This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase.

No description available.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24555号 / 薬科博第172号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 小野 正博, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Sulfated low molecular weight lignins (LMWLs), CDSO3 and FDSO3, designed recently as macromolecular mimetics of heparin, were found to exhibit potent anticoagulant activity. Small molecules based on the same scaffold, SBD and SBT, showed promising thrombin inhibition. We were able to address the mechanism of the inhibition using Michaelis-Menten kinetics. All the molecules were found to be allosterically impairing thrombin activity using either noncompetitive or uncompetitive mechanism. Absence of competition with hirugen, an exosite 1 ligand, and competition with polymeric heparin points to exosite 2 as the site of interaction for these inhibitors. Yet mixed competition results with other exosite 2 ligands indicated that the molecules utilize different sub-sites within exosite 2 for interaction. Site-directed mutagenesis was used to pin point the key residues important for inhibition. All of all positively charged exosite 2 residues were mutated one at a time to alanine to abolish its charge. The data showed that Arg93 and Arg175 are the major residues involved in CDSO3 binding. FDSO3 showed a progressively greater defect in inhibition with double point mutations, the triple mutant Arg93,97,101Ala displayed a 50 fold drop in inhibition. A single mutant, Arg173Ala, displayed 22-fold reduction in IC50 of SBD, while Arg233Ala was the only mutation that impaired SBT inhibition. This proves the fact that inspite of the structural similarity between the two polymers and the two small molecules, thtey do not share the same binding space in exosite 2. To understand the types of interactions involved in thrombin interaction with the polymers, we resorted to salt-dependence studies. This showed that CDSO3 had fewer ionic contacts with thrombin, with most of its binding energy derived from non-ionic interactions. FDSO3 on the other hand had a balanced contribution of ionic and non-ionic forces. Thermodynamic studies showed that both polymers have a positive ΔCp of binding, which proves the involvement of electrostatic forces and signals the burial of the polar residues on thrombin exosite 2. These molecules offer a rare chance to study thrombin allostery. Little is known about the interplay between exosite 2, active site and sodium binding site. The allosteric nature of inhibition indicated that, for the first time, a link is proven to exist between exosite 2 and the active site that could be used to inhibit the enzyme. The presence of sodium was found to enhance the binding of FDSO3 at exosite 2, which establish the energetic coupling between exosite 2 and sodium binding site. The results identify novel binding sub-sites within exosite 2 that are energetically coupled to thrombin’s catalytic function and linked to the sodium binding site. The design of high affinity small molecules based on LMWLs scaffold presents major opportunities for developing clinically relevant, allosteric modulators of thrombin.

FORTES, S.S. Estudo de metabolismo in vitro e inibição enzimática do produto natural Licarina A empregando microssomas hepático de humanos. 2017. Tese (Doutorado) - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2017. Muitos fármacos comercializados tiveram sua origem em produtos naturais e seus derivados. Devido ao grande potencial farmacológico destas novas moléculas pesquisadas, uma etapa importante e inicial no desenvolvimento de um novo fármaco é a avaliação do seu comportamento frente as enzimas do citocromo P450 (CYP 450), incluindo os estudos de interações medicamentosas. Neste contexto, um substrato que merece destaque é a Licarina A (Lic A). Este composto é uma neolignana encontrada em algumas espécies de plantas e vêm demonstrando várias propriedades biológicas promissoras, dentre elas destaca-se a atividade anti-leishimania. No entanto, para que esta substância com comprovada atividade se torne um fármaco é necessário realizar, na fase pré-clínica, estudos sobre seu perfil metabólico frente às enzimas do CYP450 e estudos de interação medicamentosa. Portanto, esta Tese teve como objetivo determinar os parâmetros enzimáticos utilizando microssomas hepáticos de humanos através do estudo de metabolismo in vitro com esta molécula e realizar estudos de interação medicamentosa através dos estudos de inibição enzimática e pesquisar a isoforma do CYP450 que metaboliza predominantemente este produto natural através do emprego de enzimas recombinantes de humanos. Primeiramente, foi desenvolvido um método analítico para a determinação do produto natural Licarina A em meio microssomal. As análises foram realizadas por cromatografia liquida de alta eficiência empregando a coluna Ascentis C18 e fase móvel composta por metanol: solução aquosa de ácido fórmico 0,1% (75:25, v/v); a