Ecole Polytechnique Federale De Lausanne

Max-planck-gesellschaft Zur F&#xf6;rderung Der Wissenschaften E. V.

New England Biolabs, Inc.

Ecole Polytechnique Féderale De Lausanne (epfl)

Kai Peter Johnsson

Maik Kindermann

Alexandre Juillerat

Antje Keppler

Keitaro Umezawa

Lukinavicius Grazvydas

Arnaud Gautier

Florent Beaufils

Nathalie George

Hughes Jaccard

John Michael McCall

Eddine Saiah

Heather Tye

Hairuo Peng

Mark Joseph Tebbe

Holly Atton

Mirco Meniconi

Mark Peter Ridgill

Adrian Kotey

Mark Anthony Kerry

Christian Heinis

Thomas Gronemeyer

Alberto Schena

Christoph Bieri

Rudolf Griss

Qiuliyang Yu

Olivier Sallin

Luc Reymond

Adrian Kotei Kotey

Katarzyna Irena Gorska

Susanne Kindermann (née Gendreizig)

Craig Avery

India Christina Sielaff

Steven Mark Bromidge

Susanne Gendreizig

Jan Barnikow

Nathaniel J Monck

Christopher Chidley

Mark Kerry

The invention relates to the in vitro and in cellulo detection of the cofactors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Provided is a sensor molecule for fluorescence- or luminescence-based detection of a nicotinamide adenine dinucleotide analyte, in particular for detecting the concentrations of NAD, NADPand/or the ratios of the concentrations of NAD/NADH and NADP/NADPH, the sensor comprising (i) a binding protein (BP) for the nicotinamide adenine dinucleotide analyte, the BP being derived from sepiapterin reductase (SPR; EC 1.1.1.153) (ii) an SPR ligand (SPR-L) capable of intramolecular binding to said BP in the presence of the oxidized form of said analyte; and (iii) at least one fluorophore.

Sensors, methods and kits for detecting nicotinamide adenine dinucleotides

Inhibitors of sepiapterin reductase and uses of sepiapterin reductase inhibitors in analgesia, treatment of acute and chronic pain, anti-inflammation, and immune cell regulation are disclosed.

Sepiapterin reductase inhibitors

The invention relates to the field of in vitro detection methods using luminescence. Provided is a sensor molecule for detecting an analyte of interest in a sample using bioluminescence resonance energy transfer (BRET), the sensor molecule comprising a proteinaceous moiety tethered to a synthetic regulatory molecule. Also provided is an analytical device comprising a sensor and methods using the sensor molecule.

Means and methods for bioluminescence resonance energy transfer (BRET) analysis in a biological sample

The invention pertains to a near-infrared fluorescent dye that is cell permeable and can be attached to selected proteins in living cells. The dye has the general formula or its corresponding spirolactone wherein The dye (i) absorbs and emits light at wavelengths above 600 nm; (ii) possesses high photostability; (iii) has high extinction coefficients and high quantum yields; (iv) can be derivatized with different molecules; and (v) is membrane-permeable and shows minimal background binding to biomolecules and biomolecular structures.

Cell permeable, fluorescent dye

The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O-alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) wherein Ris an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH&#x2014;; Ris a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O-alkylguanine-DNA alkyltransferase (AGT)-benzylguanines.

Labelling of fusion proteins with synthetic probes

A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such labels are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilization of the fusion protein.

Methods for protein labeling based on acyl carrier protein

A method of using O-alkylguanine-DNA alkyltransferase (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.

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The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O-alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) wherein Ris an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH&#x2014;; Ris a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O-alkylguanine-DNA alkyltransferase (AGT)-benzylguanines. 

The invention relates to substrates for O-alkylguanine-DNA alkyltransferases (AGT) of formula R-A-X&#x2014;CH&#x2014;R&#x2014;R-L, wherein A is a group recognized by AGT as a substrate, X is oxygen or sulfur, Ris a group &#x2014;R-Lor a group R, Rand Rare, independently of each other, a linker, Ris an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH, Ris arylmethyl or heteroarylmethyl or an optionally substituted cycloalkyl, cycloalkenyl or heterocyclyl group, Lis a label, a plurality of same or different labels, a bond connecting Rto A forming a cyclic substrate, or a further group &#x2014;R&#x2014;CH&#x2014;X-A-R, and Lis a label or a plurality of same or different labels. The invention further relates to methods of transferring a label from these substrates to O-alkylguanine-DNA alkyltransferases (AGT) and AGT fusion proteins.

A method using O-alkylguanine-DNA alkyltransferases (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.

The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction; (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted to the nucleus; (c) improved expression yield as soluble protein and improved stability in various hosts; (d) improved stability under oxidising conditions; (e) improved stability within cells after reaction with a substrate; (f) improved stability outside cells before and after reaction with a substrate; (g) improved in vitro solubility; (h) improved reactivity against O-alkylguanine substrates; (1) reduced reactivity against DNA-based substrates; and (j) reduced reactivity against N-substituted O-alkylguanine substrates. Such AGT mutants with the mentioned improved properties are mutants wherein between 1 and 25 amino acids of the wild type human AGT are substituted by other amino acids, and optionally 1 to 5 amino acids out of the continuous chain at one, two or three positions are deleted or added and/or 1 to 4 amino acids at the N-terminus or 1 to 40 amino acids at the C-terminus are deleted. The invention further relates to a method for detecting and/or manipulating a protein of interest wherein the protein of interest is incorporated into a fusion protein with the AGT mutants of the invention. Another object of the invention are AGT fusion proteins comprising such AGT mutants and the protein of interest.

The invention relates to compounds of formula 1 wherein R-Ris a guanine derivative; X is oxygen or sulfur; Ris a heteroaromatic, unsaturated heterocyclyl or an alkynyl group with the double or triple bond connected to CH; Ris a linker; and L is a label, a plurality of same or different labels, a bond connecting Rto Rforming a cyclic substrate, or a further group &#x2014;R&#x2014;CH&#x2014;X&#x2014;R-R. The invention relates also to methods of manufacture of such novel AGT substrates, to intermediates useful in the synthesis of such AGT substrates, and to a method of transferring a label from such AGT substrates to O-alkylguanine-DNA alkyltransferase (AGT) fusion proteins comprising proteins of interest.

Substrates for O6-alkylguanina-DNA alkyltransferase

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Lausanne, Switzerland

Kai Peter Johnsson