110 research outputs found

    Molecular and cellular pharmacology of novel chiral and achiral CC-1065/duocarmycin analogues.

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    CC-1065 and the duocarmycins are highly potent anticancer agents, exerting their biological activity through covalently reacting with adenine-N3 in the minor groove of AT-rich sequences. The alkylation properties and cytotoxicity of a series of novel chiral analogues are reported in this thesis. Structural modifications of established pharmacophores resulting in novel alkylating functionalities as well as variations of the DNA binding domain were introduced in the analogues considered, The sequence specificity of these compounds was assessed by a Taq Polymerase stop assay, identifying the sites of covalent modification on plasmid DNA and the purine-N3 adducts probed by a thermally-induced strand cleavage assay. The cytotoxic potency of the analogues was determined against human, chronic myeloid leukemia, K562, cells, using a MTT based growth inhibition assay. The importance of the chiral centre present in the natural products was subsequently investigated with a series of achiral analogues. The studies established that the chiral centre is not absolutely required for DNA interaction and cytotoxicity. This finding offers the possibility of a new platform for the design of novel, active CC-1065/duocarmycin analogues. A key chiral and an achiral analogue were selected for DNA repair studies. The sensitivity of yeast mutants deficient in specific DNA repair pathways was assessed in order to delineate the mechanisms involved in the repair of the relevant adenine-N3 adducts. Nucleotide excision repair (NER) and post replication repair mutants were the most sensitive to the two analogues. Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution. Adduct elimination of both agents was by transcription-coupled NER, and dependent upon functional Radl8. Finally, the involvement of NER as the predominant excision pathway was further confirmed in mammalian DNA repair mutant cells

    Next generation 3D pharmacophore modeling

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    3D pharmacophore models are three‐dimensional ensembles of chemically defined interactions of a ligand in its bioactive conformation. They represent an elegant way to decipher chemically encoded ligand information and have therefore become a valuable tool in drug design. In this review, we provide an overview on the basic concept of this method and summarize key studies for applying 3D pharmacophore models in virtual screening and mechanistic studies for protein functionality. Moreover, we discuss recent developments in the field. The combination of 3D pharmacophore models with molecular dynamics simulations could be a quantum leap forward since these approaches consider macromolecule–ligand interactions as dynamic and therefore show a physiologically relevant interaction pattern. Other trends include the efficient usage of 3D pharmacophore information in machine learning and artificial intelligence applications or freely accessible web servers for 3D pharmacophore modeling. The recent developments show that 3D pharmacophore modeling is a vibrant field with various applications in drug discovery and beyond

    The HIV-1 Integrase: Modeling and Beyond

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    Mechanistic Elucidation of Protease–Substrate and Protein–Protein Interactions for Targeting Viral Infections

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    Viral infections represent an old threat to global health, with multiple epidemics and pandemics in the history of mankind. Despite several advances in the development of antiviral substances and vaccines, many viral species are still not targeted. Additionally, new viral species emerge, posing a menace without precedent to humans and animals and causing fatalities, disabilities, environmental harm, and economic losses. In this thesis, we present rational modeling approaches for targeting specific protease-substrate and protein-protein interactions pivotal for the viral replication cycle. Over the course of this work, antiviral research is supported beginning with the development of small molecular antiviral substances, going through the modeling of a potential immunogenic epitope for vaccine development, towards the establishment of descriptors for susceptibility of animals to a viral infection. Notably, all the research was done under scarce data availability, highlighting the predictive power of computational methods and complementarity between in-silico and in-vitro or in-vivo methods

    High-dimension profiling data generate a multifunctional peptide-mimic chemo-structure by connecting conserved fragments based on the neutrophil immune defense CAP37 protein as an in-silico antibacterial and woundhealing candidate agent

