Development Of Antineoplastic Agents Research Articles

Interactions between DNA and the acridine intercalator: A computational study

Cancer is a global public health problem characterized by deviations in the mechanisms that control cell proliferation, resulting in mutations and variations in the structure of DNA. The mechanisms of action of chemotherapeutic drugs are related to their interactions and binding with DNA; consequently, the development of antineoplastic agents that target DNA has extensively focused on use of acridine, a heterocyclic molecule that binds to deoxyribonucleic acid via intercalation, a process that modifies DNA and makes replication impossible. In this context, this study aimed to computationally investigate how acridine intercalators interact with DNA by evaluating the mechanism of interactions, binding, and interaction energies using quantum mechanics calculations. Molecular electrostatic potential (MEP) analysis revealed that acridine has well- distributed negative charges in the center of the molecule, indicative of a dominant electron-rich region. Acridine exhibits well-defined π orbitals (HOMO and LUMO) on the aromatic rings, suggesting that charge transfer occurs within the molecule and may be responsible for the pharmacological activity of the compound. Structural analysis revealed that acridine interacts with DNA mainly through hydrogen bonds between HAcridine… ODNA with bond lengths ranging from 2.370 Å to 3.472 Å. The Binding energy (ΔEBind) showed that acridine interacts with DNA effectively for all complexes and the electronic energy results (E+ZPE) for complexes revealed that the complexes are more stable when the DNA-centered acridine molecule. The Laplacian-analysis topological QTAIM parameter (∇2ρ(r)) and total energy (H(r)) categorized the interactions as being non-covalent in nature. The RGD peak distribution in the NCI analysis reveals the presence of van der Waals interactions, predominantly between the intercalator and DNA. Accordingly, we confirm that acridine/DNA interactions are relevant for understanding how the intercalator acts within nucleic acids.

Iridium (III) complexes induce cervical carcinoma apoptosis via disturbing cellular redox homeostasis disorder and inhibiting PI3K/AKT/mTOR pathway

Improvement of antineoplastic activity and selectivity is a main goal in the development of antineoplastic agents. Herein, we synthesized three new iridium (III) complexes: [Ir(ppy)2(FTTP)](PF6) (Ir1, ppy = 2-phenylpyridine, FTTP = 2-(3-fluoronaphthalen-2-yloxy)-1,4,8,9-tetraazatriphenylene), [Ir(bzq)2(FTTP)](PF6) (Ir2, bzq = benzo[h]quinolone), [Ir(piq)2(FTTP)](PF6) (Ir3, piq = 1-phenylisoquinoline). Ir1-3 exhibit excellent cytotoxicity against various cancer cells particularly towards human cervical carcinoma HeLa cells while remaining non-toxic to normal cell lines. Assays on 2D cell colony formation and 3D multicellular tumor spheroid model confirm that Ir1-3 can effectively inhibit the colony-forming and penetrate deeply into HeLa 3D multicellular tumor spheroid model exhibiting a notable cytotoxic effect, which was consistent with the results from the viability assays. Meanwhile, confocal microscopy shows a rapid uptake of Ir1-3 and co-localization experiments with subcellular markers reveal that Ir1-3 locate mainly at the mitochondria. Further investigation of the mechanism indicated the complexes Ir1-3 promote the excessive generation of ROS, inhibit glutathione and thioredoxin reductase that effectively interferes with the intracellular redox balance, induce oxidative stress and result in caspase-dependent apoptosis. Moreover, the ROS-mediated inactivation of the PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) pathway, DNA damage combing with suppression of the cyclin D1/CDK4/6 activity arrested cell cycle in the G0/G1 phase are involved in complexes-induced cell apoptosis. Finally, assays on xenografted cervical carcinoma mouse model confirm the excellent biocompatibility and antineoplastic efficiency of Ir3 in vivo. Collectively, this work offers building blocks for developing iridium (III) complexes as clinical application potential.

