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Background/Objectives: The synthesis of ( E )-1-(1,3-diphenylallyl)-1 H -1,2,4-triazoles and related compounds as anti-mitotic agents with activity in breast cancer was investigated. These compounds were designed as hybrids of the microtubule-targeting chalcones, indanones, and the aromatase inhibitor letrozole. Methods : A panel of 29 compounds was synthesized and examined by a preliminary screening in estrogen receptor (ER) and progesterone receptor (PR)-positive MCF-7 breast cancer cells together with cell cycle analysis and tubulin polymerization inhibition. Results : ( E )-5-(3-(1 H -1,2,4-triazol-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)-2-methoxyphenol 22b was identified as a potent antiproliferative compound with an IC 50 value of 0.39 mM in MCF-7 breast cancer cells, 0.77 mM in triple-negative MDA-MB-231 breast cancer cells, and 0.37 mM in leukemia HL-60 cells. In addition, compound 22b demonstrated potent activity in the sub-micromolar range against the NCI 60 cancer cell line panel including prostate, melanoma, colon, leukemia, and non-small cell lung cancers. G 2 /M phase cell cycle arrest and the induction of apoptosis in MCF-7 cells together with inhibition of tubulin polymerization were demonstrated. Immunofluorescence studies confirmed that compound 22b targeted tubulin in MCF-7 cells, while computational docking studies predicted binding conformations for 22b in the colchicine binding site of tubulin. Compound 22b also selectively inhibited aromatase. Conclusions : Based on the results obtained, these novel compounds are suitable candidates for further investigation as antiproliferative microtubule-targeting agents for breast cancer.

Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Purpose: Oral Squamous Cell Carcinoma (OSCC) is the 6th most common cancer worldwide. It is generally aggressive and closely associated with chemoresistance and poor survival. There is accumulating evidence for the involvement of inhibitors of apoptosis proteins (IAPs), including IAP1 and XIAP, in mediating chemotherapy resistance in OSCC. Various strategies for targeting IAPs have been designed and tested in recent years and several small molecule IAP inhibitors are in clinical trials as monotherapies as well as in combination with radiotherapy and chemotherapy. The purpose of this study was to evaluate and compare the efficacy and biological activity of three IAP inhibitors both as stand-alone and sensitising agents to cisplatin in a preclinical model of squamous cell carcinoma of the tongue., Methods: Cisplatin-sensitive SCC4 and -resistant SCC4cisR cells were utilised in this study. Apoptosis was evaluated by flow cytometric analysis of Annexin V/Propidium Iodide-stained cells. Expression of IAP proteins was determined by western blotting and knockdown of cIAP1, livin and XIAP was conducted by transfection of cells with siRNA., Results: We establish for the first time the therapeutic efficacy of the Smac mimetic, BV6 and the XIAP inhibitor Embelin, for OSCC. Both of these IAP targeting agents synergistically enhanced cisplatin-mediated apoptotic cell death in resistant cells which was mediated in part by depletion of XIAP. In addition, knockdown of XIAP using siRNA enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation., Conclusion: These findings provide pre-clinical evidence that IAP inhibition may be a valuable therapeutic option in OSCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The synthesis and biochemical activity of a series of chiral trans 3-hydroxyl β-lactams targeting tubulin is described. Synthesis of the series of enantiopure β-lactams was achieved using chiral derivatising reagent N-Boc-l-proline. The absolute configuration was determined as 3S,4S for (+) enantiomer 4EN1 and 3R,4R for (-) enantiomer 4EN2. Antiproliferative studies identified chiral 3S,4S b-lactams with subnanomolar IC 50 values across a range of cancer cell lines, improving potency with respect to the corresponding racemates. Fluoro-substituted (+)-(3S,4S)-4-(3-fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27EN1) was determined as the lead eutomer with dual antiproliferative activity in triple negative breast cancer cells (TNBC), and combretastatin A-4 resistant HT-29 colorectal cancer cells. IC 50 values were in the range of 0.26-0.7 nM across four cell lines. Tubulin polymerisation assays, confocal microscopy and molecular modelling studies indicated that 3S,4S eutomers are microtubule destabilisers, while 3R,4R distomers have lower potency as microtubule destabilisers. 27EN1 demonstrated anti-mitotic and pro-apoptotic activity in MDA-MB-231 and HT-29 cells in addition to selective toxicity toward MCF-7 breast cancer versus non-tumorigenic MCF-10-2A cells. The related 3S,4S β-lactam eutomer 4EN1 downregulated expression of key cell survival anti-apoptotic proteins Bcl-2 and Mcl-1 in MDA-MB-231 cells while 27EN1 downregulated Mcl-1 in HT-29 cells. Chiral β-lactam 27EN1 will be further developed for treatment of TNBC and CA-4 resistant colorectal cancers., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Niamh O’Boyle reports financial support was provided by Wellcome Trust. Niamh O’Boyle reports financial support was provided by Royal Society of Chemistry. Niamh O’Boyle reports financial support was provided by Trinity College Provosts Teaching Award., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Malignant rhabdoid tumour (MRT) is a rare and aggressive childhood malignancy that occurs in the kidneys or central nervous system and is associated with very poor prognosis. Chemoresistance is a major issue in the treatment of this malignancy leading to an urgent need for a greater understanding of its underlying mechanisms in MRT and novel treatment strategies for MRT patients. The balance between oxidative stress mediated by reactive oxygen species (ROS) and the antioxidant system has become a subject of interest in cancer therapy research. Studies have implicated key players of the antioxidant system in chemotherapeutic including the well-known antioxidant glutathione (GSH) and the transcription factor nuclear erythroid-related factor-2 (Nrf2).   METHODS: This study evaluated the role of these components in the response of MRT cells to treatment with the commonly used chemotherapeutic agent, cisplatin., Results: This study characterised the basal levels of GSH, ROS and Nrf2 in a panel of MRT cell lines and found a correlation between the expression profile of the antioxidant defence system and cisplatin sensitivity. Results showed that treatment with ROS scavenger N-acetylcysteine (NAC) protected cells from cisplatin-induced ROS and apoptosis. Interestingly, depleting GSH levels with the inhibitor buthionine sulphoximine (BSO) enhanced cisplatin-induced ROS and sensitised cells to cisplatin. Lastly, targeting Nrf2 with the small molecule inhibitor ML385 or by siRNA diminished GSH levels, enhanced ROS and sensitised resistant MRT cells to cisplatin., Conclusions: These results suggest that targeting the Nrf2/GSH antioxidant system may present a novel therapeutic strategy to combat chemoresistance in rhabdoid tumours., (© 2023. The Author(s).)

