Opitz GBBB syndrome with total anomalous pulmonary venous connection: A new MID1 gene variant.

BACKGROUND: Opitz GBBB syndrome (GBBB) is an X-linked disease characterized by midline defects, including congenital heart defects. We present our diagnostic approach to the identification of GBBB in a consanguineous family in which two males siblings were concordant for a total anomalous connection of pulmonary veins and minor facial dysmorphias. METHODS: Targeted exome sequencing analysis of a 380-gene panel associated with cardiovascular disease was performed on the propositus. Interpretative analysis of the exome results was conducted, and 3D models of the protein changes were generated. RESULTS: We identified a NM_000381.4:c.608G>A;p.(Arg203Gln) change in MID1, affecting the conformation of the B-box 2 domain of the protein, with a zinc finger structure and associated protein interactions. This clinical phenotype is consistent with GBBB; however, the type of congenital heart disease observed in this case has not been previously reported. CONCLUSION: A new likely pathogenic variant on MID1 c.608G>A was found to be associated with Opitz GBBB syndrome.

Cell-Permeable Bak BH3 Peptide Induces Chemosensitization of Hematologic Malignant Cells.

Hematologic malignancies such as leukemias and lymphomas are among the leading causes of pediatric cancer death worldwide, and although survival rates have improved with conventional treatments, the development of drug-resistant cancer cells may lead to patient relapse and limited possibilities of a cure. Drug-resistant cancer cells in these hematologic neoplasms are induced by overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein families, such as Bcl-XL, Bcl-2, and Mcl-1. We have previously shown that peptides from the BH3 domain of the proapoptotic Bax protein that also belongs to the Bcl-2 family may antagonize the antiapoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. Furthermore, cell-permeable Bax BH3 peptides also elicit antitumor activity and extend survival in a murine xenograft model of human B non-Hodgkin's lymphoma. However, the activity of the BH3 peptides of the proapoptotic Bak protein of the Bcl-2 family against these hematologic malignant cells requires further characterization. In this study, we report the ability of the cell-permeable Bak BH3 peptide to restore apoptosis and induce chemosensitization of acute lymphoblastic leukemia and non-Hodgkin's lymphoma cell lines, and this event is enhanced with the coadministration of cell-permeable Bax BH3 peptide and represents an attractive approach to improve the patient outcomes with relapsed or refractory hematological malignant cells.

Molecular dynamics of the histamine H3 membrane receptor reveals different mechanisms of GPCR signal transduction.

In this work, we studied the mechanisms of classical activation and inactivation of signal transduction by the histamine H3 receptor, a 7-helix transmembrane bundle G-Protein Coupled Receptor through long-time-scale atomistic molecular dynamics simulations of the receptor embedded in a hydrated double layer of dipalmitoyl phosphatidyl choline, a zwitterionic polysaturated ordered lipid. Three systems were prepared: the apo receptor, representing the constitutively active receptor; and two holo-receptors-the receptor coupled to the antagonist/inverse agonist ciproxifan, representing the inactive state of the receptor, and the receptor coupled to the endogenous agonist histamine and representing the active state of the receptor. An extensive analysis of the simulation showed that the three states of H3R present significant structural and dynamical differences as well as a complex behavior given that the measured properties interact in multiple and interdependent ways. In addition, the simulations described an unexpected escape of histamine from the orthosteric binding site, in agreement with the experimental modest affinities and rapid off-rates of agonists.

Live Attenuated Salmonella enterica Expressing and Releasing Cell-Permeable Bax BH3 Peptide Through the MisL Autotransporter System Elicits Antitumor Activity in a Murine Xenograft Model of Human B Non-hodgkin's Lymphoma.

The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl-XL, Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica, which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL.

Cloning and biochemical characterization of three glucose­6­phosphate dehydrogenase mutants presents in the Mexican population.