vazão empregada foi de 1,0 mL min-1. O método foi validado na faixa de concentração de 0,383 a 76,65 ?mol L-1, com coeficiente de correlação linear de 0,99 e limite de quantificação de 0,383 ?mol L-1. A precisão e exatidão apresentaram resultados dentro do recomendável pela ANVISA. Após validação do método, estabeleceram-se as condições lineares para a depleção da Lic A no meio microssomal e posteriormente, a cinética foi determinada em condições de velocidade inicial utilizando para tanto 0,20 mg mL-1 de concentração de proteínas microssomais e 20 minutos de tempo de incubação. O comportamento observado na cinética enzimática para a depleção da Lic A foi um comportamento atípico, caracterizada pelo modelo cinético de Hill. Os valores de Vmax, S50 e coeficiente de Hill foram, 1,651 ?mol mg-1 min-1, 3,87 ?mol L-1 e 2,0 respectivamente. A partir dos parâmetros cinéticos o valor de clearance intrínseco (CLint) para a Lic A foi de 0,22 mL min-1 mg-1. Posteriormente, a correlação in vitro in vivo foi realizada e foi observado um clareance hepático (CLhep) de 20 mL min-1 kg-1 e taxa de extração hepática (E) de 1. As isoformas do CYP450 envolvidas no metabolismo da Lic A foram CYP 1A2 e 2B6. Os estudos de inibição mostraram que a Lic A é um inibidor fraco frente as isoformas do CYP450 estudadas, com valores de IC50 maiores do que 80 ?mol L-1. Embora já tenha sido estudada diferentes vias metabólicas da licarina A com vários metabólitos identificados, esta foi a primeira vez que foi observado a formação de um metabólito in vitro com o uso de microssomas hepático humano. Com o auxílio da espectrometria de massa foi possível a identificação do metabólito de m/z 343 [M+H]+, possivelmente um composto epoxidado, da licarina A. / FORTES, S.S. In vitro metabolism and enzymatic inhibition study of the natural product Licarin A employing human liver microsomes. 2017. Thesis (Doctoral) - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2017. Many marketed drugs had their origin in natural products and their derivatives. Due to the biological potential of these new molecules, an important initial step in the development of a new drug is the evaluation of its behavior in front of cytochrome P450 enzymes (CYP 450), including studies of drug interactions. In this context, a substrate that deserves attention is Licarin A (Lic A). This compound is a neolignan found in some species of plants and several promising biological properties have been describing for this natural product, among them anti-leishimania activity. However, for this substance to become a drug, it is necessary to perform, in the preclinical phase, studies regarding its metabolic profile and drug interactions. Therefore, this thesis aimed to determine the enzymatic parameters by using human liver microsomes through in vitro metabolism study with this molecule and to conduct drug interaction studies through the enzyme inhibition studies and finally, to investigate the CYP450 isoforms that metabolize predominantly this natural product through the use of recombinant human enzymes. Firstly, an analytical method was developed for the quantification of the natural product Licarin A in microsomal medium. The analyzes were performed by high performance liquid chromatography employing an Ascentis C18 column and mobile phase composed of methanol: 0.1% formic acid aqueous solution (75:25, v / v); the flow rate used was 1.0 mL min-1. The method was validated in the concentration range of 0.333 to 76.65 ?mol L-1, with a linear correlation coefficient of 0.99 and a quantification limit of 0.333 ?mol L-1. Accuracy and precision showed results in agreement with ANVISA guidelines. After method validation, the linear conditions for depletion of Lic A in the microsomal medium were established. Subsequently, the kinetics were determined under initial velocity conditions using 0.20 mg mL-1 of microsomal protein concentration and 20 minutes of incubation time. The behavior observed in the enzymatic kinetics for the depletion of Lic A was an atypical behavior, characterized by the Hill kinetic model. The values of Vmax, S50 and Hill coefficient were 1.651 ?mol mg-1 min-1, 3.87 ?mol L-1 and 2.0, respectively. From the kinetic parameters, the intrinsic clearance (CLint) for Lic A was 0.22 mL min-1 mg-1. Subsequently, in vitro in vivo correlation was performed and a hepatic clareance (CLhep) of 20 mL min-1 kg-1 and a hepatic extraction rate (E) of 1 was observed. The CYP450 isoforms involved in the metabolism of Lic A were CYP 1A2 and 2B6. Inhibition studies have shown that Lic A is a weak CYP450 inhibitor, with IC50 values greater than 80 ?mol L-1. Although different metabolic pathways of licanin A have been studied and several metabolites were identified, this is the first report about the formation of an in vitro metabolite after metabolism by human liver microsomes. With the aid of mass spectrometry it was possible to identify the metabolite of m/z 343 [M+H]+, possibly an epoxidized compound, of licanin A.