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    CAP37, a protein constitutively EXPRESSED in human neutrophils and induced in responseto infection in corneal epithelial cells, plays a significant role in host defense against infection. Initiallyidentified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Delineation of thedomains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use have alsobeen explored. Novel findings of a multifunctional domain between a 120 and 146 have also been reported.Here, in Biogenea Pharmaceuticals Ltd we for the first time generated a multifunctional peptide-mimicchemo-structure by connecting conserved fragments based on the neutrophil immune defense CAP37 proteinas an in-silico antibacterial and wound-healing canditate agent. This in silico effort was accomplished byutilizing various generated descriptors of proteins, compounds and their interactions resulting in aperformance/cost evaluation study for a GPU-based drug discovery application on volunteer computingapproaches based on Automated Structure-Activity Relationship Minings in Connecting Chemical Structureto Biological Profiles for the generation of novel Computational biomodeling of 3D drug-protein binding freeenergy evaluation

    In silico studies of nucleic acid complexes with proteins, and therapeutic small molecules.

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    In silico approaches to nucleic acid targeted drug discovery have been used in order to study duplex DNA, in complexes with proteins as well as more unusual form of G-rich DNA folded into higher-order structures termed as G-quadruplexes, in complexes with therapeutic small molecules. The overall aim of this work has been to provide insight into the stability, recognition, energetics of binding and dynamic behavior of these DNAs in complexes with the STAT3βtc homodimer:DNA complex and with therapeutic small molecules in G-quadruplex/pyridostatin and G-quadruplex/fragment complexes by means of combined in silico approaches. The techniques of explicit solvent molecular dynamics (MD) simulations, and subsequent calculations of the free energies of binding, molecular docking, and 3D-pharmacophore modeling have been applied to study STAT3 and G-quadruplex DNA, promising targets for anticancer therapeutic intervention. Analysis of the data obtained from multiple 50-ns MD simulations of the STAT3-DNA complexes has suggested how the transcription factor STAT3 interacts with duplex DNA, the nature of the conformational changes, and ways in which func- tion may be affected. A majority of known pathologic mutations affecting the DNA-biding region of the STAT3 have been found at the protein-DNA interface, and they have been mapped in detail. The STAT3 conformations obtained from these MD simulations have been subsequently used as a basis for a comparative multiple-target molecular docking study with an in-house library of potential STAT3 inhibitors, providing a rational of their binding in the absence of structural data. A novel “dynamic docking” approach (robust platform of numerous MD simulations) has been developed to address the G-quadruplex receptor and ligand flexibility issue, and subsequent conformational change upon binding. The strength of binding at different regions and both sites of the G-quadruplex were then closely examined. An in silico study of a fragment-based approach towards G-quadruplex stabilizing ligands has also been explored, in parallel with experimental studies, to assess whether this could provide a reliable rapid approach to finding hit fragments in the case of the c-MYC promoter quadruplex

    Determination of in silico rules for predicting small molecule binding behavior to nucleic acids in vitro.

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    The vast knowledge of nucleic acids is evolving and it is now known that DNA can adopt highly complex, heterogeneous structures. Among the most intriguing are the G-quadruplex structures, which are thought to play a pivotal role in cancer pathogenesis. Efforts to find new small molecules for these and other physiologically relevant nucleic acid structures have generally been limited to isolation from natural sources or rationale synthesis of promising lead compounds. However, with the rapid growth in computational power that is increasingly becoming available, virtual screening and computational approaches are quickly becoming a reality in academia and industry as an efficient and economical way to discover new lead compounds. These computational efforts have historically almost entirely focused on proteins as targets and have neglected DNA. We present research here showing that not only can software be utilized for targeting DNA, but that selectivity metrics can be developed to predict the binding mechanism of a small molecule to a DNA target. The software Surflex and Autodock were chosen for evaluation and were demonstrated to be able to accurately reproduce the known crystal structures of several small molecules that bind by the most common nucleic acid interacting mechanisms of groove binding and intercalation. These software were further used to rationalize known affinity and selectivity data of a 67 compound library of compounds for a library of nucleic acid structures including duplex, triplex and quadruplexes. Based upon the known binding behavior of these compounds, in silica metrics were developed to classify compounds as either groove binders or intercalators. These rules were subsequently used to identify new triplex and quadruplex binding small molecules by structure and ligand-based virtual screening approaches using a virtual library consisting of millions of commercially available small molecules. The binding behavior of the newly discovered triplex and quadruplex binding compounds was empirically validated using a number of spectroscopic, fluorescent and thermodynamic equilibrium techniques. In total, this research predicted the binding behavior of these test compounds in silica and subsequently validated these findings in vitro. This research presents a novel approach to discover lead compounds that target multiple nucleic acid morphologies

