Structure-function studies of class I aldolases - exploring novel activities : mechanism, moonlighting, and inhibition

La fructose-1,6-bisphosphate aldolase de classe I est une enzyme glycolytique (EC 4.1.2.13) qui catalyse le clivage réversible du fructose-1,6-bisphosphate (FBP) en dihydroxyacétone phosphate (DHAP) et glycéraldéhyde-3-phosphate (G3P). Des années de recherche sur FBP aldolase ont permis d’identifier les résidus impliqués dans son mécanisme réactionnel, ont tracé en grande partie les coordonnées de la réaction, ont révélé de nouvelles fonctions dites « moonlighting », et ont validé l’aldolase comme une cible attrayante pour des applications anti-glycolytiques tel que le cancer. Il existe néanmoins des questions en suspens relatives à ces activités que nous avons étudiées. Tout d'abord, la trajectoire détaillée de l'aldéhyde relatif à sa liaison au site actif allant jusqu’à la formation du lien carbone-carbone par condensation aldolique est indéfini. Pour élucider les détails moléculaires liés à ces événements, nous avons déterminé des structures cristallographiques à hautes résolution de l’aldolase de classe I chez Toxoplasma gondii, qui porte une identité de séquence élevée avec l’aldolase humaine (57%), en complexe avec l’intermédiaire ternaire de pré-condensation. Le complexe ternaire révèle un mode de liaison non-productive inhabituel pour G3P dans une configuration cis qui permet l’alignement de l'aldéhyde à proximité du nucléophile naissant. La configuration compétente pour la condensation aldolique provient d'une transposition cis-trans de l'aldéhyde qui produit une liaison hydrogène courte permettant la polarisation de l'aldéhyde et le transfert de proton au niveau de Glu-189. Nos résultats informent les chimistes synthétiques qui cherchent à développer l’aldolase comme biocatalyseur pour des réactions stéréo-contrôlées. Le rôle présumé de l’aldolase dans la production du méthyglyoxal (MGO), un métabolite dicarbonyle hautement réactif qui génère des « advanced glycation end products » (AGES) a également été étudié structurellement et enzymatiquement. Une enquête structurelle cristallographique de MGO générée par décomposition enzymatique chez l’aldolase de classe I a révélé que, contrairement aux indications préliminaires, l'apparition hypothétique de MGO et de phosphate inorganique (Pi) résultant de la décomposition enzymatique de DHAP dans le site actif de l’aldolase est mieux interprétée par une population mixte de DHAP et de molécules d'eau. Une étude enzymatique a révélé que la décomposition spontannée des trioses-phosphate est une source majeure de la production de MGO, alors qu’une production catalysée par l’aldolase est peu concluante. L’identification des sources de production de MGO continue d'être une priorité afin de développer des stratégies pour atténuer les manifestations cliniques de pathologies associées au MGO. La FBP aldolase est également reconnu pour ses activités « moonlighting » - du fait qu’elle effectue plus d'une activité sans rapport avec sa fonction glycolytique. Divers partenaires de l’aldolase sont rapportés dans la littérature, y compris les adhésines de surface cellulaire chez les parasites apicomplexes, dans lequel l’aldolase exécute une fonction d'échafaudage entre le complexe actomyosine et les adhésines - une interaction qui est décisive pour la motilité et l'invasion des cellules hôte. Le mode de liaison de cette interaction a été étudié et nos résultats sont compatibles avec une liaison au site actif. Les détails précis de cette interaction ont des implications thérapeutiques, étant donné que le ciblage de celui-ci réduit l'invasion des cellules hôte par les parasites. Enfin, l’aldolase de classe I est de plus en plus reconnu pour son potentiel comme cible anti-glycolytique dans les cellules qui sont fortement tributaires du flux glycolytique, comme les cellules cancéreuses et les parasites protozoaires. Le développement de nouveaux inhibiteurs de haute affinité est donc non seulement avantageux pour des études mécanistiques, mais représente un potentiel pharmacologique sans fin. Nous avons développé une nouvelle classe d’inhibiteurs de haute affinité de type inhibition lente