Development, evaluation and application of 3D QSAR Pharmacophore model in the discovery of potential human renin inhibitors

<p>Abstract</p> <p>Background</p> <p>Renin has become an attractive target in controlling hypertension because of the high specificity towards its only substrate, angiotensinogen. The conversion of angiotensinogen to angiotensin I is the first and rate-limiting step of renin-angiotensin system and thus designing inhibitors to block this step is focused in this study.</p> <p>Methods</p> <p>Ligand-based quantitative pharmacophore modeling methodology was used in identifying the important molecular chemical features present in the set of already known active compounds and the missing features from the set of inactive compounds. A training set containing 18 compounds including active and inactive compounds with a substantial degree of diversity was used in developing the pharmacophore models. A test set containing 93 compounds, Fischer randomization, and leave-one-out methods were used in the validation of the pharmacophore model. Database screening was performed using the best pharmacophore model as a 3D structural query. Molecular docking and density functional theory calculations were used to select the hit compounds with strong molecular interactions and favorable electronic features.</p> <p>Results</p> <p>The best quantitative pharmacophore model selected was made of one hydrophobic, one hydrogen bond donor, and two hydrogen bond acceptor features with high a correlation value of 0.944. Upon validation using an external test set of 93 compounds, Fischer randomization, and leave-one-out methods, this model was used in database screening to identify chemical compounds containing the identified pharmacophoric features. Molecular docking and density functional theory studies have confirmed that the identified hits possess the essential binding characteristics and electronic properties of potent inhibitors.</p> <p>Conclusion</p> <p>A quantitative pharmacophore model of predictive ability was developed with essential molecular features of a potent renin inhibitor. Using this pharmacophore model, two potential inhibitory leads were identified to be used in designing novel and future renin inhibitors as antihypertensive drugs.</p

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

Drug Repurposing

This book focuses on various aspects and applications of drug repurposing, the understanding of which is important for treating diseases. Due to the high costs and time associated with the new drug discovery process, the inclination toward drug repurposing is increasing for common as well as rare diseases. A major focus of this book is understanding the role of drug repurposing to develop drugs for infectious diseases, including antivirals, antibacterial and anticancer drugs, as well as immunotherapeutics

Molecular and dynamic mechanisms of prokaryotic and eukaryotic flavoenzymes: insights into their implication in human metabolism and health

Las flavoenzimas y flavoproteínas son biomoléculas versátiles y diversas que están implicadas en el metabolismo energético y otros procesos celulares como la transducción de señales, la síntesis de nucleótidos, el plegamiento de proteínas o la defensa frente al estrés oxidativo. Estas proteínas tienen como cofactores los derivados de riboflavina (RF, vitamina B2), mononucleótido de flavina (FMN) y/o dinucleótido de flavina y adenina (FAD), que les confieren propiedades únicas y versátiles. Todos los organismos contienen flavoproteínas y flavoenzimas clave, y muchas de ellas se han convertido en interesantes dianas terapéuticas o herramientas biotecnológicas. En esta tesis, se ha indagado en los mecanismos moleculares de flavoenzimas y flavoproteínas con funciones metabólicas clave en procariotas y eucariotas, como las enzimas humanas RF quinasa (Publicación I), FAD sintetasa (FADS) (Publicación II) o NAD(P)H:quinona oxidorreductasa 1 (Publicación III), o las FADS procariotas (Publicaciones IV y V). La caracterización detallada de estas enzimas contribuye a la mejor comprensión de sus patologías asociadas, y sienta las bases de nuevas estrategias terapéuticas y del diseño de compuestos dirigidos a estas dianas. Por ejemplo, aquí presentamos una primera aproximación a la búsqueda de inhibidores de las FADS procariotas que puede contribuir a su explotación farmacológica como potencial agentes antimicrobianos (Publicaciones IV y V).Esta Tesis Doctoral, presentada en la modalidad de compendio de publicaciones, incluye las siguientes publicaciones:− Publicación I. Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in thebiosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886.JCR Impact Factor 2019: 4.966. Rank: Q1 (57/297) Biochemistry and Molecular Biology; D1 (9/93) Biology; Q2 (58/195) Cell Biology.− Publicación II. Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FADsynthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203.JCR Impact Factor 2019: 4.556. Rank: Q1 (74/297) Biochemistry and Molecular Biology; Q2 (48/177) Chemistry (multidisciplinary). − Publicación III. Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. JCR Impact Factor 2019: 5.014. Rank: Q1 (56/297) Biochemistry and Molecular Biology; Q1 (7/61) Medicinal Chemistry; D1 (10/139) Food Science & Technology.− Publicación IV. Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. JCR Impact Factor 2018: 4.027. Rank: Q1 (10/61) Medicinal Chemistry; Q2 (84/299) Biochemistry and Molecular Biology.− Publicación V. Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation ofligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898. JCR Impact Factor 2019: 4.700. Rank: Q1 (9/77) Biochemical Research Methods; Q1 (6/59) Mathematical & Computational Biology.Flavoenzymes and flavoproteins are versatile and diverse biomolecules that are implicated in the energetic metabolism and other cellular processes such as signalling, nucleotide synthesis, protein folding or defense against oxidative stress. These proteins have as cofactors the riboflavin (RF, vitamin B2) derivatives flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which confer them their unique and versatile properties. All organisms contain key flavoproteins and flavoenzymes, and many of them are becoming interesting as therapeutic targets or biotechnological tools. In the present thesis, we have delved into the molecular mechanisms of flavoenzymes with key metabolic functions in prokaryotes and eukaryotes, such as the human RF kinase (Publication I) and FAD synthase (FADS) (Publication II), human NAD(P)H:quinone oxidoreductase 1 (Publication III), and prokaryotic FADS (Publications IV and V). The detailed characterization of these enzymes contributes to the better understanding of their associated pathologies, and provides a framework to novel therapeutic strategies and to the design of compounds targeting them. For instance, here we show a first approximation for identification of inhibitors of the prokaryotic FADS that might contribute to exploit them as pharmacological antimicrobial drugs (Publications IV and V). This Doctoral Thesis, presented in the form of a compendium of publications, comprises the following publications: − Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in the biosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886. − Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203. − Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1 enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. − Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. − Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation of ligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898.<br /