A series of novel 3-(prop-1-en-2-yl)azetidin-2-one, 3-allylazetidin-2-one and 3-(buta-1,3-dien-1-yl)azetidin-2-one analogues of combretastatin A-4 (CA-4) were designed and synthesised as colchicine-binding site inhibitors (CBSI) in which the ethylene bridge of CA-4 was replaced with a β-lactam (2-azetidinone) scaffold. These compounds, together with related prodrugs, were evaluated for their antiproliferative activity, cell cycle effects and ability to inhibit tubulin assembly. The compounds demonstrated significant in vitro antiproliferative activities in MCF-7 breast cancer cells, particularly for compounds 9h, 9q, 9r, 10p, 10r and 11h , with IC 50 values in the range 10-33 nM. These compounds were also potent in the triple-negative breast cancer (TBNC) cell line MDA-MB-231, with IC 50 values in the range 23-33 nM, and were comparable with the activity of CA-4. The compounds inhibited the polymerisation of tubulin in vitro, with significant reduction in tubulin polymerization, and were shown to interact at the colchicine-binding site on tubulin. Flow cytometry demonstrated that compound 9q arrested MCF-7 cells in the G 2 /M phase and resulted in cellular apoptosis. The antimitotic properties of 9q in MCF-7 human breast cancer cells were also evaluated, and the effect on the organization of microtubules in the cells after treatment with compound 9q was observed using confocal microscopy. The immunofluorescence results confirm that β-lactam 9q is targeting tubulin and resulted in mitotic catastrophe in MCF-7 cells. In silico molecular docking supports the hypothesis that the compounds interact with the colchicine-binding domain of tubulin. Compound 9q is a novel potent microtubule-destabilising agent with potential as a promising lead compound for the development of new antitumour agents.

The stilbene combretastatin A-4 (CA-4) is a potent microtubule-disrupting agent interacting at the colchicine-binding site of tubulin. In the present work, the synthesis, characterisation and mechanism of action of a series of 3-fluoro and 3,3-difluoro substituted β-lactams as analogues of the tubulin-targeting agent CA-4 are described. The synthesis was achieved by a convenient microwave-assisted Reformatsky reaction and is the first report of 3-fluoro and 3,3-difluoro β-lactams as CA-4 analogues. The β-lactam compounds 3-fluoro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxy phenyl)azetidin-2-one 32 and 3-fluoro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) 33 exhibited potent activity in MCF-7 human breast cancer cells with IC 50 values of 0.075 µM and 0.095 µM, respectively, and demonstrated low toxicity in non-cancerous cells. Compound 32 also demonstrated significant antiproliferative activity at nanomolar concentrations in the triple-negative breast cancer cell line Hs578T (IC 50 0.033 μM), together with potency in the invasive isogenic subclone Hs578Ts(i)8 (IC 50 = 0.065 μM), while 33 was also effective in MDA-MB-231 cells (IC 50 0.620 μM). Mechanistic studies demonstrated that 33 inhibited tubulin polymerisation, induced apoptosis in MCF-7 cells, and induced a downregulation in the expression of anti-apoptotic Bcl2 and survivin with corresponding upregulation in the expression of pro-apoptotic Bax. In silico studies indicated the interaction of the compounds with the colchicine-binding site, demonstrating the potential for further developing novel cancer therapeutics as microtubule-targeting agents.

Aims: We have recently described a novel guanidinium-based compound, VP79s, which induces cytotoxicity in various cancer cell lines. Here, we aim to investigate the activity of VP79s and associated mechanisms of action in multiple myeloma (MM) cells in vitro and ex vivo., Main Methods: The effects of VP79s on cell viability and induction of apoptosis was examined in a panel of drug-sensitive and drug-resistant MM cell lines, as well as ex vivo patient samples and normal donor lymphocytes and platelets. Cell signaling pathways associated with the biological effects of VP79s were analysed by immunoblotting and flow cytometry. Gene expression changes were assessed by quantitative real-time PCR analysis., Key Findings: VP79s was found to rapidly inhibit both constitutively active and IL-6-induced STAT3 signaling with concurrent downregulation of the IL-6 receptors, CD130 and CD126. VP79s induced a rapid and dose-dependent downregulation of anti-apoptotic Bcl-2 family member, myeloid cell leukaemia-1 (MCL-1). VP79s enhanced bortezomib induced cell death and was also found to overcome bone marrow stromal cell induced drug resistance. VP79s exhibited activity in ex vivo patient samples at concentrations which had no effect on peripheral blood mononuclear cells, lymphocytes and platelets isolated from healthy donors., Significance: As VP79s resulted in rapid inhibition of the key IL-6/STAT3 signaling pathway and downregulation of MCL-1 expression with subsequent selective anti-myeloma activity, VP79s may be a potential therapeutic agent with a novel mechanism of action in MM cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric malignancy most commonly diagnosed in those below the age of three. MRTs can arise in soft tissue but are more often associated with the central nervous system or kidney. Unfortunately, the prognosis upon diagnosis with MRT is poor. Given the resistance of MRT to current treatment protocols including cisplatin, and the vulnerability of this young patient population to aggressive therapies, there is a need for novel treatment options. Several members of the inhibitor of apoptosis protein (IAP) family including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2), livin and survivin have been implicated in chemotherapy resistance in various malignancies. We have previously demonstrated expression of these IAP family members in a panel of MRT cell lines. In the present study, sensitivity of this same panel of MRT cell lines to small-molecule mediated inhibition of the IAPs via the survivin inhibitor YM155 and the XIAP/cIAP1/cIAP2 inhibitor BV6 was demonstrated. Additionally, both BV6 and the XIAP inhibitor embelin synergistically enhanced cisplatin mediated apoptotic cell death in MRT cell lines, with enhanced caspase-3 cleavage. Importantly, we have demonstrated, for the first time, expression of XIAP, its target caspase-3 and its endogenous inhibitor SMAC in rhabdoid tumour patient tissue. In conclusion, this study provides pre-clinical evidence that IAP inhibition may be a new therapeutic option in MRT., (Copyright © 2022. Published by Elsevier Ltd.)