Antimitotic and toxicogenetic action of Stevia urticifolia aerial parts on proliferating vegetal and mammalian cells: in vitro and in vivo traditional and replacement methods

ABSTRACT Stevia urticifolia Thunb. is an underexploited herb possessing bioactive flavonoids, saponins, and terpenoids. The aim of this study was to examine the antiproliferative and toxicogenetic properties of the ethyl acetate extract from Stevia urticifolia aerial parts (EtAcSur) upon Artemia salina, erythrocytes, Allium cepa and sarcoma 180 cells and fibroblasts, as well as in vivo studies on mice to determine systemic, macroscopic, and behavioral alterations and bone marrow chromosomal damage. The assessment using A. salina larvae and mouse blood cells revealed LC50 and EC50 values of 68.9 and 113.6 µg/ml, respectively. Root growth and mitosis were inhibited by EtAcSur, and chromosomal aberrations were detected only at 100 μg/ml. EtAcSur exhibited potent concentration-dependent viability reduction of S180 and L-929 cells and antioxidant capacity employing ABTS• and DPPH•. No previous in vivo studies were performed before with the EtAcSur. Signals of acute toxicity were not observed at 300 mg/kg. Physiological and toxicological investigations at 25 and 50 mg/mg/day i.p. for 8 days did not markedly change body or organ relative weights, nor patterns of spontaneous locomotor and exploratory activities. In contrast, clastogenic effects on bone marrow were found at 50 mg/mg/day. EtAcSur was found to (1) produce toxicity in microcrustaceans, (2) capacity as free radical scavenger, (3) antimitotic, cytotoxic and clastogenic activties upon vegetal and mammalian cells, and (4) lethality on both tumor and normal murine cells indistinctly. In vivo damage systemic effects were not remarkable and clinical signals of toxicity were not observed, suggesting the significant pharmacological potential of S. urticifolia for the development of antineoplastic agents. Abbreviations: ABTS: 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DMSO: dimethylsulfoxide; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EC50: effective concentration 50%; EtAcSur: ethyl acetate extract from Stevia urticifolia aerial parts; Hb, hemoglobin; IC50: inhibitory concentration 50%; LC50,: lethal concentration 50%; MI: mitotic index; RBC, red blood cells; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

Abstract 5992: Targeting glucose metabolism in cancer

Abstract Positron emission tomographic studies with 2-18F-fluoro-2-deoxy-glucose (FDG-PET) consistently demonstrate that tumors take up significantly more glucose than adjacent normal tissues in vivo. Tumors rely on glucose metabolism to provide the energy and biosynthetic intermediates required to fuel their rapid growth and metastasis. Tumor glucose metabolism is stimulated in part by fructose-2,6-bisphosphate (F26BP), the product of the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4), which allosterically activates a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). We have found that the PFKFB4 family member plays a significant role in regulating glucose metabolism in neoplastic cells. We determined that PFKFB4 is highly expressed in multiple cancer types and strongly induced by the transcription factor HIF-1α and that its suppression markedly reduced F26BP, glycolytic flux and the growth of xenograft tumors providing strong rationale for the development of anti-neoplastic agents selectively targeting PFKFB4. By the use of virtual screening, we discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits PFKFB4 activity with a resultant decrease in F26BP, glycolysis and cancer cell growth in vitro and a marked suppression of growth in established tumors, importantly without systemic toxicity. During our studies, we unexpectedly observed that inhibition of a frequently co-expressed PFKFB family member, PFKFB3, increased PFKFB4 expression, indicating that PFKFB4 may compensate for decreased PFKFB3 expression. This observation is highly relevant given that a PFKFB3 inhibitor (PFK158) has recently been clinically evaluated in a Phase I trial and compensation by PFKFB4 may limit the efficacy of this and other PFKFB3 inhibitors. We examined the effects of simultaneous silencing of PFKFB4 and PFKFB3 on cell lines derived from multiple tumor types and found that co-knockdown led to a significant decrease in cell viability and glycolysis and a near-complete abrogation of anchorage independent growth in vitro. We also have now evaluated the effects of simultaneous inhibition of PFKFB4 and PFKFB3 on cancer cells using small molecule inhibitors of both enzymes and found that co-inhibition led to a significant decrease in F26BP production, a synergistic increase in cell death in vitro and marked suppression of xenograft tumor growth in vivo relative to either inhibitor alone. Taken together, our data indicate that targeting PFKFB4 may be a viable therapeutic option against cancer and strongly support the further exploration of co-targeting PFKFB4 and PFKFB3 as a potential therapeutic strategy for the successful treatment of cancer. Citation Format: Evan Meiman, Bryana Braxton, John Trent, Jason Chesney, Sucheta Telang. Targeting glucose metabolism in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5992.

