Evaluation of a new chemotype of tubulin modulators of the vinca domain

173 p.-60 fig.-10 tab.-2 anexos.Nowadays, one of the major public health issues in developed countries is cancer and, for this reason, the study of new treatments against this disease receives today special attention. Among all existing therapies, chemotherapy is preferential choice in most cases because of their high success expectations. Chemotherapy involves the block of a specific cellular function by administering a chemical agent. The two cellular pathways, on which chemotherapy usually acts, are the genetic material replication and the cell division. Within the group of treatments that act on cell division, those using chemotherapy drugs that interact with the cytoskeleton, are at the forefront of the fight against cancer. Microtubules, the major components of cytoskeleton, are dynamic polymers, which look like hollow cylinders. They are made up of tubulin protofilaments, which establish lateral contacts with other protofilaments. Besides, such protofilaments are formed by the head-tail association of αβ-tubulin heterodimers. Balance between dimeric and polymeric state is regulated by a dynamics, which is GTP-hydrolysis dependent. GTP-heterodimers are more likely to polymerize, while GDP-heterodimers drive balance toward free species. There is a large collection of chemical compounds that are able to regulate this dynamics, either promoting tubulin assembly and/or stabilizing microtubules, or promoting disassembly and/or destabilizing polymers. In both cases, proper function of these structures is altered and thus, the whole functions they are responsible for, such as chromosome segregation or division of nuclear and cell envelopes during the replication process, are also altered. The work presented in this report, entitled “Evaluation of a new chemotype of tubulin modulators of the vinca domain”, shows the characterization of the binding process and the mechanism of action of a synthetic compound and its structural derivatives, from a biological, biochemical and structural point of view. Obtained information was further used in order to determine the implication of the different structural determinants in the activity of each compound. On one hand, performed tests on human cell lines demonstrated that the studied compounds have cytotoxic activity and they also demonstrated that the compounds interact with the microtubule network, being able to arrest cell cycle at G2/M phase. In addition, it was observed that none of the ligands is a substrate for the P-glycoprotein (PGP). This fact causes that these ligands are able to maintain, approximately, the same level of activity in cells that show the multidrug resistance phenotype (MDR), by overexpression of PGP. On the other hand, the study of the activity on the microtubule network of Aspergillus nidulans allowed to determine that both ligand binding and the consequent produced effect are reversible actions. Additionally, by in vitro biochemical experiments, it was analyzed the effect of compounds on tubulin. Its assembly capacity significantly increased as a result of the presence of studied compounds, so it was concluded that these compounds behave as tubulin polymerizers. Afterwards, by DOSY-NMR and analytical ultracentrifugation experiments, it was concluded that studied molecules induce the formation of oligomers of various sizes, without actually reaching microtubule generation.Furthermore, it was observed that binding occurs, neither to tubulin dimers nor to preformed microtubules. However, ligands are able to bind to tetramers. This, coupled with the fact that ligands compete for the binding site with model drugs, which bind in the interfacial region, indicates that the region of contact with the tubulin molecule is located in the vinca domain. Since interfacial ligands often require self-assembly of tubulin in order to bind, and since such self-assembly depends on the concentration of magnesium, it was determined the relation between ligand binding to target and magnesium concentration. The result showed a clear dependence between these two parameters, which means that binding and self-assembly are two coordinated processes in the studied system. Binding stoichiometry for all the studied compounds, measured in glycerol-buffer, fluctuates between 0.6:1 and 1.15:1, indicating the average oligomerization state induced by each ligand. This average oligomerization state tends to the tetrameric state, the more the value approaches to the relation 0.5:1, while it tends to big polymers, the more the value approaches to 1:1. Moreover, a method for determining equilibrium constants of ligands which bind to tubulin, by experiments of sedimentation velocity of analytical ultracentrifugation, has been optimized. This method is based on the assumption that the analyzed system follows the isodesmic condition. In such systems, each oligomerization step has the same binding constant as the previous step. The developed method allows to determine affinity constants for any ligand, as long as the studied ligand interacts with proteins, which assemble in a isodesmic manner, and changes the affinity of the protein self-assembly when it binds. Obtained results show that compounds coded as 1 (parental) and 2 are the most actives of the series. Finally, the structural characterization studies involved X-ray diffraction, electron microscopy, STD-NMR and bioinformatics. The tubulin tetramer structure, crystallized in the presence of the parental compound revealed the real conformation of the bound ligand, and it also allowed to determine its mechanism of action. When binding happens, the tetrameric curved apo-structure slightly straightens, what makes possible some degree of tubulin assembly. From the results obtained by electron microscopy, it is found that all the compounds induce the formation of helical polymers, while from the STDNMR analysis it was concluded that they contact with the vinca domain through the triazole ring, the trifluorobenzene ring and the substituent of the 3-position of the azabicyclo. Additionally, molecular modelling of the ligands showed that all of them bind to the tubulin in a very similar way, mainly through two aromatic stacks with two tubulin residues. It was also observed an inverse relationship between the polarity of the substituents at 3-position of the azabicyclo and the binding constant of the compounds. Finally, the effects induced by the studied ligands and the effects induced by vinblastine were compared. Vinblastine is a widely used antitumor drug which, as well as the studied compounds do, it binds to tubulin in the vinca domain. Comparison between data obtained in this work and those available in the literature about vinblastine, revealed several differences. While vinblastine, which is a drug with larger volume than the studied compounds, induces a wedge that promotes tubulin disassembly, these studied compounds access the vinca domain and straighten the tetramer structure they contact with. This fact suggests that the difference in the specific volume of the ligands of the vinca domain determines the induced effect on tubulin.Peer reviewe

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)- 2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-tostraight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.M-DC thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This work has received the Ramón Madroñero award for young researchers (to M-DC and OB) in the XVII call www.impactjournals.com/oncotarget 17 Oncotarget sponsored by the Spanish Society of Medicinal Chemistry (SEQT). This project has been supported by the Spanish Ministerio de Economia y Competitividad (SAF2012- 39760-C02-01 to M-JC, M-JP-P, SV and E-MP; and BIO2013-42984-R to JFD), Comunidad de Madrid (BIPEDD2; ref. P2010/BMD-2457 to M-JC and J-FD), the Swiss National Science Foundation (310030B_138659 and 31003A_166608; to MOS). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” and COST action CM1470.Peer reviewe

Cancer mortality inequalities in urban areas: a Bayesian small area analysis in Spanish cities

Background: Intra-urban inequalities in mortality have been infrequently analysed in European contexts. The aim of the present study was to analyse patterns of cancer mortality and their relationship with socioeconomic deprivation in small areas in 11 Spanish cities. Methods: It is a cross-sectional ecological design using mortality data (years 1996-2003). Units of analysis were the census tracts. A deprivation index was calculated for each census tract. In order to control the variability in estimating the risk of dying we used Bayesian models. We present the RR of the census tract with the highest deprivation vs. the census tract with the lowest deprivation. Results: In the case of men, socioeconomic inequalities are observed in total cancer mortality in all cities, except in Castellon, Cordoba and Vigo, while Barcelona (RR = 1.53 95%CI 1.42-1.67), Madrid (RR = 1.57 95%CI 1.49-1.65) and Seville (RR = 1.53 95%CI 1.36-1.74) present the greatest inequalities. In general Barcelona and Madrid, present inequalities for most types of cancer. Among women for total cancer mortality, inequalities have only been found in Barcelona and Zaragoza. The excess number of cancer deaths due to socioeconomic deprivation was 16,413 for men and 1,142 for women. Conclusion: This study has analysed inequalities in cancer mortality in small areas of cities in Spain, not only relating this mortality with socioeconomic deprivation, but also calculating the excess mortality which may be attributed to such deprivation. This knowledge is particularly useful to determine which geographical areas in each city need intersectorial policies in order to promote a healthy environment.This article was partially supported by Fondo de Investigaciones Ssanitarias (FIS) projects numbers PI042013, PI040041, PI040170, PI040069, PI042602 PI040388, PI040489, PI042098 , PI041260, PI040399, PI081488 and by the CIBER en Epidemiología y Salud Pública (CIBERESP), Spain and by the program of “Intensificación de la Actividad Investigadora (Carme Borrell)” funded by the “Instituto de Salud Carlos III” and “Departament de Salut. Generalitat de Catalunya”

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Zampanolide binding to tubulin indicates crosstalk of taxane site with colchicine and nucleotide sites

41 p.-8 fig.-2 tab. Field, Jessica J. et al.The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) from Ministerio de Economia y Competitividad and the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (JJF & JHM). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. The CNIC is supported by the Ministerio de Ciencia e Innovación and the Fundación Pro CNIC.Peer reviewe

Quinolin-6-yloxyacetamides are microtubule destabilizing agents that bind to the colchicine site of tubulin

Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells.This work was supported by grants from Spanish Ministerio de Economía y Competitividad (BFU2016-75319-R (AEI/FEDER, UE) to J. Fernando Díaz) and from the Swiss National Science Foundation (31003A_166608 to Michel O. Steinmetz). The authors acknowledge networking contribution by the COST (European Cooperation in Science and Technology) Action CM1407 “Challenging organic syntheses inspired by nature—from natural products chemistry to drug discovery”. X-ray data were collected at beamline X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland). Ashwani Sharma was supported by an EMBO (European Molecular Biology Organization) Long Term Fellowship, J. Fernando Díaz is member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET)

Quinolin-6-Yloxyacetamides Are Microtubule Destabilizing Agents That Bind to the Colchicine Site of Tubulin

Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells

Novel colchicine-site binders with a cyclohexanedione scaffold identified through a ligand-based virtual screening approach

Vascular disrupting agents (VDAs) constitute an innovative anticancer therapy that targets the tumor endothelium, leading to tumor necrosis. Our approach for the identification of new VDAs has relied on a ligand 3-D shape similarity virtual screening (VS) approach using the ROCS program as the VS tool and as query colchicine and TN-16, which both bind the α,β-tubulin dimer. One of the hits identified, using TN-16 as query, has been explored by the synthesis of its structural analogues, leading to 2-(1-((2-methoxyphenyl)amino)ethylidene)-5-phenylcyclohexane-1,3-dione (compound 16c) with an IC50 = 0.09 ± 0.01 μM in HMEC-1 and BAEC, being 100-fold more potent than the initial hit. Compound 16c caused cell cycle arrest in the G2/M phase and interacted with the colchicine-binding site in tubulin, as confirmed by a competition assay with N,N'-ethylenebis(iodoacetamide) and by fluorescence spectroscopy. Moreover, 16c destroyed an established endothelial tubular network at 1 μM and inhibited the migration and invasion of human breast carcinoma cells at 0.4 μM. In conclusion, our approach has led to a new chemotype of promising antiproliferative compounds with antimitotic and potential VDA properties.status: publishe