    Molecular Modelling of a novel G-quadruplex structure and its interaction with ligands

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    DNA can exist under many different forms. Lately, G-quadruplexes, which are one example of the non-canonical DNA forms, have been getting a lot of attention due to the role they play in certain biological processes and as potential targets for therapeutic interventions. For example, these structures can exist in certain parts of the telomeres, structures responsible for cell replication. In cancer cells, if the enzyme telomerase could be inhibited, by inducing the formation of a G-quadruplex structure in guanine-rich telomere sequences, the spread of cancer cells would cease. For this and other reasons, it becomes important to be able to induce the formation of G-quadruplex structures and/or stabilize them, and one of the ways of doing so consists of targeting these sequences with ligands that have good affinity to G-quadruplex structures. However, few G-quadruplex ligands demonstrated the needed properties to fulfill the clinical needs, and further efforts to determine which would be better suited to target any particular sequence are needed. This work aimed at comparing the affinity to the pre-miR-149 G-quadruplex structure of seven promising ligands found in the literature, through the latest techniques fit for that purpose. The seven ligands tested were: [16]phenN2, [32]phen2N4, phen-DC3, pyridostatin, acridine orange derivatives C8 and C8-NH2 and L-arginine. Firstly, they underwent computational tests, with the molecular structure of the quadruplex and the ligand being simulated, and their optimal binding site and conformation found. Their binding energies were compared, and they underwent molecular dynamics runs to simulate their behavior in an environment with solvent, followed by another binding energy comparison. The trend obtained in order of decreasing binding affinity was: pyridostatin > [32]phen2N4 > [16]phenN2 > Phen-DC3 > L-arginine > C8 > C8-NH2. Biophysical techniques were then performed, to determine the binding affinities experimentally. First, circular dichroism spectroscopy and melting studies (performed on four ligands) established the following trend: C8 > pyridostatin > C8-NH2 > [16]phenN2. Fluorescence spectroscopy titration (performed on three) revealed a similar trend: C8 > C8-NH2 > [16]phenN2. Lastly, affinity chromatography experiments were held to test how other DNA sequences would bind to C8-NH2. The results revealed that the ligand has better binding affinity with parallel quadruplexes over antiparallel ones, and poor binding with a duplex sequence. Overall, the best ligands identified for binding to the G-quadruplex structure were the acridine orange derivatives C8 and C8-NH2, and pyridostatin. These three ligands should be considered prime candidates for further research in this area.ADN pode existir sob a forma de diversas estruturas, contrariamente ao que a vasta maioria da população pensa, ao imaginar a dupla hélice de Watson e Crick. Uma das formas que tem sido mais investigada ultimamente consiste no G-quadruplex. Esta estrutura não canónica do DNA ocorre quando guaninas se emparelham e organizam em estruturas cíclicas através de pontes de hidrogénio Hoogsteen, chamadas G-quartetos. Estas estruturas formam-se por empilhamento p-p entre elas próprias, originando o G-quadruplex, desde que haja um catião (preferivelmente K+) para assumir uma localização central entre todos os quartetos. Estas estruturas desempenham funções importantes a nível de regulação da transcrição e replicação do DNA. Alguns estudos indicam também que podem ser relevantes a nível de manutenção do DNA, e que várias secções do DNA humano se encontram num estado de equilíbrio entre a forma de G-quadruplex e duplex. São também considerados alvos para certas abordagens terapêuticas a nível do cancro. Por exemplo, vários oncogenes como c-kit e c-myc têm a capacidade de formar G-quadruplexes nos seus promotores. Controlando a forma que estes genes assumem, seria possível controlar a sua transcrição, e possivelmente impedir a formação de cancro. Outra possibilidade cinge-se à inibição da telomerase, uma enzima responsável pela replicação celular, que está sobreexpressa em células cancerígenas. Se uma parte do telómero assumir uma estrutura em G-quadruplex, a