Identification of a small-molecule compound that inhibits homodimerization of oncogenic NAC1 protein and sensitizes cancer cells to anticancer agents

Nucleus accumbens-associated protein-1 (NAC1) is a transcriptional repressor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and plays critical roles in tumor development, progression, and drug resistance. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. However, effective approaches for effective targeting of this nuclear protein remain elusive. In this study, we identified a core unit consisting of Met7 and Leu90 in NAC1's N-terminal domain (amino acids 1-130), which is critical for its homodimerization and stability. Furthermore, using a combination of computational analysis of the NAC1 dimerization interface and high-throughput screening (HTS) for small molecules that inhibit NAC1 homodimerization, we identified a compound (NIC3) that selectively binds to the conserved Leu-90 of NAC1 and prevents its homodimerization, leading to proteasomal NAC1 degradation. Moreover, we demonstrate that NIC3-mediated down-regulation of NAC1 protein sensitizes drug-resistant tumor cells to conventional chemotherapy and enhances the antimetastatic effect of the antiangiogenic agent bevacizumab both in vitro and in vivo These results suggest that small-molecule inhibitors of NAC1 homodimerization may effectively sensitize cancer cells to some anticancer agents and that NAC1 homodimerization could be further explored as a potential therapeutic target in the development of antineoplastic agents.

Abstract 5487: Targeting glycolysis for the treatment of cancer

Abstract Tumors exhibit markedly increased glucose uptake and glycolysis and this metabolic phenotype serves to satisfy their increased requirement for energy and biosynthetic intermediates. Tumor glucose metabolism is stimulated in part by fructose-2,6-bisphosphate (F26BP), which is the product of a family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and allosterically activates a key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). Previous studies have found the PFKFB3 family member to be a dominant source of F26BP in cancer cells due to a high kinase activity, expression in multiple tumor types and the inhibition of tumor growth by specific PFKFB3 inhibitors including a compound, PFK158, that is currently under evaluation in a phase I trial. Recent data, however, indicate that the PFKFB4 enzyme plays a significant role in regulating glucose metabolism in neoplastic cells. We have found that PFKFB4 is highly expressed in multiple cancers, strongly induced by HIF-1α, and that its suppression markedly reduced F26BP and glycolytic flux, increased apoptosis and inhibited growth of xenograft tumors in vivo, thus providing strong rationale for the development of antineoplastic agents selectively targeting PFKFB4. Using virtual screening, we have discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity with a resultant decrease in F26BP, glycolytic flux and cancer cell growth in vitro and markedly reduces the growth of established tumors without systemic toxicity. During our studies, we unexpectedly observed that PFKFB4 expression is increased by PFKFB3 inhibition, suggesting that PFKFB4 may compensate for decreased PFKFB3 expression and activity. We examined the effects of simultaneous silencing of PFKFB4 and PFKFB3 on cancer cell lines and found that co-knockdown led to a significant decrease in cell viability and glycolysis and to a near-complete abrogation of anchorage-independent growth in vitro. We additionally examined the effect of simultaneous inhibition of PFKFB4 and PFKFB3 using 5MPN and several PFKFB3 inhibitors in vitro and found a synergistic increase in cell death. Taken together, our data indicate that targeting PFKFB4 may be a viable therapeutic option against cancer and strongly support the further development of PFKFB4 small-molecule inhibitors as well as the co-targeting of PFKFB4 and PFKFB3 as potential strategies for the treatment of cancer. Citation Format: Jason Chesney, Aubrey Mojesky, Yoannis Imbert-Fernandez, John Trent, Sucheta Telang. Targeting glycolysis for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5487.