Diseño y evaluación de inhibidores peptídicos dirigidos a la interfaz de Dimerización de la Tripanotjón Reductasa de Leishmania Infantum

Entre las muchas peculiaridades que se encuentran al estudiar los organismos tripanosomátidos de interés clínico, una de las más interesantes desde el punto de vista de la identificación de nuevas dianas terapéuticas es la forma como este parásito destoxifica su ambiente intracelular de especies oxidantes. Al carecer del ubicuo sistema glutatión/glutatión reductasa, en su lugar, el parásito cuenta con un sistema análogo modificado que emplea el tripanotión (T[SH]2), que no es más que dos moléculas de glutatión unidas por un puente espermidina. Esta unión permite que los tioles activos de la molécula se encuentren en más proximidad haciéndola mejor reductor que el glutatión. En el centro del metabolismo del T(SH)2 se encuentra la tripanotión reductasa (TryR), una enzima esencial para la supervivencia del parásito que está encargada de mantener en todo momento un pool de tripanotión reducido dentro de la célula. Esta enzima es un homodímero que cuenta con dos centros activos en los que participan residuos de ambas subunidades, haciendo de su configuración dimérica una característica fundamental para su actividad. Al sintetizar un péptido que emula una de las hélices que hacen parte de la interfaz de dimerización ubicada donde ambas subunidades hacen contacto, hemos sido capaces de desestabilizar el dímero de la enzima y minimizar su actividad. Se han desarrollado distintas variantes de este péptido con el fin de mejorar su estabilidad y potenciar su actividad. Entre las modificaciones realizadas se encuentran (1) la sustitución por alanina de cada uno de sus aminoácidos para evaluar su relevancia, (2) el acortamiento del péptido para determinar la secuencia mínima que conserva la actividad deseada y (3) la creación de péptidos estructuralmente restringidos en los que se intentan mantener las características estructurales de la hélice original. A partir de las exploraciones realizadas se obtuvieron dos péptidos especialmente relevantes: TRL35, (Ac-PKIIQSVGISNLEKNLE-NH2) y TRL38 (Ac-PKIIQSVGI-NH2), de 13 y 9 aminoácidos, respectivamente. Además, basándonos en las interacciones que se dan entre la enzima y el péptido corto deducidas de las simulaciones de dinámica molecular, se desarrolló un modelo farmacofórico que permitió la identificación de moléculas pequeñas con capacidad de inhibir la actividad enzimática. Para finalizar, hemos caracterizado bioquímicamente la interacción entre TRL35 y TryR, logrando identificar el modelo mediante el cual se da esta inhibición y las implicaciones de esta en la estabilidad de la estructura globular de la enzima

Synthesis of new pyrazolium based tunable aryl alkyl ionic liquids and their use in removal of methylene blue from aqueous solution

In this study, two new pyrazolium based tunable aryl alkyl ionic liquids, 2-ethyl-1-(4-methylphenyl)-3,5- dimethylpyrazolium tetrafluoroborate (3a) and 1-(4-methylphenyl)-2-pentyl-3,5-dimethylpyrazolium tetrafluoroborate (3b), were synthesized via three-step reaction and characterized. The removal of methylene blue (MB) from aqueous solution has been investigated using the synthesized salts as an extractant and methylene chloride as a solvent. The obtained results show that MB was extracted from aqueous solution with high extraction efficiency up to 87 % at room temperature at the natural pH of MB solution. The influence of the alkyl chain length on the properties of the salts and their extraction efficiency of MB was investigated