The deficiency of glucose­6­phosphate dehydrogenase (G6PD) is one of the most common inborn errors of metabolism worldwide. This congenital disorder generally results from mutations that are spread throughout the entire gene of G6PD. Three single-point mutations for G6PD have been reported in the Mexican population and named Veracruz (Arg365His), G6PD Seattle (Asp282His), and G6PD Mexico DF (Thr65Ala), whose biochemical characterization have not yet been studied. For this reason, in this work we analyzed the putative role of the three mutations to uncover the functional consequences on G6PD activity. To this end, was developed a method to clone, overexpress, and purify recombinant human G6PD. The results obtained from all variants showed a loss of catalysis by 80 to 97% and had a decrease in affinity for both physiological substrates with respect to the wild type (WT) G6PD. Our results also showed that the three mutations affected three-dimensional structure and protein stability, suggesting an unstable structure with low conformational stability that affected its G6PD functionality. Finally, based on the biochemical characterization of the unclassified G6PD Mexico DF, we suggest that this variant could be grouped as a Class I variant, because biochemical data are similar with other Class I G6PDs.

Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardialamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardialamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardialamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

Ligand recognition properties of the vasopressin V2 receptor studied under QSAR and molecular modeling strategies.

The design of new drugs that target vasopressin 2 receptor (V2R) is of vital importance to develop new therapeutic alternatives to treat diseases such as heart failure, polycystic kidney disease. To get structural insights related to V2R-ligand recognition, we have used a combined approach of docking, molecular dynamics simulations (MD) and quantitative structure-activity relationship (QSAR) to elucidate the detailed interaction of the V2R with 119 of its antagonists. The three-dimensional model of V2R was built by threading methods refining its structure through MD simulations upon which the 119 ligands were subjected to docking studies. The theoretical results show that binding recognition of these ligands on V2R is diverse, but the main pharmacophore (electronic and π-π interactions) is maintained; thus, this information was validated under QSAR results. QSAR studies were performed using MLR analysis followed by ANN analysis to increase the model quality. The final equation was developed by choosing the optimal combination of descriptors after removing the outliers. The applicability domains of the constructed QSAR models were defined using the leverage and standardization approaches. The results suggest that the proposed QSAR models can reliably predict the reproductive toxicity potential of diverse chemicals, and they can be useful tools for screening new chemicals for safety assessment.

Is there something else besides the proapoptotic AVPI-segment in the Smac/DIABLO protein? / ¿Existe algo adicional al segmento pro-apoptótico AVPI de la proteína Smac/DIABLO?

Abstract: In mammals, apoptosis is the main mechanism to eliminate unwanted cells, securing tissue homeostasis and consequently maintaining the health in the organism. Classically, apoptosis culminates with the activation of caspases, which are enzymes that display cysteine protease activity to degrade specific substrates implied in essential cellular processes. This process is highly regulated. A key regulation mechanism is mediated by the Inhibitor of Apoptosis Proteins (IAPs) family members, which inhibit the activated forms of caspases through physical interaction with them. Smac/DIABLO, a mitochondrial protein that is translocated to the cytoplasm in apoptotic conditions, derepresses the IAP-mediated caspase inhibition through physical interaction with IAPs. The first four amino acids (AVPI) of Smac/DIABLO mediate the interaction with IAPs and subsequent apoptosis induction. This interaction has lead to the creation of small molecules mimicking the AVPI segment for potential anticancer therapy. Nevertheless, several studies have pointed out the existence of AVPI-independent functions of Smac/DIABLO. The aim of this review was to provide a landscape of these underestimated AVPI-independent biological functions that have been observed using different approaches, such as the study of endogenous splice variant isoforms and truncated and mutated artificial proteins.

