Methods of detecting and measuring glutamine and analogues thereof, and methods related thereto

Patent metadata

Field	Value
Publication number	US-2016299151-A1
Application number	US-201415101883-A
Country	US
Kind code	A1
Filing date	Dec 5, 2014
Priority date	Dec 5, 2013
Publication date	Oct 13, 2016
Grant date	—

Abstract

Method for the detection of glutamine and its analogues are provided by the present disclosure. Also provided are methods for measuring the levels of glutamine and its analogues, including diagnostic methods. Further provided are methods that utilise glutamine analogues for the synthesis of coloured pigments and other useful agents. The methods comprise the use of a nonribosomal peptide synthetase (NRPS) under conditions to produce an indigoidine or indigoidine-related pigment.

First claim

1 - 43 . (canceled)

44 . A method of measuring the concentration of L-glutamine or a glutamine analogue in a sample, comprising:
incubating the sample with an activated pigment-producing NRPS under conditions to produce indigoidine pigment or an indigoidine-related pigment; and quantifying the amount of the indigoidine pigment or the an indigoidine-related pigment produced, thereby measuring the concentration of the L-glutamine or the glutamine analogue, respectively, in the sample.

45 . The method of claim 44 , wherein the pigment-producing NRPS is BpsA or a functional variant thereof, wherein the BpsA or the functional variant thereof is activated prior to isolation of the BpsA or the functional variant thereof, or the BpsA or the functional variant thereof is activated following isolation of the BpsA or the functional variant thereof.

46 . The method of claim 45 , wherein the sample is a biological sample selected from the group consisting of a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a lymph fluid sample, a eukaryotic cell culture sample, and a bacterial cell culture sample.

47 . The method of claim 45 , wherein the glutamine analogue is selected from the group consisting of glycyl-glutamine, N-trifluoroacetyl-L-glutamine, alanyl-glutamine, indoleacetyl glutamine, N-acetylglutamine, phenylacetyl-L-glutamine, and glutamine-terminating peptides or polypeptides.

48 . A method of diagnosing a condition or disorder associated with altered levels of L-glutamine or a glutamine analogue in a test subject, comprising:
providing a biological sample from the test subject; incubating the biological sample with an activated pigment-producing NRPS under conditions to produce indigoidine pigment or an indigoidine-related pigment; measuring the amount of the indigoidine pigment or the indigoidine-related pigment produced by incubation with the biological sample;
wherein an altered amount of the indigoidine pigment or the indigoidine-related pigment relative to a control or standard amount indicates altered levels of the L-glutamine or the glutamine analogue, respectively.

49 . The method of claim 48 , wherein the pigment-producing NRPS is BpsA or a functional variant thereof, wherein the BpsA or the functional variant thereof is activated prior to isolation of the BpsA or the functional variant thereof, or the BpsA or the functional variant thereof is activated following isolation of the BpsA or the functional variant thereof.

50 . The method of claim 49 , wherein the biological sample is a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a lymph fluid sample.

51 . The method of claim 49 , wherein the condition or disorder associated with altered levels of L-glutamine is selected from the group consisting of overtraining syndrome, urea cycle disorders, Alzheimer's disease and other neurodegenerative disorders, cancers, and aminoaciduria and related conditions.

52 . The method of claim 49 , wherein the glutamine analogue is selected from the group consisting of indoleacetyl glutamine, N-acetylglutamine, and phenylacetyl-L-glutamine.

53 . The method of claim 49 , wherein the condition or disorder associated with altered levels of the glutamine analogue is selected from the group consisting of Hartnup disease, autosomal dominant polycystic kidney disease, renal tubal injury, aminoacylase deficiency, and uremia.

54 . A method of identifying a bacterial cell having altered L-glutamine or glutamine analogue production, comprising:
providing a bacterial cell that synthesises L-glutamine or the glutamine analogue; incubating the bacterial cell with an activated pigment-producing NRPS under conditions to produce indigoidine pigment or indigoidine-related pigment; measuring the amount of the indigoidine pigment or the indigoidine-related pigment produced by incubation with the bacterial cell;
wherein an alteration in the amount of the indigoidine pigment or the indigoidine-related pigment relative to a control or standard amount indicates altered levels of the L-glutamine or the glutamine analogue production, respectively.

55 . The method of claim 54 , wherein the pigment-producing NRPS is BpsA or a functional variant thereof, wherein the BpsA or the functional variant thereof is activated prior to isolation of the BpsA or the functional variant thereof, or the BpsA or the functional variant thereof is activated following isolation of the BpsA or the functional variant thereof.

56 . The method of claim 55 , wherein the bacterial cell is selected from the group consisting of a mutant cell, a variant cell, and a cell produced by targeted or random mutagenesis.

57 . The method of claim 55 , wherein the glutamine analogue is selected from the group consisting of a glutamine-terminating peptide, a glutamine-terminating polypeptide, and a glutamine-terminating dipeptide.

58 . The method of claim 57 , wherein the glutamine analogue is alanyl-glutamine.

59 . A method of producing an indigoidine-related pigment molecule, comprising: incubating a glutamine analogue with an activated pigment-producing NRPS under conditions to produce the indigoidine-related pigment molecule.

60 . The method of claim 59 , wherein the pigment-producing NRPS is BpsA or a functional variant thereof, wherein the BpsA or the functional variant thereof is activated prior to isolation of the BpsA or the functional variant thereof, or the BpsA or the functional variant thereof is activated following isolation of the BpsA or the functional variant thereof.

61 . The method of claim 60 , wherein the glutamine analogue is selected from the group consisting of glycyl-glutamine, N-trifluoroacetyl-L-glutamine, alanyl-glutamine, indoleacetyl glutamine, N-acetylglutamine, phenylacetyl-L-glutamine, and glutamine-terminating peptides or polypeptides.

Assignees

• Victoria Link Ltd

Inventors

• Ackerley David Francis
• Brown Alistair Sinclair
• Robins Katherine Jane

Classifications

• C12Q1/48 Primary

  involving transferase · CPC title

• G01N2333/91171

  with definite EC number (2.5.1.-) · CPC title

• G01N33/6812 Primary

  Assays for specific amino acids · CPC title

• C12P17/165

  Heterorings having nitrogen atoms as the only ring heteroatoms · CPC title