ação desta enzima fica inibida, efetivamente parando a progressão do cancro. Portanto, torna-se necessário induzir e estabilizar a formação de estruturas do G-quadruplex. A estratégia é utilizar ligandos que interajam por interações intermoleculares de forma a estabilizar a estrutura do G-quadruplex, e outra topologia que esteja em equilíbrio. No entanto, analisando a literatura, conclui-se que apenas alguns grupos de ligandos são efetivamente ligandos de G-quadruplex. Este trabalho de investigação teve como objetivo comparar 7 ligandos promissores da estrutura de G-quadruplex designada por pre-miR-149 literatura. Os ligandos selecionados foram macrociclos derivados de fenantrolina ([16]phenN2, [32]phen2N4, Phen-DC3, e derivados de laranja de acridina C8 e C8-NH2. Determinou-se a afinidade e a estabilização destes ligandos com a estrutura do RNA G-quadruplex, a pre-miR-149. Isso será feito em duas etapas principais. Primeiro, foram realizadas simulações computacionais para determinar quais os ligandos mais promissores e quais os seus métodos de interação com a estrutura G-quadruplex. Estas dividiram-se em três passos: primeiro, foram geradas as estruturas da sequência e de cada ligando em software adequado. Segundo, foram feitas simulações de docking de modo a averiguar os locais de ligação de cada ligando ao G-quadruplex, e a conformação e interações entre o ligando e o quadruplex, sendo também calculadas energias de ligação entre o ligando e o G-quadruplex. Finalmente, foram feitas simulações de dinâmica molecular sobre como essa conformação evoluiria num ambiente fisiológico simulado e calculadas novas energias de ligação, que comparadas entre si, revelam diferenças de afinidades entre os ligandos. Após estas técnicas computacionais, foram executadas técnicas biofísicas, como espetroscopia de dicroísmo circular e estudos de desnaturação térmica, e espectroscopia de fluorescência para determinar experimentalmente as afinidades de cada ligando para com a estrutura escolhida. Foram também executadas experiências de cromatografia de afinidade para determinar o comportamento de um ligando para com sequência do RNA G-quadruplex, a pre-miR-149. O programa usado para avaliar as conformações iniciais gerou estruturas demasiado rígidas e pouco flexíveis com os ligandos macrocíclicos [16]phenN2 e [32]phen2N4. As energias de ligação obtidas revelaram a nível de afinidade a seguinte ordem decrescente: piridostatina > [32]phen2N4 > [16]phenN2 > PhenDC3 > L-arginina > C8 > C8-NH2. Esta tendência não foi a mesma verificada experimentalmente, e logo, foi descartada. A nível destas experiências, retiram-se maioritariamente apenas as conformações dos ligandos que não são macrociclos. A nível das experiências de dicroísmo circular mencionadas, as variações de temperatura de desnaturação térmica ligando-quadruplex foram diferentes,verificando-se a seguinte ordem: C8 > piridostatina > C8-NH2 > [16]phenN2. Seguidamente, foram realizadas titulações por espectroscopia de fluorescência as quais revelaram a seguinte tendência: C8 > C8-NH2 > [16]phenN2. De notar que apenas quatro dos sete ligandos ([16]phenN2, [32]phen2N4, C8 and C8-NH2) possuíam fluorescência intrínseca, e que desses, apenas estes três puderam ser selecionados. Estes resultados mostraram que a piridostatina, e derivados de laranja de acridina C8 e C8-NH2 apresentaram maior afinidade para esta estrutura de G-quadruplex. Por último, os resultados de cromatografia de afinidade revelaram que o ligando C8-NH2 tem maior afinidade com o RNA G-quadruplex pre-miR-149 . Das seis sequências testadas, três delas (c-myc, c-kit e pre-miR-149) formam G-quadruplexes com topologia paralela, e tiveram tempos de retenção mais altos. Outras sequências (TBA e AG23) formam G-quadruplexes com topologia antiparalela, e mostram tempos de retenção mais baixos. A sequência ds26 (duplex) teve o tempo de retenção mais baixo. Conclui-se que este ligando tem maior especificidade para com G-quadruplexes com topologia paralela em detrimento do duplex. As simulações de docking corroboram esta conclusão. Deste modo, conclui-se que os melhores ligandos a nível de afinidade para com a sequência pre-miR-149 são os derivados de laranja de acridina C8 e C8-NH2 e a piridostatina, de modo que futura investigação nesta área deve considerar estes três como fortes candidatos a ligandos de RNA G-quadruplex