Mechanism of anti-glioma activity and in vivo efficacy of the cannabinoid ligand KM-233

Glioblastoma multiforme (GBM) is the most common and devastating form of primary central nervous system malignancy. The prognosis for patients diagnosed with GBM is poor, having a median survival rate of 12-15 months. Despite modern advances in the development of antineoplastic agents, the efficacy of newer anti-cancer agents in the treatment of GBM is yet to be determined. Thus, there remains a significant unmet need for new therapeutic strategies against GBM. A promising chemotherapeutic intervention has emerged from studies of cannabinoid receptor agonists wherein tetrahydrocannabinol has been the most extensively studied. The novel cannabinoid ligand KM-233 was developed as a lead platform for future optimization of biopharmaceutical properties of classical based cannabinoid ligands. Treatment of U87MG human GBM cells with KM-233 caused a time dependent change in the phosphorylation profiles of MEK, ERK1/2, Akt, BAD, STAT3, and p70S6K. Almost complete mitochondrial depolarization was observed 6 h post-treatment followed by a rapid increase in cleaved caspase 3 and significant cytoskeletal contractions. Treatment with KM-233 also resulted in a redistribution of the Golgi-endoplasmic reticulum structures. Dose escalation studies in the orthotopic model using U87MG cells revealed an 80 % reduction in tumor size after 12 mg/kg daily dosing for 20 days. The evaluation of KM-233 against primary tumor tissue in the side flank model revealed a significant decrease in the rate of tumor growth. These findings indicate that structural refinement of KM-233 to improve its biopharmaceutical properties may lead to a novel and efficacious treatment for GBM.

Synthetic and Application Perspectives of Azapodophyllotoxins: Alternative Scaffolds of Podophyllotoxin

Podophyllotoxin (1) has been known to possess anti-tumor activity and is still considered an important lead for research and development of antineoplastic agents. Derivatives of podophyllotoxin, namely etoposide (2), etopophos (3) and teniposide (4) have been developed and are currently used in clinic for the treatment of a variety of malignancies. These agents are also used in combination therapies with other drugs. Due to the drug resistance developed by cancer cells as well as side effects associated with the use of these agents in clinic, the search for new effective anticancer analogues of podophyllotoxin remains an intense area of research. The structural complexity of podophyllotoxin, arising from the presence of four stereogenic carbons in ring C has restricted most of the structural activity relationship (SAR) studied by derivatization of the parent natural product rather than by de novo multi-step chemical synthesis. These issues provide strong impetus to a search for analogues of 1 with simplified structures, which can be accessible via short synthetic sequences from simple starting materials. Even if such initial compounds might have diminished cytotoxic potencies compared with the parent cyclolignan, the ease of preparation of carefully designed libraries of analogues would lead to more informative SAR studies and expeditious structure optimization. In this regard, during the last two decades considerable efforts have been made to synthesize aza- analogs of podophyllotoxin, i. e. aza-podophyllotoxins, with hetero atoms at different positions of the podophyllotoxin skeleton, while keeping the basic podophyllotoxin structure. Recently, there have been significant efforts towards the convenient synthesis of aza-analogs of 1. The use of multicomponent reactions (MCRs) and the synergies of ultrasound and microwave irradiations have increased the synthetic speed and variety of azapodophyllotoxins which are and will be available to be tested against a diverse population of carcinomas and other diseases. It has been reported that several aza-podophyllotoxins retain a great fraction of the cytotoxicity associated with the parent lignan. This review focuses on the strategies towards synthesis of various aza-podophyllotoxin analogues and their biological activities.

New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid.

The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, beta-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol. The presence of the hydrated aldehyde was confirmed in solution by (13)C-(1)H NMR spectroscopy of the ternary GAR Tfase-beta-GAR-10-formyl-TDAF complex using the (13)C-labeled 10-formyl-TDAF. This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents.

