scholarly journals Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis

2012 ◽  
Vol 444 (3) ◽  
pp. 529-535 ◽  
Author(s):  
Zhaohua Zheng ◽  
Syazwani I. Amran ◽  
Jiuxiang Zhu ◽  
Oleg Schmidt-Kittler ◽  
Kenneth W. Kinzler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
In Vitro Mutagenesis ◽  
Selective Inhibitors ◽  
New Class ◽  
Phosphoinositide 3 Kinase ◽  
Conserved Amino Acids

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser773, A-66S bound the region 1 non-conserved amino acid p110α Gln859, and J-32 binding had an indirect interaction with Lys776 and Ile771. The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.

scholarly journals Reduced Susceptibility of Human Immunodeficiency Virus Type 1 (HIV-1) from Patients with Primary HIV Infection to Nonnucleoside Reverse Transcriptase Inhibitors Is Associated with Variation at Novel Amino Acid Sites

2000 ◽  
Vol 74 (22) ◽  
pp. 10269-10273 ◽  
Author(s):  
Andrew J. Leigh Brown ◽  
Heather M. Precious ◽  
Jeannette M. Whitcomb ◽  
Joseph K. Wong ◽  
Marlynne Quigg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Hiv Infection ◽  
Reverse Transcriptase ◽  
Acid Sites ◽  
Antiretroviral Drugs ◽  
In Vitro Mutagenesis ◽  
Immunodeficiency Virus

ABSTRACT Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

scholarly journals In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

1994 ◽  
Vol 14 (4) ◽  
pp. 2722-2730 ◽  
Author(s):  
J Yang ◽  
J M Kramer
Keyword(s):  
Amino Acids ◽  
Caenorhabditis Elegans ◽  
Cleavage Site ◽  
Normal Function ◽  
In Vitro Mutagenesis ◽  
Disulfide Bonding ◽  
Collagen Genes ◽  
Cuticle Collagens ◽  
Conserved Amino Acids

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly

1994 ◽  
Vol 14 (4) ◽  
pp. 2722-2730
Author(s):  
J Yang ◽  
J M Kramer
Keyword(s):  
Amino Acids ◽  
Caenorhabditis Elegans ◽  
Cleavage Site ◽  
Normal Function ◽  
In Vitro Mutagenesis ◽  
Disulfide Bonding ◽  
Collagen Genes ◽  
Cuticle Collagens ◽  
Conserved Amino Acids

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

scholarly journals FRUCTOSE-AMINO ACIDS IN LIVER: STIMULI OF AMINO ACID INCORPORATION IN VITRO

1955 ◽  
Vol 215 (1) ◽  
pp. 111-124 ◽  
Author(s):  
Henry Borsook ◽  
Adolph Abrams ◽  
Peter H. Lowy
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

scholarly journals Transdermal Delivery Systems for Ibuprofen and Ibuprofen Modified with Amino Acids Alkyl Esters Based on Bacterial Cellulose

2021 ◽  
Vol 22 (12) ◽  
pp. 6252
Author(s):  
Paula Ossowicz-Rupniewska ◽  
Rafał Rakoczy ◽  
Anna Nowak ◽  
Maciej Konopacki ◽  
Joanna Klebeko ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacterial Cellulose ◽  
Acid Ester ◽  
Active Pharmaceutical Ingredient ◽  
Amino Acid Ester ◽  
Isopropyl Ester ◽  
Isopropyl Esters ◽  
Transdermal Application

The potential of bacterial cellulose as a carrier for the transport of ibuprofen (a typical example of non-steroidal anti-inflammatory drugs) through the skin was investigated. Ibuprofen and its amino acid ester salts-loaded BC membranes were prepared through a simple methodology and characterized in terms of structure and morphology. Two salts of amino acid isopropyl esters were used in the research, namely L-valine isopropyl ester ibuprofenate ([ValOiPr][IBU]) and L-leucine isopropyl ester ibuprofenate ([LeuOiPr][IBU]). [LeuOiPr][IBU] is a new compound; therefore, it has been fully characterized and its identity confirmed. For all membranes obtained the surface morphology, tensile mechanical properties, active compound dissolution assays, and permeation and skin accumulation studies of API (active pharmaceutical ingredient) were determined. The obtained membranes were very homogeneous. In vitro diffusion studies with Franz cells were conducted using pig epidermal membranes, and showed that the incorporation of ibuprofen in BC membranes provided lower permeation rates to those obtained with amino acids ester salts of ibuprofen. This release profile together with the ease of application and the simple preparation and assembly of the drug-loaded membranes indicates the enormous potentialities of using BC membranes for transdermal application of ibuprofen in the form of amino acid ester salts.

scholarly journals Phylogenetic analysis and predicted functional effect of protein mutations of E6 and E7 HPV16 strains isolated in Indonesia

2015 ◽  
Vol 24 (4) ◽  
pp. 197-205
Author(s):  
Dwi Wulandari ◽  
Lisnawati Rachmadi ◽  
Tjahjani M. Sudiro
Keyword(s):  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Asian American ◽  
Protein Function ◽  
Single Amino Acid ◽  
Hpv16 E6 ◽  
E6 And E7 ◽  
Neutral Mutations

Background: E6 and E7 are oncoproteins of HPV16. Natural amino acid variation in HPV16 E6 can alter its carcinogenic potential. The aim of this study was to analyze phylogenetically E6 and E7 genes and proteins of HPV16 from Indonesia and predict the effects of single amino acid substitution on protein function. This analysis could be used to reduce time, effort, and research cost as initial screening in selection of protein or isolates to be tested in vitro or in vivo.Methods: In this study, E6 and E7 gene sequences were obtained from 12 samples of  Indonesian isolates, which  were compared with HPV16R (prototype) and 6 standard isolates in the category of European (E), Asian (As), Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and North American (NA) branch from Genbank. Bioedit v.7.0.0 was used to analyze the composition and substitution of single amino acids. Phylogenetic analysis of E6 and E7 genes and proteins was performed using Clustal X (1.81) and NJPLOT softwares. Effects of single amino acid substitutions on protein function of E6 and E7 were analysed by SNAP.Results: Java variants and isolate ui66* belonged to European branch, while the others belonged to Asian and African branches. Twelve changes of amino acids were found in E6 and one in E7 proteins. SNAP analysis showed two non neutral mutations, i.e. R10I and C63G in E6 proteins. R10I mutations were found in Af-2 genotype (AF472509) and Indonesian isolates (Af2*), while C63G mutation was found only in Af2*.Conclusion: E6 proteins of HPV16 variants were more variable than E7. SNAP analysis showed that only E6 protein of African-2 branch had functional differences compared to HPV16R.

scholarly journals Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1971 ◽  
Vol 121 (5) ◽  
pp. 817-827 ◽  
Author(s):  
R. C. Hider ◽  
E. B. Fern ◽  
D. R. London
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Longus Muscle ◽  
Kinetics Of

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

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