    Biophysical and computational studies of biomacromolecular systems

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    The binding of mono- and dinuclear platinum(II) complexes with both double-stranded and G-quadruplex DNA (QDNA) was explored using a combination of spectroscopic and computational techniques. The stabilising effects on G-quadruplex DNA were assessed and structure-activity relationships developed to guide the future development of QDNA selective complexes. Additionally, two pieces of software were developed, each able to process spectroscopic information. The first application calculates binding constants from various spectroscopic data including circular dichroism and fluorescence. The second program, SOMSpec, uses a machine learning approach to elucidate biomacromolecular structure from circular dichroism and infrared spectra

    Metallodrugs as inducers and inhibitors of chemical nuclease activity

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    Manipulation of DNA is both an intrinsic and essential component of molecular biology and biotechnology. Reagents capable of cutting DNA are applied within these fields as probes for DNA structure and function, with the ultimate aim being the design of target-specific—customised endonucleases—capable of modifying genomic DNA. Thus, DNA cleaving reagents are essential tools for both sequence analysis and genome engineering. Furthermore, the discovery of new molecular mechanisms by which small molecules modify DNA structure, reactivity, and biological repair contributes significantly to potential drug development. The chemical nuclease of [Cu(Phen)2]2+ (where Phen = 1,10-phenanthroline), is a well studied reagent which randomly cleaves nucleic acids in the presence of molecular oxygen (or hydrogen peroxide) upon reduction to Cu+. In addition, compounds based on this chemotype have found application in the biological field as antimicrobial and anticancer agents, DNA intercalators, and as nucleoside constituents for incorporation into the DNA backbone. [Cu(Phen)2]+ oxidises duplex DNA without specificity, predominately at the minor groove with C-H bonds at C1′, C4′, and C5′ being the main targets of hydrogen atom abstraction. The aim of this research was to extend structure-activity relationships of Cu2+-Phen complexes containing sterically functionalized pendant carboxylates and to investigate how synthetic extension of the ligated phenazine ligand within this complex model influences DNA recognition and oxidative degradation. These compounds have shown an enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2]+. Furthermore, the effects of nuclearity on DNA oxidation were elucidated using the [Cu(-terephthalate)(Phen)4]2+ cation with results showing potent DNA oxidation in the absence of exogenous reductant. Many compounds developed in this work constitute a series of novel anticancer leads capable of intracellular DNA oxidation leading to genomic double strand breaks. In addition to the application of developmental metallodrugs as inducers of chemical nuclease activity, the effects of cytotoxic trinuclear platinum(II) complexes as high-affinity DNA binders that inhibit—or block—endonuclease enzyme recognition and excision are reported through a wide variety of biophysical and molecular biological methods
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