Resumen: La apoptosis es uno de los principales mecanismos en los mamíferos para eliminar células no deseadas, asegurando la homeostasis de los tejidos y, consecuentemente, la salud de los mismos. De forma clásica, la apoptosis finaliza con la activación de las caspasas, enzimas que despliegan actividad de proteasas de cisteína, involucradas en la degradación de sustratos específicos implicados en procesos celulares esenciales. El proceso apoptótico se encuentra altamente regulado. Un mecanismo de regulación es el mediado por los miembros de la familia de las Proteínas Inhibidoras de la Apoptosis (PIA), las cuales inhiben a las formas activas de las caspasas a través de la interacción física con estas. Smac/DIABLO, proteína mitocondrial que es translocada al citoplasma en condiciones apoptóticas, antagoniza la inhibición de las caspasas mediante su interacción física con las PIA. Los cuatro primeros aminoácidos (AVPI) de Smac/DIABLO intervienen en su asociación con las PIA y la subsecuente inducción apoptótica. Esto ha guiado a la generación de pequeñas moléculas miméticas del segmento AVPI para el uso potencial como una terapia anti-cancerígena. Sin embargo, varios estudios han indicado la presencia de funciones en Smac/DIABLO independientes del AVPI. El objetivo de esta revisión fue proporcionar un panorama de estas funciones biológicas desestimadas —independientes al AVPI— las cuales se han observado utilizando diferentes aproximaciones, como el estudio de las isoformas generadas por el procesamiento alternativo del gen y la síntesis de proteínas artificialmente mutadas.

Application of computational methods for anticancer drug discovery, design, and optimization / Aplicación de métodos computacionales para el descubrimiento, diseño y optimización de fármacos contra el cáncer

Abstract: Developing a novel drug is a complex, risky, expensive and time-consuming venture. It is estimated that the conventional drug discovery process ending with a new medicine ready for the market can take up to 15 years and more than a billion USD. Fortunately, this scenario has recently changed with the arrival of new approaches. Many novel technologies and methodologies have been developed to increase the efficiency of the drug discovery process, and computational methodologies have become a crucial component of many drug discovery programs. From hit identification to lead optimization, techniques such as ligand- or structure-based virtual screening are widely used in many discovery efforts. It is the case for designing potential anticancer drugs and drug candidates, where these computational approaches have had a major impact over the years and have provided fruitful insights into the field of cancer. In this paper, we review the concept of rational design presenting some of the most representative examples of molecules identified by means of it. Key principles are illustrated through case studies including specifically successful achievements in the field of anticancer drug design to demonstrate that research advances, with the aid of in silico drug design, have the potential to create novel anticancer drugs.

Resumen: El desarrollo de un nuevo fármaco es un proceso complejo y arriesgado que requiere una enorme cantidad de tiempo y dinero. Se estima que el proceso estándar para producir un nuevo fármaco, desde su descubrimiento hasta que acaba en el mercado, puede tardar hasta 15 años y tener un costo de mil millones de dólares (USD). Por fortuna, este escenario ha cambiado recientemente con la llegada de nuevas tecnologías y metodologías. Entre ellas, los métodos computacionales se han convertido en un componente determinante en muchos programas de descubrimiento de fármacos. En un esfuerzo por incrementar las posibilidades de encontrar nuevas moléculas con potencial farmacológico, se utilizan técnicas como el cribado virtual de quimiotecas construidas con base en ligandos o estructuras para la identificación de hits y hasta para la optimización de compuestos líder. En lo que respecta al diseño y descubrimiento de nuevos candidatos a fármacos contra el cáncer, estos enfoques tienen, a la fecha, un impacto importante y aportan nuevas posibilidades terapéuticas. En este artículo se revisa el concepto del diseño racional de moléculas con potencial farmacológico, ilustrando los principios clave con algunos de los ejemplos más representativos y exitosos de moléculas identificadas mediante estas aproximaciones. Se incluyen casos desarrollados en el campo del diseño de fármacos contra el cáncer con la finalidad de mostrar cómo, con la ayuda del diseño asistido por computadora, se pueden generar nuevos fármacos que den esperanza a millones de pacientes.

