Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
J Med Chem ; 66(7): 5289-5304, 2023 04 13.
Article in English | MEDLINE | ID: mdl-36920850

ABSTRACT

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be installed in proteins by genetic encoding. In addition, we describe a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (Mpro) as a model system with three different ligands, we show that the 1H and 19F nuclei of the solvent-exposed trimethylsilyl and CF3 groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum. Their response to active-site ligands differed significantly when positioned near rather than far from the active site. Conversely, the NMR probes failed to confirm the previously reported binding site of the ligand pelitinib, which was found to enhance the activity of Mpro by promoting the formation of the enzymatically active dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive path for site-specific NMR analysis of ligand binding to large proteins of limited stability and at low concentrations.


Subject(s)
Amino Acids , COVID-19 , Humans , Amino Acids/chemistry , Binding Sites , Ligands , Lysine , Magnetic Resonance Spectroscopy , Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
2.
Chem Sci ; 13(13): 3826-3836, 2022 Mar 30.
Article in English | MEDLINE | ID: mdl-35432913

ABSTRACT

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

3.
Magn Reson (Gott) ; 3(2): 169-182, 2022.
Article in English | MEDLINE | ID: mdl-37904871

ABSTRACT

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with α-cysteine, penicillamine or ß-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with Gd3+ ions.

4.
Chem Commun (Camb) ; 58(5): 701-704, 2022 Jan 13.
Article in English | MEDLINE | ID: mdl-34927186

ABSTRACT

Arsenical probes enable structural studies of proteins. We report the first organoarsenic probes for nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to study proteins in solutions. These probes can be attached to irregular loop regions. A lanthanide-binding tag induces sizable pseudocontact shifts in protein NMR spectra of a magnitude never observed for small paramagnetic probes before.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular
5.
Bioorg Med Chem Lett ; 50: 128333, 2021 10 15.
Article in English | MEDLINE | ID: mdl-34418570

ABSTRACT

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.


Subject(s)
COVID-19/pathology , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , COVID-19/virology , Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Cysteine/metabolism , Humans , Lactams/chemistry , Lactams/metabolism , Leucine/chemistry , Leucine/metabolism , Nitriles/chemistry , Nitriles/metabolism , Peptides/chemistry , Peptides/metabolism , Peptidomimetics/chemistry , Peptidomimetics/metabolism , Proline/chemistry , Proline/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/isolation & purification , Structure-Activity Relationship , Substrate Specificity
6.
Chembiochem ; 22(8): 1480-1486, 2021 04 16.
Article in English | MEDLINE | ID: mdl-33319405

ABSTRACT

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.


Subject(s)
Maltose-Binding Proteins/analysis , Picolinic Acids/chemistry , Selenoproteins/chemistry , Molecular Structure , Selenoproteins/chemical synthesis
7.
Nat Commun ; 11(1): 5945, 2020 11 23.
Article in English | MEDLINE | ID: mdl-33230119

ABSTRACT

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.


Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Evolution, Molecular , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Catalysis , Catalytic Domain , Enzymes/genetics , Models, Molecular , Mutation , Phylogeny , Prephenate Dehydratase/chemistry , Prephenate Dehydratase/genetics , Prephenate Dehydratase/metabolism , Protein Conformation , Structure-Activity Relationship
8.
J Am Chem Soc ; 142(41): 17277-17281, 2020 10 14.
Article in English | MEDLINE | ID: mdl-32975937

ABSTRACT

SF5Phe, para-pentafluorosulfanyl phenylalanine, is an unnatural amino acid with extreme physicochemical properties, which is stable in physiological conditions. Here we present newly developed aminoacyl-tRNA synthetases that enable genetic encoding of SF5Phe for site-specific incorporation into proteins in high yields. Owing to the SF5 moiety's dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins. The potential of SF5Phe in protein research is illustrated by (i) increasing the binding affinity of a consensus pentapeptide motif toward the ß subunit of Escherichia coli DNA polymerase III holoenzyme by mutation of a phenylalanine to a SF5Phe residue, (ii) site-specifically adhering ß-cyclodextrin to the surface of ubiquitin, and (iii) selective detection of 19F-19F nuclear Overhauser effects in the Escherichia coli peptidyl-prolyl cis/trans-isomerase B following mutation of two phenylalanine residues in the core of the protein to SF5Phe. With increasing use of the SF5 moiety in pharmaceutical chemistry, this general method of functionalizing proteins with SF5 groups opens unique opportunities for structural biology and in vivo studies.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , DNA Polymerase III/metabolism , Fluorocarbons/chemistry , Phenylalanine/chemistry , Amino Acyl-tRNA Synthetases/genetics , Cyclodextrins/chemistry , DNA Polymerase III/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Fluorine/chemistry , Hydrophobic and Hydrophilic Interactions , Isomerases/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Surface Properties , Ubiquitin/chemistry
9.
ACS Med Chem Lett ; 10(2): 168-174, 2019 Feb 14.
Article in English | MEDLINE | ID: mdl-30783498

ABSTRACT

The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.

10.
Phys Chem Chem Phys ; 20(36): 23535-23545, 2018 Sep 19.
Article in English | MEDLINE | ID: mdl-30183028

ABSTRACT

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.


Subject(s)
Bacterial Proteins/analysis , Electrons , Gadolinium/chemistry , Spin Labels , Viral Nonstructural Proteins/analysis , Electron Spin Resonance Spectroscopy , RNA Helicases/analysis , Serine Endopeptidases/analysis
11.
Chem Commun (Camb) ; 53(79): 10894-10897, 2017 Oct 03.
Article in English | MEDLINE | ID: mdl-28890978

ABSTRACT

Site-selective chemical protein modification is achieved by self-assembly of a specific di-cysteine motif, trivalent pnictogens (As, Sb or Bi) and an aromatic mercaptomethyl-based probe. The strategy is demonstrated with a quaternary complex involving Zika virus protease and a lanthanide ion, enabling paramagnetic nuclear magnetic resonance spectroscopy and luminescence measurements.


Subject(s)
Antimony/chemistry , Arsenic/chemistry , Bismuth/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Hydrolases/analysis , Zika Virus/enzymology , Cysteine/chemistry , Humans , Lanthanoid Series Elements/chemistry , Luminescent Measurements/methods , Models, Molecular , Sulfhydryl Compounds/chemistry , Zika Virus Infection/virology
12.
Antiviral Res ; 142: 141-147, 2017 06.
Article in English | MEDLINE | ID: mdl-28336347

ABSTRACT

The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal ß-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.


Subject(s)
Viral Nonstructural Proteins/chemistry , Zika Virus/enzymology , Binding Sites , Boronic Acids/antagonists & inhibitors , Dipeptides/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Models, Molecular , Protease Inhibitors/chemistry , Protein Conformation , RNA Helicases/chemistry , RNA Helicases/drug effects , Serine Endopeptidases/chemistry , Serine Endopeptidases/drug effects , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...