Malignant rhabdoid tumour (MRT) is a rare and aggressive paediatric tumour that typically arises in the kidneys or central nervous system (CNS). The malignancy often affects patients under the age of three and is associated with an extremely poor survival rate, with most deaths occurring within the first year of presentation. Thus, there is an unmet and urgent medical need for novel therapeutic strategies for this malignancy. One of the major issues when treating MRT patients is the emergence of chemoresistance. Autophagy has become an area of focus in the study of chemoresistance due to its reported dual role as both a pro-survival and pro-death mechanism. The role of autophagy in the chemotherapeutic response of MRT remains largely unknown. A greater understanding of the role of autophagy may lead to the development of therapeutic strategies to enhance chemotherapeutic effect and improve the clinical outcome of MRT patients. This study evaluated the cellular response to cisplatin, a representative chemotherapeutic agent used in the treatment of MRT, and the role of autophagy in mediating cisplatin resistance. Our results demonstrated that cisplatin induced apoptosis and autophagy concomitantly in a panel of MRT cell lines. Furthermore, inhibition of caspase-induced apoptosis with Z-VAD-FMK also inhibited autophagy levels demonstrating a complex interplay between these two pathways. In addition, blocking autophagy at the early stages of the autophagic process using the pharmacological inhibitor SAR405 or through the genetic knockdown of critical autophagic protein ATG5 by siRNA did not sensitise cells to cisplatin-induced apoptosis. Collectively, these results suggest that induction of autophagy does not appear to elicit a pro-survival effect in the chemotherapeutic response of MRT cells., (Copyright © 2022. Published by Elsevier Ltd.)

Survival and expansion of malignant B cells in chronic lymphocytic leukemia (CLL) are highly dependent both on intrinsic defects in the apoptotic machinery and on the interactions with cells and soluble factors in the lymphoid microenvironment. The adaptor protein p66Shc is a negative regulator of antigen receptor signaling, chemotaxis and apoptosis whose loss in CLL B cells contributes to their extended survival and poor prognosis. Hence, the identification of compounds that restore p66Shc expression and function in malignant B cells may pave the way to a new therapeutic approach for CLL. Here we show that a novel oxazepine-based compound (OBC-1) restores p66Shc expression in primary human CLL cells by promoting JNK-dependent STAT4 activation without affecting normal B cells. Moreover, we demonstrate that the potent pro-apoptotic activity of OBC-1 in human leukemic cells directly correlates with p66Shc expression levels and is abrogated when p66Shc is genetically deleted. Preclinical testing of OBC-1 and the novel analogue OBC-2 in Eμ-TCL1 tumor-bearing mice resulted in a significantly longer overall survival and a reduction of the tumor burden in the spleen and peritoneum. Interestingly, OBCs promote leukemic cell mobilization from the spleen to the blood, which correlates with upregulation of sphingosine-1-phosphate receptor expression. In summary, our work identifies OBCs as a promising class of compounds that, by boosting p66Shc expression through the activation of the JNK/STAT4 pathway, display dual therapeutic effects for CLL intervention, namely the ability to mobilize cells from secondary lymphoid organs and a potent pro-apoptotic activity against circulating leukemic cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antimitotic drugs that target tubulin are among the most widely used chemotherapeutic agents; however, the development of multidrug resistance has limited their clinical activity. We report the synthesis and biological properties of a series of novel 3-chloro-β-lactams and 3,3-dichloro-β-lactams (2-azetidinones) that are structurally related to the tubulin polymerisation inhibitor and vascular targeting agent, Combretastatin A-4. These compounds were evaluated as potential tubulin polymerisation inhibitors and for their antiproliferative effects in breast cancer cells. A number of the compounds showed potent activity in MCF-7 breast cancer cells, e.g., compound 10n (3-chloro-4-(3-hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) and compound 11n (3,3-dichloro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one), with IC 50 values of 17 and 31 nM, respectively, and displayed comparable cellular effects to those of Combretastatin A-4. Compound 10n demonstrated minimal cytotoxicity against non-tumorigenic HEK-293T cells and inhibited the in vitro polymerisation of tubulin with significant G 2 /M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 10n caused a mitotic catastrophe by targeting tubulin. In addition, compound 10n promoted apoptosis by regulating the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2 and Mcl-1. Molecular docking was used to explore the potential molecular interactions between novel 3-chloro-β-lactams and the amino acid residues of the colchicine binding active site cavity of β-tubulin. Collectively, these results suggest that 3-chloro-2-azetidinones, such as compound 10n , could be promising lead compounds for further clinical anti-cancer drug development.