New perspectives on the computational characterization of the kinetics of binding-unbinding in drug design: implications for novel therapies / Nuevas perspectivas sobre la caracterización computacional de la cinética de unión-desunión en el diseño de fármacos: implicaciones para terapias de vanguardia

Abstract: The efficiency and the propensity of a drug to be bound to its target protein have been inseparable concepts for decades now. The correlation between the pharmacological activity and the binding affinity has been the first rule to design and optimize a new drug rationally. However, this argument does not prove to be infallible when the results of in vivo assays have to be confronted. Only recently, we understand that other magnitudes as the kinetic rates of binding and unbinding, or the mean residence time of the complex drug-protein, are equally relevant to draw a more accurate model of the mechanism of action of a drug. It is in this scenario where new computational techniques to simulate the all-atom dynamics of the biomolecular system find its valuable place on the challenge of designing new molecules for more effective and less toxic therapies.

Resumen: La eficiencia de un fármaco se ha relacionado habitualmente con su constante de afinidad, magnitud que puede ser medida experimentalmente in vitro y que cuantifica la propensión mostrada por la molécula ligando para interaccionar con su proteína diana. Este modo de entender el mecanismo de acción ha guiado durante años el desarrollo de nuevas moléculas con potencial farmacológico. Sin embargo, dicho modelo o criterio no es infalible cuando se confronta con los resultados de ensayos in vivo. Otras magnitudes, como las constantes cinéticas de asociación o disociación o el tiempo de residencia del ligando acoplado a su proteína diana, demuestran ser igualmente necesarias para comprender y predecir la capacidad farmacológica del compuesto químico. En este nuevo escenario, con ayuda de las técnicas computacionales de simulación molecular, la correcta caracterización del proceso dinámico de unión y desunión del ligando y receptor resulta imprescindible para poder diseñar racionalmente nuevas moléculas que permitan terapias más eficaces y menos tóxicas.

Is there something else besides the proapoptotic AVPI-segment in the Smac/DIABLO protein?

In mammals, apoptosis is the main mechanism to eliminate unwanted cells, securing tissue homeostasis and consequently maintaining the health in the organism. Classically, apoptosis culminates with the activation of caspases, which are enzymes that display cysteine protease activity to degrade specific substrates implied in essential cellular processes. This process is highly regulated. A key regulation mechanism is mediated by the Inhibitor of Apoptosis Proteins (IAPs) family members, which inhibit the activated forms of caspases through physical interaction with them. Smac/DIABLO, a mitochondrial protein that is translocated to the cytoplasm in apoptotic conditions, derepresses the IAP-mediated caspase inhibition through physical interaction with IAPs. The first four amino acids (AVPI) of Smac/DIABLO mediate the interaction with IAPs and subsequent apoptosis induction. This interaction has lead to the creation of small molecules mimicking the AVPI segment for potential anticancer therapy. Nevertheless, several studies have pointed out the existence of AVPI-independent functions of Smac/DIABLO. The aim of this review was to provide a landscape of these underestimated AVPI-independent biological functions that have been observed using different approaches, such as the study of endogenous splice variant isoforms and truncated and mutated artificial proteins.

New perspectives on the computational characterization of the kinetics of binding-unbinding in drug design: implications for novel therapies.

The efficiency and the propensity of a drug to be bound to its target protein have been inseparable concepts for decades now. The correlation between the pharmacological activity and the binding affinity has been the first rule to design and optimize a new drug rationally. However, this argument does not prove to be infallible when the results of in vivo assays have to be confronted. Only recently, we understand that other magnitudes as the kinetic rates of binding and unbinding, or the mean residence time of the complex drug-protein, are equally relevant to draw a more accurate model of the mechanism of action of a drug. It is in this scenario where new computational techniques to simulate the all-atom dynamics of the biomolecular system find its valuable place on the challenge of designing new molecules for more effective and less toxic therapies.

Application of computational methods for anticancer drug discovery, design, and optimization.

