Structural basis of the selective activation of enzyme isoforms: Allosteric response to activators of b1- and b2-containing AMPK complexes

AMP-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 complexes, which exhibit notable differences in the sensitivity to allosteric activators. To shed light into the molecular determinants of the allosteric regulation of this energy sensor, we have examined the structural and dynamical properties of β1- and β2-containing AMPK complexes formed with small molecule activators A-769662 and SC4, and dissected the mechanical response leading to active-like enzyme conformations through the analysis of interaction networks between structural domains. The results reveal the mechanical sensitivity of the α2β1 complex, in contrast with a larger resilience of the α2β2 species, especially regarding modulation by A-769662. Furthermore, binding of activators to α2β1 consistently promotes the pre-organization of the ATP-binding site, favoring the adoption of activated states of the enzyme. These findings are discussed in light of the changes in the residue content of β-subunit isoforms, particularly regarding the β1Asn111→β2Asp111 substitution as a key factor in modulating the mechanical sensitivity of β1- and β2-containing AMPK complexes. Our studies pave the way for the design of activators tailored for improving the therapeutic treatment of tissue-specific metabolic disorders

From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus

With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains

Understanding the mechanism of direct activation of AMP-kinase: Towards a fine allosteric tuning of the kinase activity

This research deals with the regulation of the AMPK activity by direct activators, such as compound A-769662. AMPK is a key enzyme to maintain the cellular energy homeostasis, as it regulates the levels of ATP, being an important target to metabolic diseases like obesity or diabetes MT2. It is formed by 3 subunits α, β, and γ. The activation mechanism of A-769662 is of particular interest, because it activates AMPK independently of α-Thr172 phosphorylation, the β-Ser108 being phosphorylated. Under these circumstances, binding of A-769662 enhances the AMPK activity up to >90-fold (PDB 4CFF) [1-3]. We have recently studied the chain of events implicated in the binding of this ligand to the activating binding site, which is located between the α and β subunits of AMPK. MD simulations of AMPK were run for apo, holo, and holo+ ATP systems. For each system, we ran three independent MD simulations up to 1 μs. The impact of the activator binding was studied by different analysis, such as essential dynamics and evaluation of conformational entropies, among others [4]. We concluded that A-769662 acts as a molecular glue, making an effective connection between β- and α-subunits that pre-organizes the ATP-binding site, favouring the binding of ATP, and explaining the increase of the AMPK activity. These findings pave the way to explore the structural features that underline the different sensitivity of AMPK isoforms to A-769662, i.e., try to discern why A-769662 is only active in the α2β1γ1 isoform, while other compounds are active with isoform β2

Elucidating the activation mechanism of AMPK by direct pan-activator PF-739

Adenosine monophosphate-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 heterotrimeric complexes, which exhibit notable differences in the sensitivity to direct activators. To comprehend the molecular factors of the activation mechanism of AMPK, we have assessed the changes in the structural and dynamical properties of b1- and b2-containing AMPK complexes formed upon binding to the pan-activator PF-739. The analysis revealed the molecular basis of the PF-739-mediated activation of AMPK and enabled us to identify distinctive features that may justify the slightly higher affinity towards the b1-isoform, such as the b1-Asn111 to b2-Asp111 substitution, which seems to be critical for modulating the dynamical sensitivity of b1- and b2 isoforms. The results are valuable in the design of selective activators to improve the tissue specificity of therapeutic treatment

Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK-eNOS-NO pathway

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK-eNOS-NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity

Exploring molecular mechanisms of key targets in metabolic and infectious diseases