ATP is the main cellular energy generated by the enzyme ATP synthase in almost all organisms from bacteria to vertebrates. While malfunction of the ATP synthase complex is responsible for several disease conditions, the enzyme itself can be used as a potent molecular drug target to combat many diseases including microbial infections, cancer, tuberculosis, and obesity. Recent widespread escalation of antibiotic resistant microbes in general and E. coli in particular demands novel alternative approaches to combat microbial infections. Inhibition of ATP synthase by inhibitors such as peptides is known to deprive microbes of required energy, resulting in microbial cell death. Therefore, we have examined the venom peptide induced inhibition of E. coli ATP synthase. It was found that venom peptides completely inhibited E. coli ATP synthase and the process of inhibition was found to be fully reversible. This study also links the antimicrobial properties of peptides in part to the inhibition of ATP synthase. Thus, selective use of ATP synthase as a molecular drug may have an important impact on biology and medicine.

This thesis is concerned with a range of chemical problems which are amenable to theoretical investigation via the application of current methods of computational quantum chemistry. These problems include the calculation of accurate thermochemical data, the prediction of reaction kinetics, the study of molecular potential energy surfaces, and the investigation of molecular structures and binding. The heats of formation (from both atomisation energies and isodesmic schemes) of a set of approximately 120 C1 and C2 fluorocarbons and oxidised fluorocarbons (along with C3F6 and CF3CHFCF2) were calculated with the Gaussian-3 (G3) method (along with several approximations thereto). These molecules are of importance in the flame chemistry of 2-H-heptafluoropropane, which has been proposed as a potential fire retardant with which to replace chloro- and bromofluorocarbons (CFC�s and BFC�s). The calculation of the data reported here was carried out in parallel with the modelling studies of Hynes et al.1-3 of shock tube experiments on CF3CHFCF3 and on C3F6 with either hydrogen or oxygen atoms. G3 calculations were also employed in conjunction with the experimental work of Owens et al.4 to describe the pyrolysis of CFClBr2 (giving CFCl) at a radiation wavelength of 265 nm. The theoretical prediction of the dissociation energy of the two C-Br bonds was found to be consistent with the energy at which carbene production was first observed, thus supporting the hypothesis that the pyrolysis releases two bromine radicals (rather than a Br2 molecule). On the basis of this interpretation of the experiments, the heat of formation of CFClBr2 is predicted to be 184 � 5 kJ mol-1, in good agreement with the G3 value of 188 � 5 kJ mol-1. Accurate thermochemical data was computed for 18 small phosphorus containing molecules (P2, P4, PH, PH2, PH3, P2H2, P2H4, PO, PO2, PO3, P2O, P2O2, HPO, HPOH, H2POH, H3PO, HOPO and HOPO2), most of which are important in the reaction model introduced by Twarowski5 for the combustion of H2 and O2 in the presence of phosphine. Twarowski reported that the H + OH recombination reaction is catalysed by the combustion products of PH3 and proposed two catalytic cycles, involving PO2, HOPO and HOPO2, to explain this observation. Using our thermochemical data we computed the rate coefficients of the most important reactions in these cycles (using transition state and RRKM theories) and confirmed that at 2000K both cycles have comparable rates and are significantly faster than the uncatalysed H + OH recombination. The heats of formation used in this work on phosphorus compounds were calculated using the G2, G3, G3X and G3X2 methods along with the far more extensive CCSD(T)/CBS type scheme. The latter is based on the evaluation of coupled cluster energies using the correlation consistent triple-, quadruple- and pentuple-zeta basis sets and extrapolation to the complete basis set (CBS) limit along with core-valence correlation corrections (with counterpoise corrections for phosphorus atoms), scalar relativistic corrections and spin-orbit coupling effects. The CCSD(T)/CBS results are consistent with the available experimental data and therefore constitute a convenient set of benchmark values with which to compare the more approximate Gaussian-n results. The G2 and G3 methods were found to be of comparable accuracy, however both schemes consistently underestimated the benchmark atomisation