Academic literature on the topic 'Structural Modeling - Heme Enzymes'

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B–C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

Abstract Enhanced understanding of structure-function relationships of human 21-hydroxylase, CYP21, is required to better understand the molecular causes of congenital adrenal hyperplasia. To this end, a structural model of human CYP21 was calculated based on the crystal structure of rabbit CYP2C5. All but two known allelic variants of missense type, a total of 60 disease-causing mutations and six normal variants, were analyzed using this model. A structural explanation for the corresponding phenotype was found for all but two mutants for which available clinical data are also discrepant with in vitro enzyme activity. Calculations of protein stability of modeled mutants were found to correlate inversely with the corresponding clinical severity. Putative structurally important residues were identified to be involved in heme and substrate binding, redox partner interaction, and enzyme catalysis using docking calculations and analysis of structurally determined homologous cytochrome P450s (CYPs). Functional and structural consequences of seven novel mutations, V139E, C147R, R233G, T295N, L308F, R366C, and M473I, detected in Scandinavian patients with suspected congenital adrenal hyperplasia of different severity, were predicted using molecular modeling. Structural features deduced from the models are in good correlation with clinical severity of CYP21 mutants, which shows the applicability of a modeling approach in assessment of new CYP21 mutations.

ABSTRACT The respiratory chain of the thermohalophilic bacteriumRhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream ofrcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E278, Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the CuA domain, that is characteristic ofcaa 3 oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa 3oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa 3 class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in degrading heme into biliverdin and iron. HO-1 can also enter the nucleus and regulate gene transcription independent of its enzymatic activity. Whether HO-1 can alter gene expression through direct binding to target DNA remains unclear. Here, we performed HO-1 CHIP-seq and then employed 3D structural modeling to reveal putative HO-1 DNA binding domains. We identified three probable DNA binding domains on HO-1. Using the Proteinarium, we identified several genes as the most highly connected nodes in the interactome among the HO-1 gene binding targets. We further demonstrated that HO-1 modulates the expression of these key genes using Hmox1 deficient cells. Finally, mutation of four conserved amino acids (E215, I211, E201, and Q27) within HO-1 DNA binding domain 1 significantly increased expression of Gtpbp3 and Eif1 genes that were identified within the top 10 binding hits normalized by gene length predicted to bind this domain. Based on these data, we conclude that HO-1 protein is a putative DNA binding protein, and regulates targeted gene expression. This provides the foundation for developing specific inhibitors or activators targeting HO-1 DNA binding domains to modulate targeted gene expression and corresponding cellular function.

Enzymatic halogenation and haloperoxidation are unusual processes in biology; however, a range of halogenases and haloperoxidases exist that are able to transfer an aliphatic or aromatic C–H bond into C–Cl/C–Br. Haloperoxidases utilize hydrogen peroxide, and in a reaction with halides (Cl−/Br−), they react to form hypohalides (OCl−/OBr−) that subsequently react with substrate by halide transfer. There are three types of haloperoxidases, namely the iron-heme, nonheme vanadium, and flavin-dependent haloperoxidases that are reviewed here. In addition, there are the nonheme iron halogenases that show structural and functional similarity to the nonheme iron hydroxylases and form an iron(IV)-oxo active species from a reaction of molecular oxygen with α-ketoglutarate on an iron(II) center. They subsequently transfer a halide (Cl−/Br−) to an aliphatic C–H bond. We review the mechanism and function of nonheme iron halogenases and hydroxylases and show recent computational modelling studies of our group on the hectochlorin biosynthesis enzyme and prolyl-4-hydroxylase as examples of nonheme iron halogenases and hydroxylases. These studies have established the catalytic mechanism of these enzymes and show the importance of substrate and oxidant positioning on the stereo-, chemo- and regioselectivity of the reaction that takes place.

