Improving the selectivity of engineered protease inhibitors: Optimizing the P2 prime residue using a versatile cyclic peptide library

Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact β-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2′ specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2′ preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2′ residue in standard mechanism inhibition and unveils a new approach for screening P2′ substitutions that will benefit future inhibitor engineering studies.

Functional interaction of diphenols with polyphenol oxidase - Molecular determinants of substrate/inhibitor specificity SO FEBS JOURNAL

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on o-diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His 109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants Of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.

Studies of electronic structure by x-ray Raman and emission spectroscopy: applications to MgB2 superconductors and high pressure physics

X-ray Raman scattering and x-ray emission spectroscopies were used to study the electronic properties and phase transitions in several condensed matter systems. The experimental work, carried out at the European Synchrotron Radiation Facility, was complemented by theoretical calculations of the x-ray spectra and of the electronic structure. The electronic structure of MgB2 at the Fermi level is dominated by the boron σ and π bands. The high density of states provided by these bands is the key feature of the electronic structure contributing to the high critical temperature of superconductivity in MgB2. The electronic structure of MgB2 can be modified by atomic substitutions, which introduce extra electrons or holes into the bands. X ray Raman scattering was used to probe the interesting σ and π band hole states in pure and aluminum substituted MgB2. A method for determining the final state density of electron states from experimental x-ray Raman scattering spectra was examined and applied to the experimental data on both pure MgB2 and on Mg(0.83)Al(0.17)B2. The extracted final state density of electron states for the pure and aluminum substituted samples revealed clear substitution induced changes in the σ and π bands. The experimental work was supported by theoretical calculations of the electronic structure and x-ray Raman spectra. X-ray emission at the metal Kβ line was applied to the studies of pressure and temperature induced spin state transitions in transition metal oxides. The experimental studies were complemented by cluster multiplet calculations of the electronic structure and emission spectra. In LaCoO3 evidence for the appearance of an intermediate spin state was found and the presence of a pressure induced spin transition was confirmed. Pressure induced changes in the electronic structure of transition metal monoxides were studied experimentally and were analyzed using the cluster multiplet approach. The effects of hybridization, bandwidth and crystal field splitting in stabilizing the high pressure spin state were discussed. Emission spectroscopy at the Kβ line was also applied to FeCO3 and a pressure induced iron spin state transition was discovered.

Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population

"The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5x2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2-4.9% for the S segment, 0.2-4.8% for the M segment and 0.2-9.7% for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, 'A' and 'B'. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALA or SBMBLB.. A substantial proportion (8/40, 20%) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLB or SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains."

Genetic characterization of Indian type O FMD virus 3A region in context with host cell preference

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. For example, amino acid deletions in the porcinophilic strain (O/TAW/97) at 93-102 aa of the 153 codons long 3A protein have been recognized as the determinant of species specificity. In the present study, 18 type 0 FMDV isolates from India were adapted in different cell culture systems and the 3A sequence was analyzed. These isolates had complete 3A coding sequence (153 aa) and did not exhibit growth restriction in cells based on species of origin. The 3A region was found to be highly conserved at N-terminal half (1-75 aa) but exhibited variability or substitutions towards C-terminal region (80-153). Moreover the amino acid substitutions were more frequent in recent Indian buffalo isolates but none of the Indian isolates showed deletion in 3A protein, which may be the reason for the absence of host specificity in vitro. Further inclusive analysis of 3A region will reveal interesting facts about the variability of FMD virus 3A region in an endemic environment. (C) 2010 Elsevier B.V. All rights reserved.

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine, Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments, The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in Delta C-p and positive values of both Delta Delta H degrees and aas of folding. Enthalpic effects were dominant, and the sign of Delta Delta S is the opposite of that expected from the hydrophobic effect. Values of Delta Delta G degrees and Delta Delta H degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.

M.tuberculosis Pantothenate Kinase: Dual Substrate Specificity and Unusual Changes in Ligand Locations

Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action,observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometryof the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity. (C) 2010 Elsevier Ltd.All rights reserved.