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. Chemoresistance is a critical problem in OSCC leading to therapeutic failure and tumour recurrence. Recently, autophagy has acquired an emerging interest in cancer as it has been shown to be frequently activated in tumour cells treated with chemotherapeutics. Whether drug-induced autophagy represents a mechanism that allows cancer cells to survive or a pro-death mechanism associated with apoptosis remains controversial. This study evaluated the cellular response to cisplatin and the role of autophagy in mediating cisplatin resistance in OSCC cells. Our results demonstrated that cisplatin concurrently induced apoptosis and autophagy in OSCC cell lines partially through the ROS/JNK pathway. Moreover, inhibition of cisplatin-induced apoptosis abrogated autophagy, indicating a complex interplay between these pathways. Cisplatin-induced autophagy does not appear to elicit a pro-survival effect in OSCC as early-stage autophagy inhibition, using either a pharmacological inhibitor or knockdown of the key autophagy protein ATG5, did not sensitise cells to cisplatin. Additionally, autophagy did not play a role in acquired resistance to cisplatin in our novel cisplatin-resistant OSSC cell line (SCC-4cisR) obtained by pulsed stepwise exposure of SCC-4 cells to cisplatin (~14-fold change in sensitivity). There was no change in the basal levels of autophagy in the SCC-4cisR cells compared to the SCC-4 cells. Furthermore, a significant increase in cisplatin-induced autophagy was observed only in the SCC-4 cells, but not in the derived SCC-4cisR cells. Collectively, these data indicate that autophagy may not be implicated in acquired cisplatin resistance in OSCC., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We report the synthesis and biochemical evaluation of compounds that are designed as hybrids of the microtubule targeting benzophenone phenstatin and the aromatase inhibitor letrozole. A preliminary screening in estrogen receptor (ER)-positive MCF-7 breast cancer cells identified 5-((2 H -1,2,3-triazol-1-yl)(3,4,5-trimethoxyphenyl)methyl)-2-methoxyphenol 24 as a potent antiproliferative compound with an IC 50 value of 52 nM in MCF-7 breast cancer cells (ER+/PR+) and 74 nM in triple-negative MDA-MB-231 breast cancer cells. The compounds demonstrated significant G 2 /M phase cell cycle arrest and induction of apoptosis in the MCF-7 cell line, inhibited tubulin polymerisation, and were selective for cancer cells when evaluated in non-tumorigenic MCF-10A breast cells. The immunofluorescence staining of MCF-7 cells confirmed that the compounds targeted tubulin and induced multinucleation, which is a recognised sign of mitotic catastrophe. Computational docking studies of compounds 19e , 21l , and 24 in the colchicine binding site of tubulin indicated potential binding conformations for the compounds. Compounds 19e and 21l were also shown to selectively inhibit aromatase. These compounds are promising candidates for development as antiproliferative, aromatase inhibitory, and microtubule-disrupting agents for breast cancer.

We previously identified a guanidinium-based lead compound that inhibited BRAF through a hypothetic type-III allosteric mechanism. Considering the pharmacophore identified in this lead compound (i.e., "lipophilic group", "di-substituted guanidine", "phenylguanidine polar end"), several modifications were investigated to improve its cytotoxicity in different cancer cell lines. Thus, several lipophilic groups were explored, the di-substituted guanidine was replaced by a secondary amine and the phenyl ring in the polar end was substituted by a pyridine. In a structure-based design approach, four representative derivatives were docked into an in-house model of an active triphosphate-containing BRAF protein, and the interactions established were analysed. Based on these computational studies, a variety of derivatives was synthesized, and their predicted drug-like properties calculated. Next, the effect on cell viability of these compounds was assessed in cell line models of promyelocytic leukaemia and breast, cervical and colorectal carcinomas. The potential of a selection of these compounds as apoptotic agents was assessed by screening in the promyelocytic leukaemia cell line HL-60. The toxicity against non-tumorigenic epithelial MCF10A cells was also investigated. These studies allowed for several structure-activity relationships to be derived. Investigations on the mechanism of action of representative compounds suggest a divergent effect on inhibition of the MAPK/ERK signalling pathway.

A series of novel 1,4-diaryl-2-azetidinone analogues of combretastatin A-4 (CA-4) have been designed, synthesised and evaluated in vitro for antiproliferative activity, antiapoptotic activity and inhibition of tubulin polymerisation. Glucuronidation of CA-4 by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) has been identified as a mechanism of resistance in cancer cells. Potential sites of ring B glucuronate conjugation are removed by replacing the B ring meta-hydroxy substituent of selected series of β-lactams with alternative substituents e.g. F, Cl, Br, I, CH 3 . The 3-phenyl-β-lactam 11 and 3-hydroxy-β-lactam 46 demonstrate improved activity over CA-4 in CA-4 resistant HT-29 colon cancer cells (IC 50  = 9 nM and 3 nM respectively compared with IC 50  = 4.16 μM for CA-4), while retaining potency in MCF-7 breast cancer cells (IC 50  = 17 nM and 22 nM respectively compared with IC 50  = for 4 nM for CA-4). Compound 46 binds at the colchicine site of tubulin, and strongly inhibits tubulin assembly at micromolar concentrations comparable to CA-4. In addition, compound 46 induced mitotic arrest at low concentration in both cell lines MCF-7 and HT-29 together with downregulation of expression of antiapoptotic proteins Mcl-1, Bcl-2 and survivin in MCF-7 cells. These novel antiproliferative and antiapoptotic β-lactams are potentially useful scaffolds in the development of tubulin-targeting agents for the treatment of breast cancers and chemoresistant colon cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier Ltd.)

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2‑year survival rate of 30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF‑related apoptosis‑inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase‑8 and co‑occurred with caspase‑8 and caspase‑3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase‑8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL‑mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.

Microtubules are a validated clinical target for the treatment of many cancers. We describe the design, synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain the CA-4 core structure with modifications to the stilbene linking group, and are predominantly piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC or Mukaiyama's reagent. All target compounds were screened for antiproliferative activity in MCF-7 breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC50 = 190 nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC50 values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise tubulin, induce G2/M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells, and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells. Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be developed further as novel, water-soluble molecules targeting microtubules.