ABSTRACTThe heme-containing enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and IDO-2 catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection withPlasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified 8-aminobenzo[b]quinolizinium (compound 2c) to be an inhibitor of IDO-1 with potency at nanomolar concentrations (50% inhibitory concentration, 164 nM). Twenty-one structurally modified derivatives of compound 2c were synthesized for structure-activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the roles of prominent amino acids in the catalytic mechanisms of the two isoenzymes via homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance ofl-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives (6 compounds with 50% effective concentration values between 2.1 and 6.7 nM) were found to be highly effective againstP. falciparum3D7 blood stages in cell culture with a mechanism independent of IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics; it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.

Abstract Context: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause. Objective: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function. Results: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo. Conclusion: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

Enzymes are proteins that catalyze biochemical reactions and their use report multiple advantages, as they can be very selective, low polluting (biodegradable), cheap and allow working in mild conditions compared with traditional non enzymatic processes. Despite their enormous benefits, their applications at the industrial level are still limited, mainly due to low productivity, low substrate tolerance (too specifics) and poor resistance to the industrial conditions, and for this reason, developing enhanced enzymes by means of enzyme engineering is a central research field nowadays. Notably, the application of computational chemistry in the field of enzyme engineering is increasing due to improvements in hardware and software. Moreover, this process is fast and low-priced and therefore, profitable for the application to the real problems that face industry. Therefore, motivated by this progress, the main goal of this thesis is the development of computational strategies that allow designing and evaluating modifications in enzymes, also aiming to obtain results quickly and inexpensively. This purpose was reached by the combination of different in silico methodologies that were further supported by experimental data in an interactive feedback process. As a result of this thesis, the enzymatic process in heme peroxidases was first satisfactorily described by dividing the process into two steps (from the ligand diffusion to the chemical reaction), using a combination of different computational techniques. The first step, which involves the protein/ligand recognition, was characterized with different molecular mechanics based techniques (MD, Docking and MC-PELE). On the other hand, the chemical reaction (including bond formation and electron transfer) was reproduced using QM based methods by means of energy calculation, spin density characterization, e-coupling calculations and QM/MM e-pathways descriptions. Following this procedure, the oxidation of veratryl alcohol by the enzyme lignin peroxidase was also characterized. Moreover, regarding the e-coupling calculations, a server to compute this vale faster and easy was developed. In the second part of the thesis, the results demonstrated that our protocol could reliably describe and predict enzymatic functions, not only in native enzymes but also in mutated ones, which results were in agreement with experimental data. For example, the structural implications over the reactivity in manganese peroxidase and its engineered variant obtained by cutting the last terminal residues were identified and characterized by the combination of Monte Carlo simulations (PELE) and electronic coupling calculations. The pH resistance in the mutant 2-1B (which was obtained experimentally by random directed evolution) in contrast with the wild type versatile peroxidase, were also rationalized by molecular dynamics, where the residues in the heme