[eng] This doctoral thesis is focused on disclosing molecular factors that regulate the functional mechanism of key targets in i) metabolic disorders, such as diabetes and obesity, and ii) tuberculosis (TB). These diseases have a high impact on the public health, and TB is one of the diseases that causes highest mortality rates based on the World Health Organization reports. Accordingly, gaining insight on functionally relevant protein structures with key roles in these diseases and understanding their regulatory mechanisms can pave a way to identify novel therapeutic targets. Regarding the metabolic disorders, I have specifically studied the direct activation mechanism of adenosine-monophosphate activated protein kinase (AMPK) by different activators, such as A-769662, SC4 and PF-739, since AMPK plays an important role in cellular energy homeostasis and it is directly related to diabetes type 2 and obesity. These studies were carried out to explore the molecular basis of the selective isoform activation of AMPK, especially focusing on the role played by the two different isoforms of β subunit. The first part of this research was focused on the direct activation by A-769662 compound in α2β1 isoform, and the second part addressed the important issue of isoform selectivity, it being dedicated to examine the structural and dynamical properties of β1- and β2-containing AMPK complexes formed with A-769662, SC4 and PF-739. The results revealed the mechanical sensitivity of the α2β1 complex, in contrast with a larger resilience of the α2β2 species. Moreover, binding of activators to α2β1 promotes the pre-organization of the ATP-binding site, favoring the adoption of activated states of the enzyme. In another part of the research related to AMPK, in collaboration with Prof. Ana Castro from Institute of Medicinal Chemistry and Prof. María S. Fernández-Alonso from Complutense University of Madrid, I studied a novel indolic compound synthesized and experimentally analysed by their group as modulator of endothelial AMPK that acts as a mixed-type inhibitor. It means that this modulator, IND6, may bind the ATP-binding site leading to competitive inhibition of the enzyme but also suggest that IND6 may regulate the AMPK activity through binding to an additional pocket. Regarding TB, I have studied the truncated hemoglobin N (trHbN) of Mycobacterium tuberculosis (Mtb). These studies aimed to disclose innovative therapeutic approaches by identifying the molecular basis of the nitrosative stress resistance of Mtb. Our hypothesis was that a decrease in the nitric oxide (NO) resistance should affect the survival of the bacillus, increasing the efficacy of current therapeutic treatments. Nitric oxide detoxification (NOD) is carried out by trHbN, which converts NO into the harmless nitrate anion, becoming essential for the defense mechanism of Mtb. However, two points prevent the use of this system as therapeutic target. On the one side, trHbN is not druggable and there is the risk that a drug targeting trHbN may also affect other hemoglobins. On the other side, the reductase protein that helps trHbN to restore the ferrous state required to initiate the NOD is still unknown. Our studies have led to the identification of three reductase candidates for NOD activity. We have built the 3D models for the three candidates and performed the protein-protein docking in order to obtain a reductase-trHbN complex. The important role played by one of these candidates, ndh, has been highlighted in several experimental studies. This led us to focus our attention in the trHbN-ndh complex and establish the steps that could help to advance in the definition of new therapeutic strategies for TB.[spa] Esta tesis doctoral se centra en el análisis de los factores moleculares que regulan el mecanismo funcional de dianas clave en: i) trastornos metabólicos, como diabetes y obesidad, y ii) tuberculosis (TB). Estas enfermedades tienen un elevado impacto en la salud pública, y en particular la TB se halla entre las enfermedades con una mayor tasa de mortalidad según los informes de la Organización Mundial de la Salud. Por lo tanto, conocer las estructuras proteicas que tienen una función clave en estas enfermedades y comprender sus mecanismos reguladores puede allanar el camino para identificar nuevos objetivos terapéuticos. Respecto a los trastornos metabólicos, he estudiado el mecanismo de activación directa de la proteína quinasa activada por adenosina-monofosfato (AMPK) por diferentes activadores, como A-769662, SC4 y PF-739, ya que AMPK juega un papel importante en la homeostasis energética celular y está directamente relacionada con diabetes tipo 2 y obesidad. Estos estudios se llevaron a cabo con el objetivo de explorar los factores moleculares que regulan la activación selectiva de isoformas de AMPK, centrándome especialmente en el papel que desempeñan las dos isoformas de la subunidad β. La primera parte de esta investigación se centró en la activación directa por el compuesto A-769662 en la isoforma α2β1, y la segunda parte se dedicó a examinar la base molecular de la selectividad entre isoformas, considerando específicamente las propiedades estructurales y dinámicas de los complejos de AMPK que contienen isoformas β1 y β2 formados con A- 769662, SC4 and PF-739. Los resultados revelaron la sensibilidad mecánica del complejo α2β1, en contraste con una mayor resiliencia de la especie α2β2. Además, la unión de los activadores en la isoforma α2β1 promueve la organización previa del sitio de unión de ATP, favoreciendo la adopción de estados activados de la enzima. Asimismo, en colaboración con la Prof. Ana Castro del Instituto de Química Médica y la Prof. María S. Fernández-Alonso de la Universidad Complutense de Madrid, estudiamos un novedoso compuesto indólico sintetizado y analizado experimentalmente por sus grupos, que actúa como inhibidor de tipo mixto y es capaz de modular el AMPK endotelial. Nuestros cálculos sugieren que este modulador, IND6, puede unirse al sitio de unión de ATP que conduciría a una inhibición competitiva del enzima, pero también apuntan que IND6 puede regular la actividad de AMPK mediante la unión a un bolsillo adicional. En cuanto a TB, he estudiado la hemoglobina truncada N (trHbN) de Mycobacterium tuberculosis (Mtb). El objetivo ha sido obtener un nuevo enfoque terapéutico mediante la identificación de la base molecular de la resistencia al estrés nitrosativo de Mtb. Nuestra hipótesis es que una disminución de la resistencia al óxido nítrico (NO) debería afectar la supervivencia del bacilo, aumentando la eficacia de los tratamientos terapéuticos actuales. La desintoxicación del óxido nítrico (NOD, de sus siglas en inglés) se realiza mediante trHbN, que transforma NO en el inocuo anión nitrato, convirtiéndose en un mecanismo de defensa esencial de Mtb. Sin embargo, dos puntos impiden el uso de este sistema como diana terapéutica. Por un lado, la trHbN no puede ver modificada su acción mediante ningún fármaco ya que carece de bolsillos que permitan enlazar el fármaco, y segundo, porque sería muy difícil intentar modificar únicamente esta hemoglobina en el paciente sin provocar efectos secundarios. Por otro lado, aún se desconoce la proteína reductasa que ayuda a restaurar el estado ferroso de trHbN requerido para iniciar el NOD. Mi objetivo ha sido identificar esta reductasa. Por esta razón, los estudios se han centrado en la identificación de tres posibles candidatos capaces de permitir la actividad NOD de Mtb. Una vez identificados, se han construido modelos 3D para cada uno de ellos y realizado el acoplamiento proteína-proteína para obtener un complejo reductasa-trHbN de partida. Durante esta investigación, varios trabajos experimentales apuntaron la importancia de uno de los candidatos identificados, la reductasa ndh. Este hecho nos permitió centrar nuestra atención en el complejo trHbN-ndh y establecer los pasos que podrían ayudar a avanzar en la definición de una nueva estrategia terapéutica para TB

Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK-eNOS-NO pathway

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identifcation of a novel AMPK modulator, the indolic derivative IND6, which shows afnity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical fnding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profle, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.Fundación Eugenio Rodríguez PascualSpanish Ministerio de Economía y CompetitividadSpanish Ministerio de Ciencia Innovación y UniversidadesGeneralitat de CatalunyaBarcelona Supercomputing CenterConsorci de Serveis Universitaris de CatalunyaAgencia de Gestión de Ayudas Universitarias y de InvestigaciónDepto. de Farmacología, Farmacognosia y BotánicaInstituto Pluridisciplinar (IP)Fac. de FarmaciaTRUEpu