Evidence of ferromagnetic domains in the La0.67Ca0.33Mn0.9Fe0.1O3 perovskite

The nature of the low-temperature magnetic state of polycrystalline La0.67Ca0.33Mn0.9Fe0.1O3 has been studied by magnetization, neutron diffraction, and neutron depolarization measurements. Neutron depolarization measurements indicate the existence of ferromagnetic domains with low net magnetic moments below 108 K. The substitution of Mn3+ by Fe3+ reduces the number of available hopping sites for the Mn e(g) (up) electron and suppresses the double exchange, resulting in the reduction of ferromagnetic exchange. The competition between the ferromagnetic double-exchange interactions and the coexisting antiferromagnetic superexchange interactions and its randomness due to random substitutions of Mn3+ with Fe3+ drive the system into a randomly canted ferromagnetic state at low temperatures.

Koneellisen annosjakelupalvelun aloittaminen - lääkehoidon tarkistus ja sen vaikutus potilaan lääkehoitoon

Farmaseuttisilla palveluilla tarkoitetaan apteekkien palveluita, joissa hyödynnetään apteekin farmaseuttisen henkilökunnan tietoja ja taitoja. Farmaseuttiset palvelut voidaan jakaa farmaseuttisiin perus- ja erityispalveluihin. Farmaseuttiset peruspalvelut kattavat apteekkien lakisääteiset tehtävät, kun taas farmaseuttisilla erityispalveluilla pyritään ottamaan aktiivisempi rooli asiakkaan terveyden edistämisessä. Koneellinen annosjakelupalvelu on farmaseuttinen erityispalvelu. Koneellisessa annosjakelupalvelussa lääkkeet jaetaan kerta-annospusseihin annostusajankohdan mukaan. Kun uusi asiakas aloittaa koneellinen annosjakelupalvelun, tarkistetaan asiakkaan lääkitys yhteensopimattomien ja turhien lääkkeiden osalta. Palvelun aloitushetkellä huomioidaan myös lääkevalmisteiden sopivuus koneelliseen annosjakeluun sekä tarkistetaan valmisteiden annosteluajankohdat. Koneellisessa annosjakelupalvelussa asiakkaan lääkehoidosta muodostetaan lääkityskortti, josta kokonaislääkehoito on helppo tarkistaa. Erikoistyön tavoitteena oli selvittää millainen lääkehoidon arviointi tai tarkistus koneellisen annosjakelupalvelun aloittamisen yhteydessä tehdään ja miten palvelun aloittavien asiakkaiden kokonaislääkehoitotieto saadaan selvitettyä. Lisäksi selvitettiin millaisia muutoksia lääkehoitoihin tehdään palvelun aloittamisen yhteydessä, mitkä ovat muutosten syyt sekä millainen on asiakkaan kokonaislääkehoito. Kyselylomake lähetettiin kaikkiin apteekkeihin, jotka tilasivat koneellista annosjakelua sopimusvalmistuksena Espoonlahden apteekilta syyskuussa 2010. Tutkimus suoritettiin semistrukturoidulla kirjallisella kyselyllä, joka sisälsi sekä avoimia kysymyksiä että monivalintakysymyksiä. Kyselyyn saatiin 147 vastausta ja vastausprosentiksi muodostui 45. Vastauksia kyselyyn saatiin koko Mannersuomen alueelta ja kaikkien kokoluokkien apteekeilta. Koneellisen annosjakelupalvelun aloittavat henkilöt ovat pääasiassa iäkkäitä, jotka ovat kotihoidon piirissä, asuvat hoitokodissa tai palveluasumisen yksikössä. Asiakkaiden lääkitystietojen keräämisessä hyödynnetään lääkityskorttia, mutta lääkityskortin tietoja päivitetään myös muista lähteistä. Asiakkaiden lääkityksille tehdään useimmiten lääkehoidon tarkistus moniammatillisena yhteistyönä. Lääkehoidolle tehdyt muutokset johtuvat pääasiassa lääkevaihdosta, annosjakelukoneen lääkevalikoimasta tai puolittamisen välttämisestä. Lääkehoidoissa on vain vähän yhteisvaikutuksia, jotka johtavat lääkevalmisteen käytön lopettamiseen. Lääkehoidon tarkistuksella ei ollut suurta vaikutusta asiakkaiden käyttämien lääkevalmisteiden määrään. Palvelun aloittamisen jälkeen asiakkaalla on käytössään keskimäärin 11 lääkevalmistetta, joista seitsemää jaellaan koneellisesti. Lääkeaineryhmistä eniten käytettyjä ovat hermostoon vaikuttavat sekä sydän- ja verisuonisairauksien lääkkeet, joita kumpaakin on käytössä keskimäärin kolme jokaisella uudella koneellisen annosjakelupalvelun asiakkaalla sekä palvelun aloittamista ennen että sen jälkeen.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved.