et avons déterminé la base moléculaire de leur inhibition grâce à des structures cristallographiques à haute résolution et par un profilage enzymatique. Cette étude, qui combine plusieurs disciplines, y compris la cristallographie, enzymologie et chimie organique, souligne l'intérêt et l'importance d'une approche multidisciplinaire.Class I Fructose-1,6-bisphosphate aldolases are glycolytic enzymes (EC 4.1.2.13) that catalyze the reversible cleavage of fructose-1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). Years of research on FBP aldolases has identified residues implicated in the reaction mechanism, mapped the greater part of the reaction coordinates, and revealed novel moonlighting functions. Further, FBP aldolase is recognized as an attractive target for anti-glycolytic applications such as cancer. There are nevertheless outstanding questions related to these activities that were investigated in this thesis. First, the detailed trajectory of the reaction mechanism from aldehyde binding in the active site to carbon-carbon bond formation by aldol condensation is undefined. To elucidate the molecular details related to these events, we solved high-resolution crystallographic structures of native class I aldolase from Toxoplasma gondii, which has a high sequence identity with human aldolase (57 %), in complex with the pre-condensation ternary intermediate. The ternary complex reveals a condensation-incompetent binding mode for G3P in a cis-configuration that aligns the aldehyde alongside the nascent nucleophile. The productive aldol-competent configuration arises from a cis-trans rearrangement of the aldehyde that produces a short hydrogen bond required for polarization of the aldehyde and coincident proton transfer at Glu-189. Our results inform synthetic chemists seeking to develop aldolases for stereo-controlled reactions in biosynthetic applications. The suspected role of aldolase in methylglyoxal (MGO) production, a highly reactive dicarbonyl metabolite that produces advanced glycation end-products (AGES) was also probed structurally and enzymatically. A crystallographic structural investigation of MGO generated by enzymatic decomposition in class I aldolase revealed that, contrary to preliminary indications, the appearance of MGO and inorganic phosphate (Pi) resulting from enzymatic decomposition of DHAP in the active site of aldolase is more appropriately modeled by a mixed population of DHAP and water molecules. Enzymatic investigation revealed triose-phosphate decomposition to be a major source of MGO production, whereas production by aldolase did not exceed assay background levels. Identifying the main sources of MGO production continues to be a priority for mitigating the clinical manifestations of MGO-derived pathologies. FBP aldolase is also recognized for its moonlighting properties – performing more than one activity unrelated to the glycolytic function. Diverse aldolase partners are reported, including cell surface adhesins in apicomplexan parasites, in which aldolase performs a bridging function between the actomyosin complex and the cytoplasmic domain of the adhesins – an interaction that is crucial for motility and host-cell invasion. The binding mode of this interaction was investigated and our results are consistent with active site binding. The precise details of aldolase-adhesin binding has therapeutic implications, since targeting of the latter reduces host-cell invasion by parasites. Finally, class I aldolase is gaining prominence as an anti-glycolytic target in cells that are highly dependent on glycolytic flux, such as cancer cells and protozoan parasites. Developing new high-affinity inhibitors for these enzymes is therefore not only advantageous for mechanistic studies, but has endless pharmacological potential. We developed a novel class of high-affinity aldolase inhibitors, bisphosphonates, and determined the molecular basis of their inhibition with high-resolution crystallographic structures and enzymatic profiling. This study, which combined several disciplines, including crystallography, enzymology, and organic chemistry, underscores the interest and significance of a multidisciplinary approach

