Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14alpha-demethylase (CYP51)

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14alpha-demethylase (CYP51). Structure-activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine binds within the heme binding pocket in an analogy with the azoles.     

Towards the first inhibitors of trihydroxynaphthalene reductase from Curvularia lunata: synthesis of artificial substrate, homology modelling and initial screening

Trihydroxynaphthalene reductase (3HNR) is an essential enzyme in the biosynthesis of fungal melanin and it represents an emerging target for the development of new fungicides and antimicotics. To promote the discovery of new inhibitors, an improved chemical synthesis of the artificial substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) was developed. A series of compounds were screened on 3HNR from Curvularia lunata, a known plant pathogen and an opportunistic human pathogen, and several structurally diverse hits were obtained. Homology modelling of 3HNR from C. lunata can explain their binding modes and will enable further structure-based design of new and improved inhibitors.     

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.     

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.     

Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism

The hydrolysis of substrates by cholinesterases does not follow the Michaelis–Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.     

Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus)

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.     

Transient Kinetic Approach to the Study of Acetylcholinesterase Reversible Inhibition by Eseroline

The kinetic rate constants for interaction of (?)-eseroline-(3aS-cis)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo-[2,3-b]indol-5-ol with electric eel acetylcholinesterase (EC 3.1.1.7, acetylcholine acetylhydrolase) were measured at a low substrate concentration according to a transient kinetic approach by using a rapid experimental technique. The measurements were carried out on a stopped-flow apparatus where pre-incubated samples of enzyme with various inhibitor concentrations were diluted with a buffer solution containing the substrate. The experimental data in the form of sigmoid-shaped progress curves were analysed by applying an explicit progress curve equation that described the time dependence of product released during the reaction. The kinetic parameters were evaluated by non-linear regression treatment and the values of the corresponding constants showed approximately the equal affinities of eseroline and eserine (cf. Stojan, J. and Zorko, M. (1997) Biochim. Biophys. Acta, 1337, 75-84.) for binding into the active centre of the enzyme. On the other hand, the kinetic rates for association and dissociation of eseroline were two grades of magnitude higher than those of eserine. The explanation appears to be a substantionally impaired gliding of eserine into the active site gorge by the great mobility of the carbamoyl tail as well as by its numerous possible interactions with the residues lining the gorge. Additionally, a study of the dependence of the transition phase information on the inhibitor concentration was carried out using our experimental data.     

Inhibition of Major Histocompatibility Complex Class I Antigen Presentation in Pig and Primate Cells by Herpes Simplex Virus Type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8⁺ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8⁺ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species—mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans—and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8⁺-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8⁺ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47⁻ mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.Herpes simplex virus (HSV) infection of human fibroblasts leads to inhibition of antigen presentation to CD8⁺ T cells so that the virus-infected fibroblasts are not lysed by cytotoxic T lymphocytes (CTL) (10, 12, 14). The principal reason for this resistance to CTL appears to be the expression of an HSV immediate-early protein, ICP47, which causes major histocompatibility complex (MHC) class I proteins to accumulate in infected cells in a peptide-empty form (19). ICP47 was subsequently shown to inhibit the transporter associated with antigen presentation (TAP), which functions to translocate antigenic peptides across the membrane of the endoplasmic reticulum (ER) (3, 8), and without antigenic peptides, MHC class I proteins accumulate in the ER. More recent results demonstrated that ICP47 blocks peptide binding to TAP by binding with high affinity to a domain of TAP that includes the peptide binding site (1, 15).Although HSV type 1 (HSV-1) ICP47 (ICP47-1) effectively blocks TAP in human fibroblasts, it inhibits TAP little, if at all, in a variety of mouse cells unless applied in high concentrations (1, 3, 15, 19). Similarly, HSV-2 ICP47 (ICP47-2), which has only 42% amino acid identity with ICP47-1 (4), effectively blocks human TAP but inhibits murine TAP less effectively (16). Inhibition of murine TAP with these proteins occurs at ICP47-1 and ICP47-2 concentrations 50- to 100-fold higher than those required to inhibit human TAP. ICP47-1 and ICP47-2 bind poorly to mouse TAP (15, 16), which explains their inability to block peptide transport and antigen presentation in mouse cells.We were interested in extending the study of the species specificity of ICP47 for several reasons. Firstly, we wanted to find an animal model with which to assess the effects of ICP47 in vivo, both to assess its role in virus-host interactions and to provide a model for the use of ICP47 in autoimmunity, in transplantation, and in gene therapy vectors. Secondly, we wanted to determine whether ICP47 was functional in the species currently widely used for HSV pathogenesis and vaccine studies—mice, rabbits, and guinea pigs. Thirdly, we were interested in the mechanism of the extraordinary virulence of HSV in owl monkeys (aotus), speculating that the TAP in this New World primate might be exceptionally susceptible to ICP47.In order to assess the effects of ICP47 on the TAPs of various species, cells were permeabilized, recombinant ICP47-1 and ICP47-2 were introduced into the cells, and assays of TAP activity were performed. To examine the effects of ICP47 on antigen presentation and recognition by CD8⁺ T cells, fibroblasts were infected with recombinant HSV-1 that expresses mouse class I proteins and not ICP47, and lysis of the cells by mouse anti-HSV CTL was tested. We found that ICP47-1 and ICP47-2 did not block TAP in mouse, rat, guinea pig, or rabbit skin fibroblasts but effectively inhibited TAP and antigen presentation in pig, dog, cow, and monkey fibroblasts. Therefore, pigs, dogs, and monkeys can be used to study the in vivo effects of ICP47, though for several reasons, the use of pigs might be a practical starting point.     

New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains

Two yeast isolates, a wine-making yeast first identified as a Mel⁺ strain (ex. S. uvarum) and a cider-making yeast, were characterized for their nuclear and mitochondrial genomes. Electrophoretic karyotyping analyses, restriction fragment length polymorphism maps of PCR-amplified MET2 gene fragments, and the sequence analysis of a part of the two MET2 gene alleles found support the notion that these two strains constitute hybrids between Saccharomyces cerevisiae and Saccharomyces bayanus. The two hybrid strains had completely different restriction patterns of mitochondrial DNA as well as different sequences of the OLI1 gene. The sequence of the OLI1 gene from the wine hybrid strain appeared to be the same as that of the S. cerevisiae gene, whereas the OLI1 gene of the cider hybrid strain is equally divergent from both putative parents, S. bayanus and S. cerevisiae. Some fermentative properties were also examined, and one phenotype was found to reflect the hybrid nature of these two strains. The origin and nature of such hybridization events are discussed.     

Discovery of (phenylureido)piperidinyl benzamides as prospective inhibitors of bacterial autolysin E from Staphylococcus aureus

Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target.