Discovery and Development of Antineoplastic Agents from Natural Sources

Nature has provided many effective anticancer agents in current use, such as the microbially derived drugs; dactinomycin; bleomycin and doxorubicin; and the plant-derived drugs, vinblastine, irinotecan, topotecan, etoposide, and paclitaxel. The search for novel antitumor agents from natural sources continues through collaboration among scientists worldwide in the investigation of coral reefs, rainforests, and deep subsurface thermal vents for novel bioactive compounds. The potential for drug discovery is being further enhanced by recent advances in procedures for microbial cultivation and the extraction of nucleic acids from environmental samples, resulting in the identification of novel microbes that provide a vast untapped reservoir of genetic and metabolic diversity. Manipulation of the biosynthetic pathways of microbial polyketides through genetic engineering permits the biosynthesis of bioactive polyketides not generated naturally.

Discovery and Development of Antineoplastic Agents from Natural Sources

Nature has provided many effective anticancer agents in current use, such as the microbially derived drugs; dactinomycin; bleomycin and doxorubicin; and the plant-derived drugs, vinblastine, irinotecan, topotecan, etoposide, and paclitaxel. The search for novel antitumor agents from natural sources continues through collaboration among scientists worldwide in the investigation of coral reefs, rainforests, and deep subsurface thermal vents for novel bioactive compounds. The potential for drug discovery is being further enhanced by recent advances in procedures for microbial cultivation and the extraction of nucleic acids from environmental samples, resulting in the identification of novel microbes that provide a vast untapped reservoir of genetic and metabolic diversity. Manipulation of the biosynthetic pathways of microbial polyketides through genetic engineering permits the biosynthesis of bioactive polyketides not generated naturally.

The pulmonary toxicity of nitrosoureas

Drug-induced pulmonary fibrosis in cancer chemotherapy has become a more serious problem as treatment regimens are refined, larger total doses of cytotoxic drugs are administered and patient prognosis improves. The nitrosoureas are a class of chemotherapeutic agents whose clinical use is severely limited by drug-induced toxicities. The myelosuppression caused by nitrosourea administration can be managed clinically; however, the development of irreversible pulmonary fibrosis is a more serious clinical problem. Studies are needed to identify biochemical markers for early lung injury so that the amount of pulmonary tissue damage can be assessed and monitored. Additionally, considerable research is needed to understand the mechanisms by which these agents produce lung injury, so that therapeutic regimens can be developed to minimize or prevent lung toxicity. In understanding the mechanisms of toxicity, potentially the pulmonary injury caused by nitrosourea administration can be altered without affecting the clinical antitumor activity of this class of compounds. In the future, knowledge on mechanisms of drug-induced pulmonary injury can be used in the development of antineoplastic agents which are more disease specific and less pulmonary toxic.

Predictive tests in cancer chemotherapy. A reappraisal.

The evolution of medical oncology so far owes much to the preclinical and clinical development of antineoplastic agents. Prognostic factors and empiric treatment strategies have guided the clinician in his choice of drugs. In the light of increasing ethical restrictions met with phase I-II clinical trials and major advances in propagating human tumor cells outside the donor patient, a reappraisal of predictive tests in cancer chemotherapy is warranted. Among 'short-term assays' only the determination of steroid-hormone receptor content in tumor tissues has gained clinical acceptance, whereas other methods still suffer from theoretical or practical shortcomings. Both the human tumor stem cell assay and the xenograft model have revealed unique patterns of sensitivity for each individual tumor line. While interindividual heterogeneity among tumors sharing a common site of origin justifies efforts to develop predictive tests, microheterogeneity among tumor samples from the same donor patient limits the potential of this approach. Predictive tests should be performed in conjunction with clinical trials to ensure optimal extraction of information. As additional prognostic factors, they should in the near future accelerate drug development and reduce the hazard of unnecessary drug toxicity without therapeutic benefit.