environment presented different conformation due to the mutations introduced, resulting in different pH resistance. Interestingly, the last part of the thesis was centered of engineering heme peroxidases. We engineered a peroxidase from in silico predictions to elucidate the long range electron transfer processes involved in the oxidation of the substrate veratryl alcohol by the enzyme versatile peroxidase. In this work we identify the key residues involved in the process, with further applications in engineering enhanced enzymes. Moreover, an enhanced manganese peroxidase mutant from a complete computational study was designed. First, the ligand diffusion study allowed finding the key aminoacids in the substrate/enzyme recognition and binding. Then, the chemical reaction in terms of the oxidation probability and kinetic constant for the proposed mutant were estimated, and the results were in agreement with experimental data. Therefore, the work of this thesis probed that computational biophysics and biochemistry are promising and valuable tools for enzyme engineering. In particular, in the field of rational design of heme peroxidases, they provide relevant information about the enzymatic mechanism and allow designing new enzymes, as well as checking their improvement/worsening, in an efficient way.
Las enzimas son proteínas que catalizan reacciones bioquímicas y cuyo uso aporta múltiples ventajas, ya que son en general muy selectivas, poco contaminantes (biodegradables), baratas y permiten trabajar en condiciones suaves, en comparación con los procesos tradicionales no enzimáticos. A pesar de sus enormes beneficios, sus aplicaciones a nivel industrial son todavía limitadas, debido principalmente a la baja productividad, baja tolerancia al sustrato (demasiado específicos) y una escasa resistencia a las condiciones industriales en general, y por esta razón el desarrollo de enzimas mejoradas es un campo de investigación muy importante hoy en día. En particular, la aplicación de la química computacional en el campo de la ingeniería de enzimas está en aumento debido a las mejoras en hardware y software. Motivado por este progreso, el objetivo principal de esta tesis es el desarrollo de estrategias de cálculo que, mediante la combinación de diferentes metodologías in silico permitan diseñar y evaluar modificaciones en las enzimas, centrándonos en la obtención de resultados de forma rápida y económica. La primera parte de la tesis está centrada en la descripción del mecanismo enzimático entendido como un proceso de dos pasos que incluyen la difusión ligando y la reacción química, mediante una combinación de diferentes técnicas computacionales. El primer paso, que implica el reconocimiento de la proteína / ligando, se caracterizó con diferentes técnicas basadas en la mecánica molecular (dinámica molecular, docking y Monte Carlo- PELE). Por otro lado, la reacción química (incluyendo la formación de enlaces y la transferencia de electrones) se simuló usando métodos basados en mecánica cuántica por medio de cálculos de energía, la caracterización del spin o cálculos de acoplamiento electrónico. Por ejemplo, siguiendo este procedimiento, se caracterizó la oxidación de alcohol veratrílico por medio de la enzima lignin peroxidasa. Además, con el objetivo de poder calcular los acoplamientos electrónicos de una manera más rápida y fácil, se desarrolló un servidor web: ecoupling server. En la segunda parte de la tesis, los resultados demostraron que el protocolo anterior podría describir funciones enzimáticas no sólo en las especies nativas sino también en las variantes mutadas. Por ejemplo, se identificaron las implicaciones estructurales de la reactividad en una manganeso peroxidasa de la subfamilia larga y su variante modificada obtenida mediante la reducción de los últimos residuos terminales gracias al estudio de simulaciones de Monte Carlo (PELE) y cálculos de acoplamiento electrónico. Además, la resistencia a pH ácido en el mutante 2-1B (que se había obtenido previamente por evolución dirigida al azar) se comparó con la especie nativa y también se racionalizó por dinámica molecular, donde se observó que los residuos del entorno del hemo presentaban diferente conformación debido a