Investigating and manipulating the reaction mechanism of reductive carboxylases

Efficient capture and conversion of atmospheric carbon dioxide (CO2) is a prerequisite to develop a carbon-neutral, circular future economy. Carbon fixation is the process by which inorganic carbon is fixed into biomass. In Nature, enzymes called caboxlyases are able to capture atmospheric carbon dioxide under mild conditions and catalyze its incorporation into organic molecules. It is estimated that 400 Gt of CO2 are fixed annually solely by the enzyme ribulose-1,5-bisphophate-carboxylase/oxygenase (RuBisCO), the key enzyme of photosynthesis. In comparison, CO2 utilization by chemical industries accounts for only 0.1 Gt of carbon annually and utilizes pressurized CO2, which emphasizes our need to understand the molecular mechanism that allow carboxylases to selectively interact with a CO2 at atmospheric concentrations (0.04% vol) during catalysis. Enoyl-CoA carboxylases/reductases (ECRs) represent the fastest carboxylases known to date and is, in contrast to RuBisCO, completely specific for CO2. These enzymes catalyze the reductive carboxylation of enoyl-CoAs by oxidizing one equivalent of NADPH. ECRs represent a good case study for the understanding of the CO2 chemistry that carboxylases use. In this work, we try to gain a better understanding of the underlying catalytic principles that enable ECRs to achieve high catalytic rates. Initially we focus on understanding how the precise interaction between protein and CO2 takes place at the active site of ECRs. We were able to identify and assign a function to four conserved amino acid residues found at the active site of ECRs. Three residues are responsible for the precise positioning of CO2 for nucleophilic attack by the enolate intermediate. Additionally, one residue is able to shield the active site from water thereby preventing the irreversible protonation of the enolate. These two mechanistic principles are at the base of the efficient carboxylation in ECRs. The following chapter briefly describes how the enzyme is able to accept other electrophiles than CO2. We show that ECRs can utilize formaldehyde as an alternative electrophile to CO2 thereby yielding beta-hydroxy thioesters. The exquisite stereospecificity together with the vast range of small electrophiles make ECR a potential biocatalyst for the production of various α-substituted thioesters. The last two chapters of this work focus on the structural aspects of ECR catalysis. We were able to obtain four new crystal structures of an ECR from Kitasatospora setae and to propose a model for the catalytic cycle of this enzyme. We show that the communication between and within the dimers that compose the functional homotetramer is crucial for the fast catalytic rates observed in this ECR. A separate study aims at developing an in vivo directed evolution screen to improve the catalytic properties of an ECR from Burkholderia ambifaria. Our approach yields an evolved variant, with mutations distant from the active site. The observed improved catalytic supports the importance of the residues for the catalytic rate. Both studies revealed the importance of the residues at the interface of the ECR monomers by their impact on catalytic rates of this enzyme

A powerful combination of computational methods on the road toward potent non-steroidal inhibitors of steroidogenic enzymes involved in hormone-dependent diseases

Different computational methods have been applied to the development of non-steroidal inhibitors of steroidogenic enzymes for the treatment of hormone-sensitive diseases, like breast and prostate cancer and hypertension. A high-quality homology model of CYP17 was created and used for docking studies. Three binding modes were identified and SAR for different CYP17-inhibitor classes, substantiated by ab initio calculations, could be derived and used in further drug design leading to improved potency. Docking studies and ligand cluster analysis for a series of CYP19 inhibitors resulted in a binding mode, well explaining their different inhibitory potencies. The derived SAR were used in ongoing drug design, resulting so far in highly potent CYP19 inhibitors. The kinetic cycle of 17β-HSD1 was hypothesized based on biochemical data, analysis of the crystal structures and a multi-trajectory MD approach and provided insights in protein motion. These were translated into the drug design process. An ensemble docking study was performed for bis(hydroxyphenyl)-arenes, potent 17β-HSD1 inhibitors, and two conformation-dependent binding modes were identified. MD simulations and quantum-chemical calculations identified one of them as the more plausible, suggesting this class of compounds to dysfunction the enzyme dynamics. Two pharmacophore models were derived from CYP11B2 inhibitors and combined into a ligand- and structure-based approach, which led to a new class of potent CYP11B2 inhibitors.Verschiedene computergestützte Methoden wurden in der Entwicklung von nicht-steroidalen Hemmstoffen steroidogener Enzyme angewandt. Diese Inhibitoren sollen zur Behandlung hormon-abhängiger Krankheiten eingesetzt werden. Ein qualitativ-hochwertiges CYP17 Homologiemodell wurde erstellt und in Docking Studien verwendet. Drei Bindungsmodi konnten identifiziert werden und Struktur-Wirkungsbeziehungen wurden für verschiedene CYP17 Hemmstoffklassen abgeleitet und erfolgreich in eine weitere Hemmstoffentwicklung eingebaut. Dockingstudien und Clusteranalysen einer Reihe von CYP19 Inhibitoren ergaben einen plausiblen Bindungsmodus und die daraus gewonnenen Erkenntnisse führten in laufenden Projekten zu höchst potenten Hemmstoffen. Der kinetische Zyklus von 17β-HSD1 wurde postuliert basierend auf biochemischen Literaturdaten, Kristallstrukturenanalyse und einem multiplen MD-Ansatz, welcher wichtige Einblicke in die Dynamik des Enzyms lieferte. Diese wurden als ensemble docking Ansatz in die Entwicklung einer Klasse hochpotenter 17β-HSD1 Inhibitoren eingebaut und ergaben zwei Enzymkonformations-abhängige Bindungsmodi. MD-Simulationen und quantenchemische Methoden identifizierten einen davon als plausibler. Dabei scheinen Substanzen dieser Klasse die Enzymdynamik zu stören. Zwei Pharmakophormodelle wurden basierend auf CYP11B2-Hemmstoffen erstellt und in einen Ligand- und Struktur-basierten Ansatz eingebaut. Dieser führte zu einer neuen Klasse von potenten und selektiven CYP11B2-Inhibitoren