Developing a novel drug is a complex, risky, expensive and time-consuming venture. It is estimated that the conventional drug discovery process ending with a new medicine ready for the market can take up to 15 years and more than a billion USD. Fortunately, this scenario has recently changed with the arrival of new approaches. Many novel technologies and methodologies have been developed to increase the efficiency of the drug discovery process, and computational methodologies have become a crucial component of many drug discovery programs. From hit identification to lead optimization, techniques such as ligand- or structure-based virtual screening are widely used in many discovery efforts. It is the case for designing potential anticancer drugs and drug candidates, where these computational approaches have had a major impact over the years and have provided fruitful insights into the field of cancer. In this paper, we review the concept of rational design presenting some of the most representative examples of molecules identified by means of it. Key principles are illustrated through case studies including specifically successful achievements in the field of anticancer drug design to demonstrate that research advances, with the aid of in silico drug design, have the potential to create novel anticancer drugs.

Homology modeling of the structure of acyl coA:isopenicillin N-acyltransferase (IAT) from Penicillium chrysogenum. IAT interaction studies with isopenicillin-N, combining molecular dynamics simulations and docking.

In the last step of penicillin biosynthesis, acyl-CoA:isopenicillin N acyltransferase (IAT) (E.C. 2.3.1.164) catalyzes the conversion of isopenicillin N (IPN) to penicillin G. IAT substitutes the α-aminoadipic acid side chain of IPN by a phenylacetic acid phenolate group (from phenylacetyl-CoA). Having a three-dimensional (3D) structure of IAT helps to determine the steps involved in side chain exchange by identifying the atomic details of substrate recognition. We predicted the IAT 3-D structure (α- and ß-subunits), as well as the manner of IPN and phenylacetyl-CoA bind to the mature enzyme (ß-subunit). The 3D IAT prediction was achieved by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations. Our model can reasonably interpret the results of a number of experiments, where key residues for IAT processing as well as strictly conserved residues most probably involved with enzymatic activity were mutated. Based on the results of docking studies, energies associated with the complexes, and binding constants calculated, we identified a site located in the region generated by ß1, ß2 and ß5 strands, which forms part of the central structure of ß-subunit, as the potential binding site of IPN. The site comprises the amino acid residues Cys103, Asp121, Phe122, Phe123, Ala168, Leu169, His170, Gln172, Phe212, Arg241, Leu262, Asp264, Arg302, Ser309, and Arg310. Through hydrogen bonds, the IPN binding site establishes interactions with Cys103, Leu169, Gln172, Asp264 and Arg310. Our model is also validated by a recently revealed crystal structure of the mature enzyme.

Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae: different mechanisms, similar activation enthalpies.

Yeast enolase is stabilized by its natural cofactor Mg(2+). This stabilization is ascribed to the reduced subunit dissociation of the holoprotein. Nevertheless, how Mg(2+) alters the unfolding mechanism has yet to be fully characterized. Here, we investigate the role of Mg(2+) in the denaturation mechanism and unfolding kinetics of yeast enolase. Apo-enolase unfolds through a three-state process (N(2)↔2I→2D). The intermediate species is described as a monomeric molten globule-like conformation that becomes noticeable in the presence of phosphate and is able to recover its native secondary structure when cooled down. Kinetic studies confirmed the presence of the intermediate species, even though it was not noticeable in the thermal scans. The cofactor increases the cooperativity of the unfolding transitions, while the intermediate species becomes less noticeable or nonexistent. Thus, holo-enolase follows a simple two-state mechanism (N(2)→2D). Our results indicate smaller unfolding rate-constants in the presence of Mg(2+), thus favoring the native state. The temperature dependence of the unfolding rates allowed us to calculate the activation enthalpies of denaturation. Interestingly, despite the different unfolding mechanisms of the apo and holo forms of enolase, they both have similar activation barriers of denaturation (185-190 kJ mol(-1)).

Thermal-unfolding reaction of triosephosphate isomerase from Trypanosoma cruzi.

Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate.