Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update.

The ubiquitin-proteasome pathway (UPP) is the central protein degradation system in eukaryotic cells, playing a key role in homeostasis maintenance, through proteolysis of regulatory and misfolded (potentially harmful) proteins. As cancer cells produce proteins inducing cell proliferation and inhibiting cell death pathways, UPP inhibition has been exploited as an anticancer strategy to shift the balance between protein synthesis and degradation towards cell death. Over the last few years, marine invertebrates and microorganisms have shown to be an unexhaustive factory of secondary metabolites targeting the UPP. These chemically intriguing compounds can inspire clinical development of novel antitumor drugs to cope with the incessant outbreak of side effects and resistance mechanisms induced by currently approved proteasome inhibitors (e.g., bortezomib). In this review, we report about (a) the role of the UPP in anticancer therapy, (b) chemical and biological properties of UPP inhibitors from marine sources discovered in the last decade, (c) high-throughput screening techniques for mining natural UPP inhibitors in organic extracts. Moreover, we will tell about the fascinating story of salinosporamide A, the first marine natural product to access clinical trials as a proteasome inhibitor for cancer treatment

Modulation of human apurinic/apyrimidinic endonuclease 1 (APE1) functions for breast cancer therapy

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2016DNA repair is required for the maintenance of genome stability. In the last years DNA repair pathways have emerged as important targets for cancer therapy. Since standard anticancer agents are mainly DNA-damaging drugs, its combination with DNA repair inhibitors may contribute to improve treatment outcomes. Among the multiple effectors involved in DNA repair, the multifunctional base excision repair (BER) protein apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most attractive druggable targets in this field. APE1 is the major endonuclease in BER participating in the repair of different DNA lesions including toxic abasic sites. In addition to the DNA repair activity, APE1 also acts independently as a reduction/oxidation signalling protein modulating the activation and DNA binding ability of several transcription factors implicated in cell survival and tumour promotion and progression. In this context, this thesis is focused on the combination of APE1 pharmacological inhibitors with conventional anticancer agents in the highly aggressive human breast cancer MDA-MB-231 cell line. Endonuclease activity has been the most studied function of APE1 in cancer therapy. Methoxyamine (MX), a commercially available indirect inhibitor of APE1 DNA repair function, was evaluated in combination with doxorubicin (Dox) in MDA-MB-231 cells. The chemotherapeutic drug Dox is widely used in the treatment of advanced breast cancer and may act, in part, by inducing oxidative DNA damage. MX had little effects in viability and colony formation of MDA-MB-231 cells. However, a significant increase in the frequency of micronucleated cells and an alteration in the pattern of micronuclei distribution were observed suggesting an increase in Dox genotoxicity. The differential results obtained in terms of cytotoxicity and genotoxicity showed that a therapeutic strategy based on APE1 inhibition is likely to have no relevance for the improvement of outcomes of Dox treatment. Although several putative inhibitors of APE1 endonuclease activity have been reported they still lack potential to be translated to the clinical setting. Therefore, in this thesis a structure-based virtual screening (SBVS) study based on molecular docking analysis of National Cancer Institute (NCI) database of compounds was performed to identify novel small-molecule inhibitors of APE1. The evaluation of SBVS study most promising compounds in a fluorescence-based APE1 endonuclease activity assay revealed three APE1 inhibitors. Compound 22 was a potent APE1 inhibitor showing inhibitory effects at nanomolar concentrations, while compounds 37 and 41 inhibited the enzyme in the micromolar range. These novel scaffolds for the design of more potent APE1 inhibitors did not affect the viability of non-tumourigenic human breast epithelial MCF10A cell line highlighting their promising features. The importance of APE1 modulation is beyond its functions in DNA repair. Therefore, E3330, a commercially available inhibitor of APE1 redox function, was also evaluated as single agent and in combination with the taxane drug docetaxel (DTX) in MDA-MB- 231 cells. DTX has anti-migratory and anti-angiogenic effects and is frequently used in advanced breast cancer refractory to anthracycline-based regimens. Consequently, relevant endpoints of cell migration and invasion were studied in addition to cell viability, proliferation and cell cycle profile assessment. Minor effects were observed in cell proliferation. However, E3330 alone significantly reduced the collective cell migration evaluated by the wound-healing assay without affecting chemotaxis and chemoinvasion. The combination of E3330 with DTX significantly decreased invasion of MDA-MB-231 cells suggesting a potential therapeutic role in metastatic breast cancer. The results described in this work emphasise the importance of preclinical studies of APE1 functions in cancer therapy and highlight the potential of novel drug combinations based on APE1 inhibitors reinforcing the role of targeting DNA repair in cancer treatment

Computer-driven development of an in silico tool for finding selective histone deacetylase 1 inhibitors

Histone deacetylases (HDACs) are a class of epigenetic modulators overexpressed in numerous types of cancers. Consequently, HDAC inhibitors (HDACIs) have emerged as promising antineoplastic agents. Unfortunately, the most developed HDACIs suffer from poor selectivity towards a specific isoform, limiting their clinical applicability. Among the isoforms, HDAC1 represents a crucial target for designing selective HDACIs, being aberrantly expressed in several malignancies. Accordingly, the development of a predictive in silico tool employing a large set of HDACIs (aminophenylbenzamide derivatives) is herein presented for the first time. Software Phase was used to derive a 3D-QSAR model, employing as alignment rule a common-features pharmacophore built on 20 highly active/selective HDAC1 inhibitors. The 3D-QSAR model was generated using 370 benzamide-based HDACIs, which yielded an excellent correlation coefficient value (R2 = 0.958) and a satisfactory predictive power (Q2 = 0.822; Q2F3 = 0.894). The model was validated (r2ext_ts = 0.794) using an external test set (113 compounds not used for generating the model), and by employing a decoys set and the receiver-operating characteristic (ROC) curve analysis, evaluating the Güner-Henry score (GH) and the enrichment factor (EF). The results confirmed a satisfactory predictive power of the 3D-QSAR model. This latter represents a useful filtering tool for screening large chemical databases, finding novel derivatives with improved HDAC1 inhibitory activity

Complex Systems Analysis of Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics in cell cycling network transformations involved in carcinogenesis is proposed. Carcinogenesis is a complex process that involves dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments that form numerous inter-related pathways referred to as networks.
The variable biotopology of such dynamic networks is highly complex, and has a number of interesting properties that can be formally characterized at one level of organization by mathematical structures called 'biogroupoids'. 
One such family of pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/ division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which all play major roles in carcinogenesis of many cancers. A novel theoretical analysis is thus possible based on recently published studies of cyclin signaling, with special emphasis placed on the roles of cyclins D1 and E, suggests novel clinical trials and rational therapies of cancer through reestablishment of cell cycling inhibition in metastatic cancer cells

Exploration of the structural requirements of Aurora Kinase B inhibitors by a combined QSAR, modelling and molecular simulation approach

Aurora kinase B plays an important role in the cell cycle to orchestrate the mitotic process. The amplification and overexpression of this kinase have been implicated in several human malignancies. Therefore, Aurora kinase B is a potential drug target for anticancer therapies. Here, we combine atom-based 3D-QSAR analysis and pharmacophore model generation to identify the principal structural features of acylureidoindolin derivatives that could potentially be responsible for the inhibition of Aurora kinase B. The selected CoMFA and CoMSIA model showed significant results with cross-validation values (q(2)) of 0.68, 0.641 and linear regression values (r(2)) of 0.971, 0.933 respectively. These values support the statistical reliability of our model. A pharmacophore model was also generated, incorporating features of reported crystal complex structures of Aurora kinase B. The pharmacophore model was used to screen commercial databases to retrieve potential lead candidates. The resulting hits were analyzed at each stage for diversity based on the pharmacophore model, followed by molecular docking and filtering based on their interaction with active site residues and 3D-QSAR predictions. Subsequently, MD simulations and binding free energy calculations were performed to test the predictions and to characterize interactions at the molecular level. The results suggested that the identified compounds retained the interactions with binding residues. Binding energy decomposition identified residues Glu155, Trp156 and Ala157 of site B and Leu83 and Leu207 of site C as major contributors to binding affinity, complementary to 3D-QSAR results. To best of our knowledge, this is the first comparison of WaterSwap field and 3D-QSAR maps. Overall, this integrated strategy provides a basis for the development of new and potential AK-B inhibitors and is applicable to other protein targets

Pyridine Based Antitumour Compounds Acting at the Colchicine Site

[EN]Antimitotics binding at the colchicine site of tubulin are important antitumour and vascular disrupting agents. Pyridines and azines are privileged scaffolds in medicinal chemistry and in recent years many colchicine site ligands (CSL) have incorporated them into their structures with the aim of improving their pharmacokinetic and pharmacodynamics properties. CSL have been classified according to their chemical structures and the chemical structures of the pyridine and azine containing antimitotic compounds are described. The design principles behind the structural modifications and the achieved effect on the biological activity upon inclusion of these heterocycles are also discussed. Lessons from the achievements and failures have been extracted and future perspectives delineated