Microtubule-targeted drugs are essential chemotherapeutic agents for various types of cancer. A series of 3-vinyl-β-lactams (2-azetidinones) were designed, synthesized and evaluated as potential tubulin polymerization inhibitors, and for their antiproliferative effects in breast cancer cells. These compounds showed potent activity in MCF-7 breast cancer cells with an IC 50 value of 8 nM for compound 7 s 4-[3-Hydroxy-4-methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one) which was comparable to the activity of Combretastatin A-4. Compound 7s had minimal cytotoxicity against both non-tumorigenic HEK-293T cells and murine mammary epithelial cells. The compounds inhibited the polymerisation of tubulin in vitro with an 8.7-fold reduction in tubulin polymerization at 10 M for compound 7 s and were shown to interact at the colchicine-binding site on tubulin, resulting in significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 7 s is targeting tubulin and resulted in mitotic catastrophe. A docking simulation indicated potential binding conformations for the 3-vinyl-β-lactam 7 s in the colchicine domain of tubulin. These compounds are promising candidates for development as antiproiferative microtubule-disrupting agents., Competing Interests: The authors declare no conflict of interest.

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Purpose The combretastatins (CAs) are known to exhibit anti-tumour activity but the underlying mechanism remains to be fully elucidated. Inflammation plays a critical role in altering the function of cancer cells and evasion of cell death and increased proliferation are characteristics of transformed malignancies. Many of the proteins involved in these pathways are regulated by the transcription factor NF-κB which can be activated by tumour necrosis factor (TNF-α), a pro-inflammatory cytokine released by both malignant and immune cells within the tumour microenvironment. In this study, we examined the ability of combretastatin A-4 (CA-4) and its novel, cis-restricted analogue CA-432 to target the NF-κB signalling pathway in T cells. Methods Effects of the CAs on the viability of DND-41 leukaemia and Jurkat lymphoma T-cell lines was assessed by the alamar blue assay. Induction of apoptosis and effects on expression levels of key apoptotic proteins was established though flow cytometry and western blotting. Modulation of the NF-κB signalling pathway was determined through western blotting and through assessment of NF-κB reporter gene activity. Results CA-4 and CA-432 reduced cell viability and induced apoptosis in DND-41 and Jurkat T cells and sensitised the cells to TNF-α-induced apoptosis through inhibition of the NF-κB signalling pathway. Suppression of the NF-κB pathway downregulated NF-κB-dependent gene products involved in cell survival (IAPs, Bcl-2 and Mcl-1), proliferation (cyclin D1) and inflammation (COX-2). Furthermore, both CA-4 and CA-432 inhibited TNF-α-induced NF-κB activation through the inhibition of IκBα degradation and p65 nuclear translocation and decreased NF-κB reporter gene activity. Conclusions Our data indicate that the anti-cancer properties of comebretastatins may be mediated in part through targeting the NF-κB pathway. This study provides new insights into the molecular mechanisms of CA compounds and a potential application of combretastatins for inflammatory diseases such as cancers, which are associated with abnormal NF-κB activation.

Considering our hypothesis that the guanidinium moiety in the protein kinase type III inhibitor 1 interacts with a phosphate of ATP within the hinge region, the nature of the interactions established between a model isouronium and the phosphate groups of ATP was computationally analysed indicating that an isouronium derivative of 1 will interact in a similar manner with ATP. Thus, a number of compounds were prepared to assess the effect of the guanidinium/isouronium substitution on cancer cell growth; additionally, the molecular shortening and conformational change induced by replacing the di-substituted guanidine-linker of 1 by an amide was explored. The effect of these compounds on cell viability was tested in human leukaemia, breast cancer and cervical cancer cell lines and the resulting IC 50 values were compared with those of the lead compound 1 . Replacement of the di-substituted guanidine-linker by an amide results in the loss of cytotoxicity; however, substitution of the mono-substituted guanidinium by an isouronium cation seems to be beneficial for cell growth inhibition. Additionally, the effect of these compounds on the MAPK/ERK pathway was studied by means of Western blotting and the results indicate that the isouronium derivative 2 decreases the levels of phosphorylated, and thus activated, ERK (pERK) both in leukaemia and breast cancer cells, whereas lead compound 1 only shows an effect on pERK levels in breast cancer cells. This confirms that both compounds could interfere with the MAPK/ERK pathway although other targets cannot be ruled out.

Estrogen receptor α (ERα) is an important target for the design of drugs such as tamoxifen (2a) and fulvestrant (5). Three series of ER-ligands based on the benzoxepin scaffold structure were synthesized: series I containing an acrylic acid, series II with an acrylamide, and series III with a saturated carboxylic acid substituent. These compounds were shown to be high affinity ligands for the ER with nanomolar IC 50 binding values. Series I acrylic acid ligands were generally ERα selective. In particular, compound 13e featuring a phenylpenta-2,4-dienoic acid substituent was shown to be antiproliferative and downregulated ERα and ERβ expression in MCF-7 breast cancer cells. Interestingly, from series III, the phenoxybutyric acid derivative compound 22 was not antiproliferative and selectively downregulated ERβ. A docking study of the benzoxepin ligands was undertaken. Compound 13e is a promising lead for development as a clinically relevant SERD, while compound 22 will be a useful experimental probe for helping to elucidate the role of ERβ in cancer cells.

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G 1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells.