energies. The performance of G3X is significantly better, having a mean absolute deviation (MAD) from the CBS results of 1.8 kcal mol-1, although the predicted atomisation energies are still consistently too low. G3X2 (including counterpoise corrections to the core-valence correlation energy for phosphorus) was found to give a slight improvement over G3X, resulting in a MAD of 1.5 kcal mol-1. Several molecules were also identified for which the approximations underlying the Gaussian-n methodologies appear to be unreliable; these include molecules with multiple or strained P-P bonds. The potential energy surface of the NNH + O system was investigated using density functional theory (B3LYP/6-31G(2df,p)) with the aim of determining the importance of this route in the production of NO in combustion reactions. In addition to the standard reaction channels, namely decomposition into NO + NH, N2 + OH and H + N2O via the ONNH intermediate, several new reaction pathways were also investigated. These include the direct abstraction of H by O and three product channels via the intermediate ONHN, giving N2 + OH, H + N2O and HNO + N. For each of the species corresponding to stationary points on the B3LYP surface, valence correlated CCSD(T) calculations were performed with the aug-cc-pVxZ (x = Q, 5) basis sets and the results extrapolated to the complete basis set limit. Core-valence correlation corrections, scalar relativistic corrections and spin orbit effects were also included in the resulting energetics and the subsequent calculation of thermochemical data. Heats of formation were also calculated using the G3X method. Variational transition state theory was used to determine the critical points for the barrierless reactions and the resulting B3LYP energetics were scaled to be compatible with the G3X and CCSD(T)/CBS values. As the results of modelling studies are critically dependent on the heat of formation of NNH, more extensive CCSD(T)/CBS calculations were performed for this molecule, predicting the heat of formation to be 60.6 � 0.5 kcal mol-1. Rate coefficients for the overall reaction processes were obtained by the application of multi-well RRKM theory. The thermochemical and kinetic results thus obtained were subsequently used in conjunction with the GRIMech 3.0 reaction data set in modelling studies of combustion systems, including methane / air and CO / H2 / air mixtures in completely stirred reactors. This study revealed that, contrary to common belief, the NNH + O channel is a relatively minor route for the production of NO. The structure of the inhibitor Nd-(N'-Sulfodiaminophosphinyl)-L-ornithine, PSOrn, and the nature of its binding to the OTCase enzyme was investigated using density functional (B3LYP) theory. The B3LYP/6-31G(d) calculations on the model compound, PSO, revealed that, while this molecule could be expected to exist in an amino form in the gas phase, on complexation in the active site of the enzyme it would be expected to lose two protons to form a dinegative imino tautomer. This species is shown to bind strongly to two H3CNHC(NH2)2+ moieties (model compounds for arginine residues), indicating that the strong binding observed between inhibitor and enzyme is partially due to electrostatic interactions as well as extensive hydrogen bonding (both model Arg+ residues form hydrogen bonds to two different sites on PSO). Population analysis and hydrogen bonding studies have revealed that the intramolecular bonding in this species consists of either single or semipolar bonds (that is, S and P are not hypervalent) and that terminal oxygens (which, being involved in semipolar bonds, carry negative charges) can be expected to form up to 4 hydrogen bonds with residues in the active site. In the course of this work several new G3 type methods were proposed, including G3MP4(SDQ) and G3[MP2(Full)], which are less expensive approximations to G3, and G3X2, which is an extension of G3X designed to incorporate additional electron correlation. As noted earlier, G3X2 shows a small improvement on G3X; G3MP4(SDQ) and G3[MP2(Full)], in turn, show good agreement with G3 results, with MAD�s of ~ 0.4 and ~ 0.5 kcal mol-1 respectively. 1. R. G. Hynes, J. C. Mackie and A. R. Masri, J. Phys. Chem. A, 1999, 103, 5967. 2. R. G. Hynes, J. C. Mackie and A. R. Masri, J. Phys. Chem. A, 1999, 103, 54. 3. R. G. Hynes, J. C. Mackie and A. R. Masri, Proc. Combust. Inst., 2000, 28, 1557. 4. N. L. Owens, Honours Thesis, School of Chemistry, University of Sydney, 2001. 5. A. Twarowski, Combustion and Flame, 1995, 102, 41.

