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1.
Angew Chem Int Ed Engl ; 61(46): e202205858, 2022 11 14.
Article in English | MEDLINE | ID: mdl-36115062

ABSTRACT

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.


Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2 , Humans , Proteome , Ligands , Drug Design
2.
FEBS Lett ; 596(12): 1503-1515, 2022 06.
Article in English | MEDLINE | ID: mdl-35397176

ABSTRACT

The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2 O2 /NaHCO3 , mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.


Subject(s)
Bacterial Proteins , Mycobacterium tuberculosis , Protein Tyrosine Phosphatases , Virulence Factors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cysteine/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Oxidation-Reduction , Prospective Studies , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatases/pharmacology , Tyrosine/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism
3.
Angew Chem Int Ed Engl ; 60(35): 19191-19200, 2021 08 23.
Article in English | MEDLINE | ID: mdl-34161644

ABSTRACT

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.


Subject(s)
Genome , RNA, Viral/metabolism , SARS-CoV-2/genetics , Small Molecule Libraries/metabolism , Drug Evaluation, Preclinical , Ligands , Molecular Structure , Nucleic Acid Conformation , Proton Magnetic Resonance Spectroscopy , RNA, Viral/chemistry , Small Molecule Libraries/chemistry
4.
Chembiochem ; 22(2): 423-433, 2021 01 15.
Article in English | MEDLINE | ID: mdl-32794266

ABSTRACT

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.


Subject(s)
DNA/metabolism , Nuclear Magnetic Resonance, Biomolecular , Proteins/metabolism , RNA/metabolism , DNA/chemistry , Fluorine/chemistry , Molecular Weight , Proteins/chemistry , RNA/chemistry
5.
J Biol Chem ; 293(30): 11823-11836, 2018 07 27.
Article in English | MEDLINE | ID: mdl-29884774

ABSTRACT

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.


Subject(s)
Bacterial Proteins/chemistry , Mycobacterium tuberculosis/enzymology , Protein-Tyrosine Kinases/chemistry , Bacterial Proteins/metabolism , Humans , Hydrogen Bonding , Models, Molecular , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/metabolism , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Conformation , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Tuberculosis/microbiology
6.
FEBS Lett ; 592(7): 1233-1245, 2018 04.
Article in English | MEDLINE | ID: mdl-29494752

ABSTRACT

Although intrinsically disordered proteins or protein domains (IDPs or IDD) are less abundant in bacteria than in eukaryotes, their presence in pathogenic bacterial proteins is important for protein-protein interactions. The protein tyrosine kinase A (PtkA) from Mycobacterium tuberculosis possesses an 80-residue disordered region (IDDPtkA ) of unknown function, located N-terminally to the well-folded kinase core domain. Here, we characterize the conformation of IDDPtkA under varying biophysical conditions and phosphorylation using NMR-spectroscopy. Our results confirm that the N-terminal domain of PtkA exists as an IDD at physiological pH. Furthermore, phosphorylation of IDDPtkA increases the activity of PtkA. Our findings will complement future approaches in understanding molecular mechanisms of key proteins in pathogenic virulence.


Subject(s)
Bacterial Proteins/chemistry , Mycobacterium tuberculosis/enzymology , Protein-Tyrosine Kinases/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/genetics , Protein Domains , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism
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