Nuclear receptors such as the estrogen receptors (ERα and ERβ) modulate the effects of the estrogen hormones and are important targets for design of innovative chemotherapeutic agents for diseases such as breast cancer and osteoporosis. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. A series of novel conjugate molecules incorporating both the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targeting agent combretastatin A-4 were synthesised and evaluated as ER ligands. A number of these compounds demonstrated pro-apoptotic effects, with potent antiproliferative activity in ER-positive MCF-7 breast cancer cell lines and low cytotoxicity. These conjugates displayed binding affinity towards ERα and ERβ isoforms at nanomolar concentrations e.g., the cyclofenil-amide compound 13e is a promising lead compound of a clinically relevant ER conjugate with IC 50 in MCF-7 cells of 187 nM, and binding affinity to ERα (IC 50 = 19 nM) and ERβ (IC 50 = 229 nM) while the endoxifen conjugate 16b demonstrates antiproliferative activity in MCF-7 cells (IC 50 = 5.7 nM) and binding affinity to ERα (IC 50 = 15 nM) and ERβ (IC 50 = 115 nM). The ER binding effects are rationalised in a molecular modelling study in which the disruption of the ER helix-12 in the presence of compounds 11e , 13e and 16b is presented These conjugate compounds have potential application for further development as antineoplastic agents in the treatment of ER positive breast cancers., Competing Interests: The authors declare no conflict of interest.

Glucuronidation by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) is a cause of intrinsic drug resistance in cancer cells. Glucuronidation of combretastatin A-4 (CA-4) was previously identified as a mechanism of resistance in hepatocellular cancer cells. Herein, we propose chemical manipulation of β-lactam bridged analogues of Combretastatin A-4 as a novel means of overcoming drug resistance associated with glucuronidation due to the expression of UGTs in the CA-4 resistant human colon cancer HT-29 cells. The alkene bridge of CA-4 is replaced with a β-lactam ring to circumvent potential isomerisation while the potential sites of glucuronate conjugation are deleted in the novel 3-substituted-1,4-diaryl-2-azetidinone analogues of CA-4. We hypothesise that glucuronidation of CA-4 is the mechanism of drug resistance in HT-29 cells. Ring B thioether containing 2-azetidinone analogues of CA-4 such as 4-(4-(methylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27) and 3-hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (45) were identified as the most potent inhibitors of tumour cell growth, independent of UGT status, displaying antiproliferative activity in the low nanomolar range. These compounds also disrupted the microtubular structure in MCF-7 and HT-29 cells, and caused G 2 /M arrest and apoptosis. Taken together, these findings highlight the potential of chemical manipulation as a means of overcoming glucuronidation attributed drug resistance in CA-4 resistant human colon cancer HT-29 cells, allowing the development of therapeutically superior analogues., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerized when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, showing potent effects in human breast carcinoma MCF-7 cells whilst being relatively non-toxic to non-tumorigenic MCF-10a cells. These compounds will be further developed as potential clinical candidates for the treatment of breast cancer., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Neuroblastoma, a paediatric malignancy of the sympathetic nervous system, accounts for 15 % of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat partly due to the development of multidrug resistance. There is thus a compelling demand for new treatment strategies that can bypass resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various tumour models. We have previously reported that PBOX compounds induce apoptosis in drug sensitive and multidrug resistant neuroblastoma cells and synergistically enhance apoptosis induced by chemotherapeutics such as carboplatin. In this study we present further data concerning the molecular basis of PBOX-induced apoptosis in neuroblastoma. We demonstrate that PBOX-6 induced AMP-activated protein kinase (AMPK) activation and downstream acetyl-CoA carboxylase phosphorylation. Increased reactive oxygen species (ROS) appeared to serve as the upstream signal for AMPK activation as pretreatment of cells with the antioxidant N-acetylcysteine inhibited both AMPK activation and PBOX-induced apoptosis. Furthermore, activation of AMPK by PBOX-6 was found to inhibit mTOR complex 1 (mTORC1) signalling. Finally, we demonstrate the efficacy of PBOX-6 in an in vivo xenograft model of neuroblastoma. This study provides new insights into understanding the molecular and cellular mechanisms involved in PBOX-induced cell death in neuroblastoma and further supports their future use as novel anti-cancer agents for the treatment of neuroblastoma.

T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N -[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL.

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC50 values of 2.71μM and 1.86μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86., (Copyright © 2016. Published by Elsevier Ltd.)

Some compounds of a series of novel pyrrolo-1,5-benzoxa(thia)zepine, a well-known group of tubulin targeting agents, display anti-tumor effects mainly inducing cell cycle arrest and apoptosis in several human cancer models. A member of this family, pyrrolo-1,5-benzoxazepine-15 (PBOX-15), has previously shown potent pro-apoptotic activity in a variety of human tumor cell types, with minimal toxicity toward normal blood and bone marrow cells. In this study, we evaluated the PBOX-15-mediated effects in human colorectal cancer cell (CRC) lines, DLD-1 and HT-29. The compound, used at concentrations equal to or greater than 1 μM, inhibited the proliferation of human CRC cells, inducing a significant cell cycle arrest in the G2/M phase. In DLD-1 cells, treatments prolonged over 48 h triggered a strong activation of the intrinsic apoptotic pathway as indicated by activation of caspase-9, caspase-3 and PARP cleavage. Moreover, nanomolar concentrations of PBOX-15, significantly improved the oxaliplatin and 5-fluouracil-induced anti-proliferative effects in DLD1 cell line. The observed synergistic interaction of both PBOX-15/Oxaliplatin and PBOX-15/5FU may involve activation of p38 MAPK and JNK pathway, which in turn significantly increased caspase-3 cleavage in DLD-1 cells, treated with PBOX-5/Oxaliplatin but not with PBOX-15/5FU. Moreover, PBOX-15/5FU-treated cells showed an increase in expression of the pro-apoptotic protein Bax. Taken together, these results show that PBOX-15 could represent a promising compound for the treatment of human CRC and a strong candidate for novel therapeutic options.