<p>Starting with the development of an enzymatic assay, where an enzyme in solution hydrolysed a solid-phase bound peptide, a model for the kinetics of enzyme action was introduced. This model allowed the estimation of kinetic parameters and enzyme activity for a system that has the peculiarity of not being saturable with the substrate, but with the enzyme. In a derivation of the model, it was found that the sensitivity of the signal to variations in the enzyme concentration had a transient increase along the reaction progress with a maximum at high substrate conversion levels. </p><p>The same behaviour was derived for the sensitivity in classical homogeneous enzymatic assays and experimental evidence of this was obtained. The impact of the transient increase of the sensitivity on the error structure, and on the ability of homogeneous end-point enzymatic assays to detect competitive inhibition, came into focus. First, a non-monotonous shape in the standard deviation of progress curve data was found and it was attributed to the random dispersion in the enzyme concentration operating through the transient increase in the sensitivity. Second, a model for the detection limit of the quantity Ki/[I] (the IDL-factor) as a function of the substrate conversion level was developed for homogeneous end-point enzymatic assays. </p><p>It was found that the substrate conversion level where the IDL-factor reached an optimum was beyond the initial velocity range. Moreover, at this optimal point not only the ability to detect inhibitors but also the robustness of the assays was maximized. These results may prove to be relevant in drug discovery for optimising end point homogeneous enzymatic assays that are used to find inhibitors against a target enzyme in compound libraries, which are usually big (>10000) and crowded with irrelevant compounds.</p>

The reaction mechanisms for the hemoglobin degrading enzymes in the Plasmodium falciparum malaria parasite, plasmepsin II (Plm II) and histo-aspartic protease (HAP), have been analyzed by molecular simulations. The reaction free energy profiles, calculated by the empirical valence bond (EVB) method in combination with molecular dynamics (MD) and free energy perturbation (FEP) simulations are in good agreement with experimental data. Additional computational methods, such as homology modelling and automated substrate docking, were necessary to generate a 3D model and a reactive substrate conformation before the reaction mechanism in HAP could be investigated. HAP is found to be an aspartic protease with a peptide cleaving mechanism similar to plasmepsin II. The major difference between these enzymes is that the negatively charged tetrahedral intermediate is stabilized by the charged histidine in HAP while in Plm II it is a neutral aspartic acid. Also the reaction mechanism for two other aspartic proteases, cathepsin D and HIV-1 protease, was simulated. These enzymes are relevant both for the inhibitor selectivity and for obtaining a general picture of catalysis in aspartic proteases. Another project involves inhibitor design towards plasmepsins. In particular, Plm II directed inhibitors based on the dihydroxyethylene scaffold have been characterized computationally. Molecular dynamics (MD) simulations were used to propagate the investigated system through time and to generate ensembles used for the calculation of free energies. The ligand binding affinities were calculated with the linear interaction energy (LIE) method. The most potent inhibitor had a Ki value of 6 nM and showed 78 % parasite inhibition when tested on red blood cells infected by malaria parasite P. falciparum. Citrate synthase is part of the citric acid cycle and is present in organisms that live in cold sea water as well as hot springs. The temperature adaptation of citrate synthase to cold and heat was investigated in terms of the difference in transition state stabilization between the psychrophilic, mesophilic and hyperthermophilic homologues. The EVB, FEP and MD methods were used to generate reaction free energy profiles. The investigated energetics points toward the electrostatic stabilization during the reaction as the major difference between the different citrate synthase homologues. The electrostatic stabilization of the transition state is most effective in the following order of the citrate synthase homologues: hyperthermophile, mesophile, psycrophile. This could be a general rule for temperature adaptation of enzyme catalysis.

No description available.