las mutaciones introducidas, resultando en una diferente resistencia a pH ácido. La última parte de la tesis se centra en la ingeniería racional de hemo peroxidasas. A partir de predicciones in silico se diseñaron variantes de peroxidasa versátil para tratar de entender los procesos de transferencia electrónica de largo alcance que participan en la oxidación del sustrato de alcohol veratrílico, mediante la identificación de los residuos intermedios involucrados en el proceso. Además, a partir de un estudio computacional completo, se diseñó un mutante mejorado de manganeso peroxidasa, cuyos valores cinéticos estimados computacionalmente se encontraban de acuerdo con los resultados experimentales. En conclusión, en esta tesis se ilustra cómo los métodos biofísicos y bioquímicos computacionales son herramientas prometedoras y valiosas para la ingeniería de enzimas, en particular en el campo del diseño racional.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2002.
Includes bibliographical references.
Chapter I. Modeling Dioxygen-Activating Centers in Non-Heme Diiron Enzymes: Carboxylate Shifts in Diiron(II) Complexes Supported by Sterically Hindered Carboxylate Ligands General synthetic routes are described for a series of diiron(II) complexes supported by sterically demanding carboxylate ligands 2,6-di(p-tolyl)benzoate (ArTolCO2-) and 2,6-di(4-fluorophenyl)benzoate (Ar4-FPhCO2-). The interlocking nature of the m-terphenyl units in self-assembled [Fe2(p-O2CArTol)2(O2CArTol)2L2] (L = C5H5N (4); 1-MeIm (5)) promotes the formation of coordination geometries analogous to those of the non-heme diiron cores in the enzymes RNR-R2 and A9D. Magnetic susceptibility and MOssbauer studies of 4 and 5 revealed properties consistent with weak antiferromagnetic coupling between the high-spin iron(II) centers. Structural studies of several derivatives obtained by ligand substitution reactions demonstrated that the [Fe2([mu]O2CAr')2L2] (Ar'=ArTol; Ar 4-FPh) module is geometrically quite flexible. Details of the core rearrangement within the tetracarboxylate diiron framework, facilitated by carboxylate shifts, were probed by solution VT 19F-NMR spectroscopic studies of [Fe2(ap-O2CAr4-FPh)2(O2CAr4-FPh)2(THF)2] (8) and [Fe2(p-O2CAr4-FPh)4(4-tBuC5H4N)2] (12). The dynamic motion in the primary coordination sphere controls the positioning of open sites and regulates the access of exogenous ligands, processes that also occur at the catalytic sites of non-heme diiron enzymes. Chapter II. Structural Flexibility within a Sterically Hindered Ligand Platform: Mononuclear Iron(II) Carboxylate Complexes as Subsite Models for Diiron(II) Centers The synthesis and characterization of a series of mononuclear iron(II) carboxylate complexes are described.
(cont.) By using sterically hindered carboxylate ligands, 2,6-di(p-tolyl)benzoate (ArTolCO2-) and 2,6-di(4-tert-butylphenyl)benzoate (Ar4-tBPhCO2-), series of four-, five-, and six-coordinate iron(II) complexes were synthesized. The compounds are [Fe(O2CArTol)2(1-BnIm)2] (3), [Fe(O2CArTol)2(1-MeBzIm)2] (4), [Fe(02C-Ar4-tBuPh)2(2,2'-bipy)2] (5), [Fe(O2CArTol)2(TMEDA)] (6), and [Fe(O2CArTol)2(BPTA)] (7). Structural analyses of 3-7 revealed that the overall stereochemistry of the [Fe(O2CAr')2Ln] units is dictated by electronic and steric factors of the N-donor ligands (L), as well as by the flexible coordination of the carboxylate ligands. Distinctive MOss-Bauer parameters obtained for these and related compounds facilitated the spectral assignment of a diiron(II) complex having asymmetric metal sites, [Fe2(p-02CArTol)3(2CArTol)(2,6-lutidine)] (2). Well-defined mononuclear iron carboxylate complexes thus may serve as subsite models for higher nuclearity species in both synthetic and biological systems. Chapter III. Functional Mimic of Dioxygen-Activating Centers in Non-Heme Diiron Enzymes: Mechanistic Implications of Paramagnetic Intermediates in the Reactions between Diiron(II) Complexes and Dioxygen Tetracarboxylate diiron(II) complexes, [Fe2(-O02CArTOl)2(02CArToll)2(C5H5N)2] (la) and [Fe2(Pl-02CArTol)4(4-tBuC5H4N)2] (2a), where ArTloCO2- = 2,6-di(p-tolyl)benzoate, react with 02 in CH2C12 at -78 C to afford deep green intermediates ...
by Dongwhan Lee.
Ph.D.