Structure-Activity Relationship of Photocytotoxic Iron(III) Complexes of Modified Dipyridophenazine Ligands

Iron(III) complexes FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido3,2-a:2',3'-c]phenazine (dppza in 4) and benzoi]dipyridro3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized, and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five. electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about 0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage Of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO center dot radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC50 value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total Cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAFT nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.

Swelling pressure of sodium montmorillonites

Determination of the swelling pressure of montmorillonitic clays is required in many situations concerned with stability problems of foundations, retaining walls, slope stability of embankments and excavations in expansive soils. Recently expansive soils such as bentonite have been used as a mixture backfill material, for example as backfill material for nuclear waste disposal systems, for which a knowledge of the swelling pressure is desirable. This is the pressure required to keep the clay-water system at the required void ratio when it is allowed to absorb water or electrolyte. If the pressure is less than the swelling pressure, volume expansion occurs; if the pressure is more than the swelling pressure, volume compression occurs. Because of isomorphous substitutions in the crystal lattice, in general the clay particles carry negative charges at the surfaces of the platelets. Exchangeable cations in the clay media are attracted to these negative charges, but this attraction is opposed by the tendency of ions to be distributed. As a result, an electric diffuse double layer is formed (Gouy, 1910).

Improvement of protein structure comparison using a structural alphabet

The three dimensional structure of a protein provides major insights into its function. Protein structure comparison has implications in functional and evolutionary studies. A structural alphabet (SA) is a library of local protein structure prototypes that can abstract every part of protein main chain conformation. Protein Blocks (PBS) is a widely used SA, composed of 16 prototypes, each representing a pentapeptide backbone conformation defined in terms of dihedral angles. Through this description, the 3D structural information can be translated into a 1D sequence of PBs. In a previous study, we have used this approach to compare protein structures encoded in terms of PBs. A classical sequence alignment procedure based on dynamic programming was used, with a dedicated PB Substitution Matrix (SM). PB-based pairwise structural alignment method gave an excellent performance, when compared to other established methods for mining. In this study, we have (i) refined the SMs and (ii) improved the Protein Block Alignment methodology (named as iPBA). The SM was normalized in regards to sequence and structural similarity. Alignment of protein structures often involves similar structural regions separated by dissimilar stretches. A dynamic programming algorithm that weighs these local similar stretches has been designed. Amino acid substitutions scores were also coupled linearly with the PB substitutions. iPBA improves (i) the mining efficiency rate by 6.8% and (ii) more than 82% of the alignments have a better quality. A higher efficiency in aligning multi-domain proteins could be also demonstrated. The quality of alignment is better than DALI and MUSTANG in 81.3% of the cases. Thus our study has resulted in an impressive improvement in the quality of protein structural alignment. (C) 2011 Elsevier Masson SAS. All rights reserved.

Quaternary association in beta-prism I fold plant lectins: Insights from X-ray crystallography, modelling and molecular dynamics

Dimeric banana lectin and calsepa, tetrameric artocarpin and octameric heltuba are mannose-specific beta-prism I fold lectins of nearly the same tertiary structure. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions that undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Energy minimization and simulations were also carried out on models using all possible oligomers by employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also led to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding.

NMR Structure Implications of Enhanced Efficacy of Obestatin Fragment Analogs

Obestatin is a more recently discovered hormone that is encoded by the ghrelin gene and produced in the stomach and gut. We report NMR analysis on synthetic Obestatin (OB23), a 23 residue peptide, along with three overlapping fragments of the same in methanol solvent as a first step towards structure activity relationship. Selective substitutions on the promising N-terminal and middle fragments of obestatin have been carried out in order to improve the efficacy and potency. In the N-terminal fragment two peptides were obtained by the replacement of Gly (8) with a-aminoisobutyric acid (Aib, U) and Phe (F5) with Cyclohexylalanine (Cha). In case of the middle fragment both Gly (3) and Gly (8) were replaced with Aib residues. The rationale being, these unusual amino acids could provide protection from immediate degradation and aid structure stabilization. Our previous studies showed that the N-terminal and the middle fragment were unstructured and hence this substitution would directly evaluate the effect of structure on the activity of these fragment analogs. Detailed NMR analysis clearly demonstrates formation of helical secondary structure in all the peptide analogues and provides justification for relative activities reported by our group previously (Nagaraj et al. 2009).