Based on the results obtained from a computational study on the suitability of the isouronium and N-hydroxyguanidinium cations as hydrogen bond donors/acceptors, the DNA binding of a series of isouronium derivatives was assessed by DNA thermal denaturation experiments and compared to related N-hydroxyguanidines. Due to the poor DNA binding observed, the nature of the diaromatic linker was explored by preparing the corresponding amide-linked bis-isouronium derivative and measuring its DNA affinity. Next, the inhibitory effects of the isouronium derivatives on cell viability were evaluated in two different cancer cell lines providing IC50 values in the range of 36.9-57.4 μM (HL-60, leukemia), and 17.3-33.9 μM (Kelly, neuroblastoma). These values are comparable to those previously found for the N-hydroxyguanidine series. Compounds with the -S- linker (3, 6, and 10) proved to be considerably active in the HL-60 cells and even more active in the Kelly cell line. No correlation was found between DNA minor groove binding and cell growth inhibition; hence, activity may depend on different modes of action. Further studies into the apoptotic potential of these compounds indicated that, besides inhibiting cell viability and proliferation, derivatives 9 and 10, are significant apoptosis-inducers in both cell lines. Results obtained with HL-60 cells suggest that G2/M arrest and subsequent apoptosis induced by compound 10 are associated with microtubular depolymerisation, loss of mitochondrial membrane potential and activation of the caspase cascade. Moreover, the effects of compound 10 on cell viability and apoptosis in two non-cancereous cell lines (NIH3T3 and MCF-10A) indicate none or minimal toxicity., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Our recent finding that paclitaxel behaves as a peptidomimetic of the endogenous protein Nur77 inspired the design of two peptides (PEP1 and PEP2) reproducing the effects of paclitaxel on Bcl-2 and tubulin, proving the peptidomimetic nature of paclitaxel. Starting from these peptide-hits, we herein describe the synthesis and the biological investigation of linear and cyclic peptides structurally related to PEP2. While linear peptides (2a,b, 3a,b, 4, 6a-f) were found inactive in cell-based assays, biological analysis revealed a pro-apoptotic effect for most of the cyclic peptides (5a-g). Cellular permeability of 5a (and also of 2a,b) on HL60 cells was assessed through confocal microscopy analysis. Further cellular studies on a panel of leukemic cell lines (HL60, Jurkat, MEC, EBVB) and solid tumor cell lines (breast cancer MCF-7 cells, human melanoma A375 and 501Mel cells, and murine melanoma B16F1 cells) confirmed the pro-apoptotic effect of the cyclic peptides. Cell cycle analysis revealed that treatment with 5a, 5c, 5d or 5f resulted in an increase in the number of cells in the sub-G0/G1 peak. Direct interaction with tubulin (turbidimetric assay) and with microtubules (immunostaining experiments) was assessed in vitro for the most promising compounds., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.

The C-KIT receptor tyrosine kinase is constitutively activated in the majority of gastrointestinal stromal tumours (GIST). Imatinib (IM) a selective inhibitor of C-KIT, is indicated for the treatment of KIT-positive unresectable and/or metastatic GIST, and has tripled the survival time of patients with metastatic GIST. However, the majority of patients develop IM-resistance and progress. Although IM elicits strong antiproliferative effects, it fails to induce sufficient levels of apoptosis; acquired IM-resistance and disease recurrence remain an issue, a more effective drug treatment is greatly needed. We examined the effect of a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-15 in combination with IM on GIST cells. PBOX-15 decreased viability and in combination with IM synergistically enhanced apoptosis in both IM-sensitive and IM-resistant GIST cells, decreased the anti-apoptotic protein Mcl-1, and enhanced activation of pro-caspase-3 and PARP cleavage. The combination treatment also led to an enhanced inhibition of C-KIT-phosphorylation and inactivation of C-KIT-dependent signalling in comparison to either drug alone; CDC37, a key regulator of C-KIT in GIST was also dramatically decreased. Furthermore, PBOX-15 reduced CKII expression, an enzyme which regulates the expression of CDC37. In conclusion, our findings indicate the potential of PBOX-15 to improve the apoptotic response of IM in GIST cells and provide a more effective treatment option for GIST patients.

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated, leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl "A" ring and 4-phenyl "B" ring for potent antiproliferative activity and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC50 values of 38 and 19 nM, respectively, in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular structure in MCF-7 cells as visualized by confocal microscopy, and caused G2/M arrest and apoptosis. Compound 90b possessed a mean GI50 value of 22 nM in the NCI60 cell line screen, displayed minimal cytotoxicity, and was shown to interact at the colchicine-binding site on β-tubulin. Phosphate and amino acid prodrugs of both 78b and 90b were synthesized, of which the alanine amide 102b retained potency and is a promising candidate for further clinical development.

The estrogen receptors (ERα and ERβ) which are ligand inducible nuclear receptors are recognized as pharmaceutical targets for diseases such as osteoporosis and breast cancer. There is an increasing interest in the discovery of subtype Selective Estrogen Receptor Modulators (SERMs). A series of novel β-lactam compounds with estrogen receptor modulator properties have been synthesized. The antiproliferative effects of these compounds on human MCF-7 breast tumor cells are reported, together with binding affinity for the ERα and ERβ receptors. The most potent compound 15g demonstrated antiproliferative effects on MCF-7 breast tumor cells (IC 50  =   186 nM) and ERα binding (IC 50  =   4.3 nM) with 75-fold ERα/β receptor binding selectivity. The effect of positioning of the characteristic amine containing substituted aryl ring (on C-4 or N-1 of the β-lactam scaffold) on the antiproliferative activity and ER-binding properties of the β-lactam compounds is rationalized in a molecular modeling study.