A Doença de Chagas, causada pelo parasita Trypanosoma cruzi, atinge cerca de um quarto da população da América Latina. Os fármacos disponíveis para o tratamento desta doença são inapropriados, apresentam baixa eficácia e sérios efeitos colaterais que limitam o seu uso. Esse grave panorama torna urgente a descoberta de novos agentes quimioterápicos para o tratamento seguro e eficaz da doença. A via glicolítica é principal forma de obtenção de energia de tripanosomatídeos. Um alvo molecular atrativo desta via bioquímica que desempenha papel essencial no controle do fluxo glicolítico do Trypanosoma cruzi, a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH), foi selecionada neste trabalho de Tese para estudos em biologia estrutural e química medicinal visando à identificação e planejamento de novos inibidores enzimáticos. Neste contexto, triagens biológicas resultaram na identificação de compostos de origem natural e sintética com atividade inibitória in vitro frente à GAPDH de T. cruzi, ampliando a diversidade química de moduladores seletivos deste alvo. Estudos cinéticos e estruturais demonstraram o comportamento não cooperativo entre os sítios ativos da enzima GAPDH de T. cruzi em relação à interação com o cofator NAD+, fornecendo importantes evidências mecanísticas e estruturais para uma melhor compreensão das bases moleculares envolvidas no processo de reconhecimento molecular. Os estudos das relações quantitativas entre a estrutura e atividade (QSAR 2D e QSAR 3D) resultaram na geração de modelos com elevada consistência estatística interna e externa, além de alto poder preditivo da propriedade-alvo. Além disso, estudos de modelagem molecular e de QSAR 3D revelaram aspectos estruturais relevantes para o planejamento de inibidores seletivos da enzima GAPDH de tripanosomatídeos. Por fim, uma estratégia de triagem virtual baseado na estrutura do receptor foi empregada para a identificação de novos inibidores da GAPDH de T. cruzi, consistindo, entre outros, na aplicação de filtros hierárquicos sucessivos envolvendo restrições farmacofóricas e estudos de docagem molecular que resultaram na seleção de 35 candidatos a inibidores da enzima-alvo. Os trabalhos integrando estudos em química medicinal e biologia estrutural apresentados nessa Tese de Doutorado significam importantes contribuições no desenvolvimento de bases científicas sólidas para o planejamento de novos inibidores potentes e seletivos da enzima GAPDH de T. cruzi, um alvo molecular de alta prioridade em nosso grupo de pesquisa.
Parasitic diseases are the foremost threat to human health and welfare around the world. Chagas\' disease (also called American trypanosomiasis) is a tropical parasitic disease which occurs in Latin America, particularly in South America. The currently available drugs for this parasitic disease have severe limitations, including poor efficacy and high toxicity. The crucial dependence of trypanosomatids on glycolysis as a source of energy makes the glycolytic enzymes promising targets for drug design. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi, a key enzyme in the glycolytic cascade, has been selected as an attractive drug target in this PhD Thesis work for studies in structural biology and medicinal chemistry for the identification and design of new enzyme inhibitors. In this context, compounds from both natural and synthetic sources with in vitro inhibitory activity against T. cruzi GAPDH were identified by screening assays, improving the chemical diversity of selective modulators of the target. Kinetic and structural studies have demonstrated the non-cooperative behavior between the T. cruzi active sites in the interaction with the NAD+ cofactor, shedding some light on the mechanistic and structural determinants underlying the biochemical recognition phenomenon. Quantitative structure-activity relationships (2D QSAR 2D and 3D QSAR) were successfully created, resulting in statistically significant models with good predictive ability for untested compounds. In addition, molecular modeling and 3D QSAR studies highlighted important structural aspects to assist the design of novel trypanosomatid GAPDH inhibitors. Finally, a structure-based virtual screening approach was employed for the identification of novel inhibitors of T. cruzi GAPDH, consisting of several consecutive hierarchical, fast pharmacophore matching and molecular docking, which afforded 35 inhibitor candidates for the target enzyme. The integration of structural biology and medicinal chemistry studies presented in this PhD Thesis are important contributions in the development of strong scientific basis for the design of new selective and potent inhibitors of GAPDH from T. cruzi, a molecular target of highest priority in our research group.

Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.

Photodynamic Therapy (PDT) involves administration of a photosensitizer (PS) either systemically or locally, followed by illumination of the lesion with visible light. PDT of cancer is now evolving from experimental treatment to a therapeutic alternative. Clinical results have shown that PDT is at least as efficacious as standard treatments of malignancies of the skin and Barrett’s esophagus. Hemes and heme proteins are vital components of essentially every cell in virtually all eukaryote organisms. Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway from the substrate aminolevulinic acid (ALA). Exogenous administration of ALA induces accumulation of (PpIX), which can be used as a photosensitiser for tumor detection or photodynamic therapy. Although the basis of the selectivity of ALA-based PDT or photodiagnosis is not fully understood, it has sometimes been correlated with the metabolic rate of the cells, or with the differential enzyme expressions along the heme biosynthetic pathway in cancer cells. An in silico analysis by modeling may be performed in order to determine the functional roles of genes coding enzymes of the heme biosynthetic pathway like ferrochelatase. Modeling and simulation systems are a valuable tool for the understanding of complex biological systems. With PyBioS, an object-oriented modelling software for biological processes, we can analyse porphyrin metabolism pathways.

The severe acute respiratory syndrome coronavirus 2 (SAR-Cov-2) caused the coronavirus (COVID-19) pandemic. The global concern is the discovery of a new target drug for the total cure. Recently, some research showed that a few COVID-19 enzymes may have been contemplated to be potential drug targets, but not much is known about its structural biology. This research investigates the 3-D structure of protease SAR-CoV-2. The tertiary structure was determined by homology modeling. The Swiss-Model workspace and the basic local alignment search tool (BLAST) were employed for modeling, and the resulted model was validated with programs that include PROCHECK, Verify3D, and QMEAN to ascertain reliability. To establish the structures that fitted, HHBlits software was employed. To verify the structure quality, a Ramachandran plot was exploited. The binding site was determined using CASTp and DeepSite algorithms, which showed that this protein can be exploited as a prospective pharmaceutical target. Albeit further experimentation is required as a COVID-19 virus vaccine-design/target-drug.

Vitamin B6 (vitB6) is a generic term that comprises six interconvertible pyridine compounds. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate (PLP). VitB6 is an essential nutrient for all living organisms, but only microorganisms and plants can carry out de novo synthesis of this vitamin. Other organisms obtain vitB6 from dietary sources and interconvert its different forms according to their needs via a biochemical pathway known as the salvage pathway. PLP is the biologically active form of vitB6 which is important for maintaining the biochemical homeostasis of the body. In the human body, PLP serves as a cofactor for more than 140 enzymatic reactions, mainly associated with synthesis, degradation and interconversion of amino acids and neurotransmitter metabolism. PLP-dependent enzymes are also involved in various physiological processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways. PLP is an important vitamer for normal brain function since it is required as a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA), serotonin, epinephrine, norepinephrine, histamine and dopamine. Intracellular levels of PLP are tightly regulated and conditions that disrupt this homeostatic regulation can cause disease. In humans, genetic and dietary (intake of high doses of vitB6) conditions leading to increase in PLP levels is known to cause motor and sensory neuropathies. Deficiency of PLP in the cell is also implicated in several diseases, the most notable example of which are the vitB6-dependent epileptic encephalopathies. VitB6-dependent epileptic encephalopathies (B6EEs) are a clinically and genetically heterogeneous group of rare inherited metabolic disorders. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but are well-controlled by the administration of PN or PLP. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities. Five main types of B6EEs are known to date, these are: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290). This chapter provides a review of vitB6 and its different vitamers, their absorption and metabolic pathways in the human body, the diverse physiological roles of vitB6, PLP homeostasis and its importance for human health. Finally, the chapter reviews the inherited neurological disorders affecting PLP homeostasis with a special focus on vitB6-dependent epileptic encephalopathies (B6EEs), their different subtypes, the pathophysiological mechanism underlying each type, clinical and biochemical features and current treatment strategies.

Collagens strengthen tissues throughout the body and are generally resistant to degradation, except by a small number of collagenolytic enzymes. As structural proteins, collagens often experience mechanical forces in vivo including expansion and contraction of blood vessels, tension on tendons and ligaments, and compression of cartilage.