PhD

dissertationI discovered that certain electrophilic prostaglandins inhibit the ubiquitin-specific protease (USP) activity of the proteasome pathway. Herein, evidence is presented that supports the hypothesis that the cross-conjugated α,β-unsaturated dienone is a molecular determinant for the potency of this activity, and that this chemical feature causes an alteration in cellular ubiquitin dynamics, resulting in decreased free ubiquitin and decreased protein degradation. I show that this decrease in protein degradation activates the unfolded-protein response (UPR) of both the cytoplasm and the endoplasmic reticulum, likely due to the accumulation of deranged/misfolded proteins. I make the novel observation that as an attempt to compensate for the loss in protein degradation by the proteasome pathway, the lysosomal degradation pathway is activated in USP inhibitor treated cells. Lastly I show that, ultimately, cell death occurs due to the build-up of toxic levels of cellular protein. These data reconcile previously known effects of prostaglandin treatment, namely that heat-shock proteins are up-regulated and that a number of short-lived proteins are stabilized, and in so doing, establish a cohesive model for prostaglandin-induced apoptosis. The potential that components of the ubiquitin-proteasome pathway may be useful targets for cancer chemotherapy has been realized only recently with the success, in human clinical trials, of the proteasome inhibitor, VELCADE™ (PS-341). Given that several hundred other potential molecular targets reside within the proteasome pathway, there is intense interest in discovering novel points for drug intervention. Our data suggest that inhibition of USP activity represents a legitimate target for chemotherapeutic development

Complex Systems Analysis of Arrested Neural Cell Differentiation during Development and Analogous Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics of arrested neural cell Differentiation--induced cell proliferation during organismic development and the analogous cell cycling network transformations involved in carcinogenesis is proposed. Neural tissue arrested differentiation that induces cell proliferation during perturbed development and Carcinogenesis are complex processes that involve dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments. Such 'dynamically inter-connected' biomolecules form numerous inter-related pathways referred to as 'molecular networks'. One such family of signaling pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which play major roles in various neoplastic transformations of many tissues. The novel theoretical analysis presented here is based on recently published studies of arrested cell differentiation that normally leads to neural system formation during early developmental stages; the perturbed development may involve cyclin signaling and cell cycling responsible for rapidly induced cell proliferation without differentiation into neural cells in such experimental studies; special emphasis in this modular model is placed upon the roles of cyclins D1 and E, and does suggest novel clinical trials as well as rational therapies of cancer through re-establishment of cell cycling inhibition in metastatic cancer cells. Cyclins are proteins that are often over-expressed in cancerous cells (Dobashi et al., 2004). They may also be over-expressed in cells whose differentiation is arrested during the early stages of organismic development, leading to increased cell proliferation instead of differentiation into specialized tissues such as those forming the neural system. Cyclin-dependent kinases (CDK), their respective cyclins, and inhibitors of CDKs (CKIs) were identified as instrumental components of the cell cycle-regulating machinery. In mammalian cells the complexes of cyclins D1, D2, D3, A and E with CDKs are considered motors that drive cells to enter and pass through the “S” phase. Cell cycle regulation is a critical mechanism governing cell division and proliferation, and it is finely regulated by the interaction of cyclins with CDKs and CKIs, among other molecules (Morgan et al., 1995). A categorical and Topos framework for Łukasiewicz Algebraic Logic models of nonlinear dynamics in complex functional genomes and cell interactomes is also proposed. Łukasiewicz Algebraic Logic models of genetic networks and signaling pathways in cells are formulated in terms of nonlinear dynamic systems with n-state components that allow for the generalization of previous logical models of both genetic activities and neural networks. An algebraic formulation of varying 'next-state' functions is extended in a Łukasiewicz-Topos with an n-valued Łukasiewicz Algebraic Logic subobject classifier description that represents non-random and nonlinear network activities as well as their transformations in developmental processes and carcinogenesis. Important aspects of Cell Cycling, the Control of Cell Division,and the Neoplastic Transformation in Carcinogenesis are being considered and subjected to algebraic-logico- relational, and computer-aided investigations. The essential roles of various levels of c-Myc, p27 quasi-complete inhibition/blocking, TP53 and/or p53 inactivation, as well as the perpetual hTERT activation of Telomerase biosynthesis are pointed out as key conditions for Malignant Cell transformations and partial re-differentiation leading to various types of cancer such as lung, breast,skin, prostate and colon. Rational Clinical trials, Individualized Medicine and the potential for optimized Radio-, Chemo-, Gene-, and Immuno- therapies of Cancers are suggested on the basis of integrated complex systems biology modeling of oncogenesis, coupled with extensive genomic/proteomic and interactomic High-throughput/high-sensitivity measurements of identified, sorted cell lines that are being isolated from malignant tumors of patients undergoing clinical trials with adjuvant signaling drug therapies. The implications of the cyclin model for abnormal neural development during early development are being considered in this model that may lead to explanations of subsequent cognitive changes associated with abnormal neural cell differentiation in environmentally-affected embryos. This new model may also be relevant to detecting the onset of senescing neuron transformations in Alzheimer's and related diseases of the human brain in ageing populations at risk