Several prodrugs of the naturally occurring combretastatins have undergone extensive clinical evaluation as vascular targeting agents (VTAs). Their increased selectivity toward endothelial cells together with their innate ability to rapidly induce vascular shutdown and inhibit tumor growth at doses up to 10-fold less than the maximum tolerated dose led to the clinical evaluation of combretastatins as VTAs. Tubulin is well established as the molecular target of the combretastatins and the vast majority of its synthetic derivatives. Furthermore, tubulin is a highly validated molecular target of many direct anticancer agents routinely used as front-line chemotherapeutics. The unique vascular targeting properties of the combretastatins have somewhat overshadowed their development as direct anticancer agents and the delineation of the various cell death pathways and anticancer properties associated with such chemotherapeutics. Moreover, the ongoing clinical trial of OXi4503 (combretastatin-A1 diphosphate) together with preliminary preclinical evaluation for the treatment of refractory acute myelogenous leukemia has successfully highlighted both the indirect and direct anticancer properties of combretastatins. In this review, we discuss the development of the combretastatins from nature to the clinic. The various mechanisms underlying combretastatin-induced cell cycle arrest, mitotic catastrophe, cell death, and survival are also reviewed in an attempt to further enhance the clinical prospects of this unique class of VTAs., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based studies. Compounds 5e, l, p and 6a, b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562 and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for compounds 5l and 6b., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Oral cancer (OC) is a largely asymptomatic disease, resulting in one of the highest mortality rates of any cancer. OC is currently ranked as the sixth most common cancer in the world, according to a recent World Health Organization analysis, and its prevalence is increasing, both in western and developing regions. Depending on the stage of OC, treatment strategies include surgery, radiation therapy and chemotherapy, or a combination thereof. As with numerous other types of cancer, resistance to conventional chemotherapeutic drugs is increasing in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the use of a novel group of compounds, the pyrrolo‑1,5‑benzoxazepines (PBOXs), as a therapeutic alternative for the treatment of OC. PBOXs are microtubule‑targeting agents that are able to induce apoptosis in numerous cancer cell types, thereby preventing tumour cell proliferation. Ca9.22 gingival and TR146 buccal cell lines were used as models for OSSC. Cell viability and proliferation in the presence of two PBOXs: PBOX‑6 and PBOX‑15, was monitored using an AlamarBlueTM assay. Flow cytometric analysis of propidium iodide‑stained cells was used to determine the DNA content, and therefore the percentage of cells in each phase of the cell cycle. Microtubule disruption was determined by indirect immunofluorescence staining. Changes in protein expression and degradation were determined by western blotting. The results of the present study indicated that both PBOX‑6 and ‑15 were able to induce apoptotic cell death by disrupting the microtubule network in both cell lines. The EC50 values were subsequently calculated for both PBOX‑6 and ‑15, and PBOX‑15 was shown to possess a higher potency. Both compounds displayed anti‑proliferative effects mediated through sustained G2/M arrest accompanied by tubulin disruption, and a decrease in DNA repair protein poly (ADP ribose) polymerase expression. These findings suggest that PBOXs may prove useful, either alone or in combination with other agents, in the treatment of chemotherapeutic resistant OSCC.

The capacity of T-lymphocytes to migrate and localise in tissues is important in their protective function against infectious agents, however, the ability of these cells to infiltrate the tumour microenvironment is a major contributing factor in the development of cancer. T-cell migration requires ligand (ICAM-1)/integrin (LFA-1) interaction, activating intracellular signalling pathways which result in a distinct polarised morphology, with an actin-rich lamellipodium and microtubule (MT)-rich uropod. Combretastatin (CA)-4 is a MT-destabilising agent that possesses potent anti-tumour properties. In this study, the effect of CA-4 and its novel analogue CA-432 on human T-cell migration was assessed. Cellular pretreatment with either of CA compounds inhibited the migration and chemotaxis of the T-cell line HuT-78 and primary peripheral blood lymphocyte (PBL) T-cells. This migration-inhibitory effect of CA compounds was due to the disruption of the MT network of T-cells through tubulin depolymerisation, reduced tubulin acetylation and decreased MT stability. In addition, both CA compounds induced the RhoA/RhoA associated kinase (ROCK) signalling pathway, leading to the phosphorylation of myosin light chain (MLC). Furthermore, the siRNA-mediated depletion of GEF-H1, a MT-associated nucleotide exchange factor that activates RhoA upon release from MTs, in T-cells prevented CA-induced phosphorylation of MLC and attenuated the formation of actin-rich membrane protrusions and cell contractility. These results suggest an important role for a GEF-H1/RhoA/ROCK/MLC signalling axis in mediating CA-induced contractility of T-cells. Therapeutic agents that target cytoskeletal proteins and are effective in inhibiting cell migration may open new avenues in the treatment of cancer and metastasis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Twelve novel β-lactams were synthesized and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERβ, were determined. β-Lactams 23 and 26 had the strongest binding affinities for ERα (IC50 values: 40 and 8 nM, respectively) and ERβ (IC50 values: 19 and 15 nM). β-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G2/M phase (mitotic blockade) and depolymerization of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation of the expression of pro-survival proteins Bcl-2 and Mcl-1. Computational modeling predicted binding preferences for the dual ER/tubulin ligand 26. This series is an important addition to the known pool of ER antagonists and β-lactam 26 is the first reported compound that has dual-targeting properties for both the ER and tubulin.

Considering the structural similarities between the kinase inhibitor sorafenib and 4,4'-bis-guanidinium derivatives previously prepared by Rozas and co., which display interesting cytotoxicity in cancer cells, we have studied whether this activity could result from kinase inhibition. Five new families have been prepared consisting of unsubstituted and aryl-substituted 3,4'-bis-guanidiniums, 3,4'-bis-2-aminoimidazolinium and 3-acetamide-4'-(4-chloro-3-trifluoromethylphenyl)guanidinium derivatives. Cytotoxicity (measuring the IC50 values) and apoptosis studies in human HL-60 promyelocytic leukemia cells were carried out for these compounds. Additionally, their potential inhibitory effect was explored on a panel of kinases known to be involved in apoptotic pathways. The previously prepared cytotoxic 4,4'-bis-guanidiniums did not inhibit any of these kinases; however, some of the novel 3,4'-substituted derivatives showed a high percentage inhibition of RAF-1/MEK-1, for which the potential mode of binding was evaluated by docking studies. The interesting antitumour properties showed by these compounds open up new exciting lines of investigation for kinase inhibitors as anticancer agents and also highlights the relevance of the guanidinium moiety for protein kinase inhibitors chemical design., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)