In silico study of Plasmodium 1-deoxy-dxylulose 5-phosphate reductoisomerase (DXR) for identification of novel inhibitots from SANCDB

Expected release date-April 201

The Conformational Universe of Proteins and Peptides: Tales of Order and Disorder

Proteins represent one of the most abundant classes of biological macromolecules and play crucial roles in a vast array of physiological and pathological processes. The knowledge of the 3D structure of a protein, as well as the possible conformational transitions occurring upon interaction with diverse ligands, are essential to fully comprehend its biological function.In addition to globular, well-folded proteins, over the past few years, intrinsically disordered proteins (IDPs) have received a lot of attention. IDPs are usually aggregation-prone and may form toxic amyloid fibers and oligomers associated with several human pathologies. Peptides are smaller in size than proteins but similarly represent key elements of cells. A few peptides are able to work as tumor markers and find applications in the diagnostic and therapeutic fields. The conformational analysis of bioactive peptides is important to design novel potential drugs acting as selective modulators of specific receptors or enzymes. Nevertheless, synthetic peptides reproducing different protein fragments have frequently been implemented as model systems in folding studies relying on structural investigations in water and/or other environments.This book contains contributions (seven original research articles and five reviews published in the journal Molecules) on the above-described topics and, in detail, it includes structural studies on globular folded proteins, IDPs and bioactive peptides. These works were conducted usingdifferent experimental methods

Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening

The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds

Molecular studies of two methylerythritol 4-phosphate pathway enzymes of isoprenoid biosynthesis : the 4-diphosphocytidyl-2C-methyl-D-erythritol kinase and the 1-deoxy-D-xylulose 5-phosphate synthase = Estudios moleculares de dos enzimas de la ruta del metileritritol 4-fosfato de biosíntesis de isoprenoides : la 4-difosfocitidil-2C-metil-D-eritritol quinasa y la 1-dexosi-D-xilulosa 5-fosfato sintasa

Los isoprenoides son una de las mayores familias de compuestos descritos en la naturaleza. Estos compuestos están presentes en todos los organismos vivos y se sintetizan a partir de dos moléculas de 5 átomos de carbono: el isopentenil difosfato (IPP) y el dimetilalil difosfato (DMAPP). Actualmente se conoce que arqueobacterias, hongos y animales presentan la ruta del mevalonato de síntesis de estos precursores, mientras que eubacterias, algún protozoo (como el causante de la malaria) y protistas presentan la ruta del metileritritol 4-fosfato (MEP) de síntesis de IPP y DMAPP. Estas rutas coexisten separadas espacialmente en plantas, helechos y algunas algas. La ruta del MEP de biosíntesis de los precursores de isoprenoides se muestra como una atractiva diana para la búsqueda de nuevos compuestos antimaláricos, antibióticos y herbicidas debido a su presencia en los principales agentes patogénicos y su ausencia en animales, además del carácter esencial de los isoprenoides para la vida. En esta tesis se ha realizado la búsqueda asistida por ordenador de compuestos que puedan interferir en la formación del complejo homodimérico del cuarto paso enzimático de la ruta del MEP. La metodología utilizada es muy útil en la búsqueda de inhibidores específicos. Se han caracterizado la unión de diferentes compuestos obtenidos con la enzima. Además se ha caracterizado el estado de oligomerización de la enzima. Paralelamente también se ha caracterizado un homólogo del primer paso enzimático de la ruta del MEP de un organismo termofílico caracterizando sus principales parámetros cinéticos y residuos importantes para la actividad enzimática mediante mutagénesis dirigida. Como último punto, se ha caracterizado el proceso de proteólisis de diferentes homólogos de este primer paso enzimático de la ruta del MEP asociándolo a modificaciones postraduccionales intramoleculares de las mismas proteínas, abriendo la posibilidad de un proceso de regulación posttraduccional de la actividad enzimática en este tipo de enzimas