinically applied topoisomerase I/IIα poisons both in vitro and in vivo, having the great advantage of alleviating poison-related toxicities.

Immune Infiltration, Cancer Stemness, and Targeted Therapy in Gastrointestinal Stromal Tumor.

Objective: To investigate the characteristics of the tumor immune microenvironment in patients with gastrointestinal stromal tumor (GIST) and identify cancer stem-like properties of GIST to screen potential druggable molecular targets. Methods: The gene expression data of 60 patients with GIST was retrieved from the Array Express database. CIBERSORT was applied to calculate the level of immune infiltration. ssGSEA and ESTIMATE were used to calculate the cancer stemness index and tissue purity. The Connectivity Map (CMAP) database was implemented to screen targeted drugs based on cancer stem-like properties of GIST. Result: There was a difference in the level of immune infiltration between the metastasis and non-metastasis GIST groups. The low level of T-cell infiltration was correlated with high tumor purity and tumor stemness index, and the correlation coefficients were -0.87 and -0.61 (p < 0.001), respectively. Furthermore, there was a positive correlation between cancer stemness index and cell purity (p < 0.001). The cancer stemness index in the metastasis group was higher than that in the non-metastasis group (p = 0.0017). After adjusting for tumor purity, there was no significant correlation between T-cell infiltration and cancer stemness index (p = 0.086). Through the pharmacological mechanism of topoisomerase inhibitors, six molecular complexes may be the targets of GIST treatment. Conclusion: Immune infiltration in GIST patients is related to cancer stem-like properties, and the correlation relies on tumor purity. Cancer stemness index can be used as a new predictive biomarker of tumor metastasis and targets of drug therapy for GIST patients.

N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents: Synthesis and biological evaluation.

Glioblastoma is one of the most lethal brain tumors. The crucial chemotherapy is mainly alkylating agents with modest clinical success. Given this desperate need and inspired by the encouraging results of a phase II trial via concomitant Topo I inhibitor plus COX-2 inhibitor, we designed a series of N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents based on structure modification on 1,5-naphthyridine derivatives (Topo I inhibitors). Notably, the target compounds I-1 (33.61 ± 1.15 µM) and I-8 (45.01 ± 2.37 µM) were confirmed to inhibit COX-2, while a previous reported compound (1,5-naphthyridine derivative) resulted nearly inactive towards COX-2 (IC50 > 150 µM). Besides, I-1 and I-8 exhibited higher anti-proliferation, anti-migration, anti-invasion effects than the parent compound 1,5-naphthyridine derivative, suggesting the success of modification based on the parent. Moreover, I-1 obviously repressed tumor growth in the C6 glioma orthotopic model (TGI = 66.7%) and U87MG xenograft model (TGI = 69.4%). Besides, I-1 downregulated PGE2, VEGF, MMP-9, and STAT3 activation, upregulated E-cadherin in the orthotopic model. More importantly, I-1 showed higher safety than temozolomide and different mechanism from temozolomide in the C6 glioma orthotopic model. All the evidence demonstrated that N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents could be promising for the glioma management.

Design, Synthesis and Anticancer Evaluation of Substituted Cinnamic Acid Bearing 2-Quinolone Hybrid Derivatives.

A new series of hybrid molecules containing cinnamic acid and 2-quinolinone derivatives were designed and synthesized. Their structures were confirmed by 1H-NMR, 13C-NMR and mass analyses. All the synthesized hybrid molecules were assessed for their in vitro antiproliferative activity against more than one cancer cell lines. Compound 3-(3,5-dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-phenylacrylic acid (5a) with IC50 = 1.89 µM against HCT-116 was proved to the most potent compound in this study, as compared to standard drug staurosporin. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed that the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a displays potent inhibitory activity compared with control.

Iridium(iii) complexes as mitochondrial topoisomerase inhibitors against cisplatin-resistant cancer cells.

Herein, we developed the first metal-based mitochondrial topoisomerase inhibitors to achieve an effective therapeutic outcome for the therapy of cisplatin-resistant tumour cells.

Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells.

Approximately 5% of cancers are caused by high-risk human papillomaviruses. Although very effective preventive vaccines will reduce this cancer burden significantly over the next several decades, they have no therapeutic effect for those already infected and remaining at risk for malignant progression of hrHPV lesions. HPV-associated cancers are dependent upon the expression of the viral E6 and E7 oncogenes. The oncogenic function of hrHPV E6 relies partially on its ability to induce p53 degradation. Since p53 is generally wildtype in hrHPV-associated cancers, p53 stabilization arrests proliferation, induces apoptosis and/or results in senescence. Here we describe a live cell, image-based high-throughput screen to identify compounds that stabilize p53 and/or affect viability in HPV-positive cancer HeLa cells. We validate the robustness and potential of this screening assay by assessing the activities of approximately 6,500 known bioactive compounds, illustrating its capability to function as a platform to identify novel therapeutics for hrHPV.

Functional interrogation of DNA damage response variants with base editing screens.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Oxocrebanine: A Novel Dual Topoisomerase inhibitor, Suppressed the Proliferation of Breast Cancer Cells MCF-7 by Inducing DNA Damage and Mitotic Arrest.

BACKGROUND: DNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo. PURPOSE: To identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential. STUDY DESIGN: This study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms. METHODS: The growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest. RESULTS: Oxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC50 of 16.66 µmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics. CONCLUSION: Oxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.

Camptothecin and Topotecan, Inhibitors of Transcription Factor Fli-1 and Topoisomerase, Markedly Ameliorate Lupus Nephritis in (NZB × NZW)F1 Mice and Reduce the Production of Inflammatory Mediators in Human Renal Cells.

OBJECTIVE: To examine the therapeutic effects of camptothecin (CPT) and topotecan (TPT), inhibitors of transcription factor Fli-1 and topoisomerase, on lupus nephritis in (NZB × NZW)F1 (NZBWF1) mice, and to examine the effects of CPT and TPT on inflammatory mediators in human renal cells. METHODS: Female NZBWF1 mice were treated with vehicle, cyclophosphamide (CYC), CPT (1 mg/kg or 2 mg/kg), or TPT (0.03 mg/kg, 0.1 mg/kg, or 0. 3 mg/kg) by intraperitoneal injection twice a week, beginning at the age of 25 weeks (n = 8-10 mice per group). Blood and urine were collected for monitoring autoantibodies and proteinuria. Mice were euthanized at 40 weeks, and renal pathology scores were assessed. Human renal endothelial and mesangial cells were treated with CPT or TPT, and cytokine expression was measured. RESULTS: None of the NZBWF1 mice treated with 1 mg/kg or 2 mg/kg of CPT or 0.3 mg/kg of TPT had proteinuria >100 mg/dl at the age of 40 weeks. One of 8 mice treated with 0.1 mg/kg of TPT and 1 of 10 mice treated with CYC had proteinuria >300 mg/dl, whereas 90% of the mice treated with vehicle had proteinuria >300 mg/dl. Compared to vehicle control, mice treated with 1 mg/kg or 2 mg/kg of CPT, 0.1 mg/kg or 0.3 mg/kg of TPT, or CYC had significantly prolonged survival, attenuated renal injury, diminished splenomegaly, reduced anti-double-stranded DNA autoantibody levels, and reduced IgG and C3 deposits in the glomeruli (all P < 0.05). Human renal cells treated with CPT or TPT had reduced expression of Fli-1 and decreased monocyte chemotactic protein 1 production following stimulation with interferon-α (IFNα) or IFNγ. CONCLUSION: Our findings indicate that low-dose CPT and TPT could be repurposed to treat lupus nephritis.

Novel urea linked ciprofloxacin-chalcone hybrids having antiproliferative topoisomerases I/II inhibitory activities and caspases-mediated apoptosis.

A novel series of urea-linked ciprofloxacin (CP)-chalcone hybrids 3a-j were synthesized and screened by NCI-60 cancer cell lines as potential cytotoxic agents. Interestingly, compounds 3c and 3j showed remarkable antiproliferative activities against both colon HCT-116 and leukemia SR cancer cells compared to camptothecin, topotecan and staurosporine with IC50 = 2.53, 2.01, 17.36, 12.23 and 3.1 µM for HCT-116 cells, respectively and IC50 = 0.73, 0.64, 3.32, 13.72 and 1.17 µM for leukemia SR cells, respectively. Also, compounds 3c and 3j exhibited inhibitory activities against Topoisomerase (Topo) I with % inhibition = 51.19% and 56.72%, respectively, compared to camptothecin (% inhibition = 60.05%) and Topo IIß with % inhibition = 60.81% and 60.06%, respectively, compared to topotecan (% inhibition = 71.09%). Furthermore, compound 3j arrested the cell cycle of leukemia SR cells at G2/M phase. It induced apoptosis both intrinsically and extrinsically via activation of proteolytic caspases cascade (caspases-3, -8, and -9), release of cytochrome C from mitochondria, upregulation of proapoptotic Bax and down-regulation of Bcl-2 protein level. Thus, the new ciprofloxacin derivative 3j could be considered as a potential lead for further optimization of antitumor agent against leukemia and colorectal carcinoma.