The Glitazone Class of Drugs as Carbonic AnhydraseInhibitors—A Spin-Off Discovery from Fragment Screening

The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen–deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Download Full-text

Mapping the Binding Interface in a Noncovalent Size Variant of a Monoclonal Antibody Using Native Mass Spectrometry, Hydrogen–Deuterium Exchange Mass Spectrometry, and Computational Analysis

Journal of Pharmaceutical Sciences ◽

10.1016/j.xphs.2017.06.009 ◽

2017 ◽

Vol 106 (11) ◽

pp. 3222-3229 ◽

Cited By ~ 6

Author(s):

Yuetian Yan ◽

Hui Wei ◽

Sutjano Jusuf ◽

Stanley R. Krystek ◽

Jie Chen ◽

...

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Computational Analysis ◽

Native Mass Spectrometry ◽

Hydrogen Deuterium Exchange ◽

Exchange Mass Spectrometry ◽

Binding Interface ◽

Size Variant ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry

Download Full-text

Monothiol and dithiol glutaredoxin-1 from Clostridium oremlandii: identification of domain-swapped structures by NMR, X-ray crystallography and HDX mass spectrometry

IUCrJ ◽

10.1107/s2052252520011598 ◽

2020 ◽

Vol 7 (6) ◽

pp. 1019-1027

Author(s):

Kitaik Lee ◽

Kwon Joo Yeo ◽

Sae Hae Choi ◽

Eun Hye Lee ◽

Bo Keun Kim ◽

...

Keyword(s):

Mass Spectrometry ◽

Disulfide Bond ◽

Protein Function ◽

Single Point ◽

Cysteine Residue ◽

Single Point Mutation ◽

Protein Oligomerization ◽

X Ray ◽

X Ray Crystallography ◽

Hydrogen Deuterium

Protein dimerization or oligomerization resulting from swapping part of the protein between neighboring polypeptide chains is known to play a key role in the regulation of protein function and in the formation of protein aggregates. Glutaredoxin-1 from Clostridium oremlandii (cGrx1) was used as a model to explore the formation of multiple domain-swapped conformations, which were made possible by modulating several hinge-loop residues that can form a pivot for domain swapping. Specifically, two alternative domain-swapped structures were generated and analyzed using nuclear magnetic resonance (NMR), X-ray crystallography, circular-dichroism spectroscopy and hydrogen/deuterium-exchange (HDX) mass spectrometry. The first domain-swapped structure (β3-swap) was formed by the hexameric cGrx1–cMsrA complex. The second domain-swapped structure (β1-swap) was formed by monothiol cGrx1 (C16S) alone. In summary, the first domain-swapped structure of an oxidoreductase in a hetero-oligomeric complex is presented. In particular, a single point mutation of a key cysteine residue to serine led to the formation of an intramolecular disulfide bond, as opposed to an intermolecular disulfide bond, and resulted in modulation of the underlying free-energy landscape of protein oligomerization.

Download Full-text

Mass Spectrometry Reveals the Assembly Pathway of Encapsulated Ferritins and Highlights a Dynamic Ferroxidase Interface

10.26434/chemrxiv.9901346.v1 ◽

2019 ◽

Author(s):

Jennifer Ross ◽

Thomas Lambert ◽

Cecilia Piergentili ◽

Didi He ◽

Kevin J. Waldron ◽

...

Keyword(s):

Mass Spectrometry ◽

Storage System ◽

Native Mass Spectrometry ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Iron Storage ◽

Exchange Mass Spectrometry ◽

Assembly Pathway ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry

Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn.

Download Full-text

Methods in the Study of PTEN Structure: X-Ray Crystallography and Hydrogen Deuterium Exchange Mass Spectrometry

PTEN - Methods in Molecular Biology ◽

10.1007/978-1-4939-3299-3_14 ◽

2015 ◽

pp. 215-230 ◽

Cited By ~ 2

Author(s):

Glenn R. Masson ◽

John E. Burke ◽

Roger L. Williams

Keyword(s):

Mass Spectrometry ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

X Ray ◽

X Ray Crystallography ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry

Download Full-text

Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.5b01940 ◽

2016 ◽

Vol 59 (5) ◽

pp. 2192-2204 ◽

Cited By ~ 28

Author(s):

Lucy A. Woods ◽

Olan Dolezal ◽

Bin Ren ◽

John H. Ryan ◽

Thomas S. Peat ◽

...

Keyword(s):

Mass Spectrometry ◽

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Native State ◽

X Ray ◽

X Ray Crystallography ◽

Fragment Screening

Download Full-text

Zinc Binding to Tau Influences Aggregation Kinetics and Oligomer Distribution

International Journal of Molecular Sciences ◽

10.3390/ijms20235979 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5979 ◽

Cited By ~ 4

Author(s):

Moreira ◽

Cristóvão ◽

Torres ◽

Carapeto ◽

Rodrigues ◽

...

Keyword(s):

Mass Spectrometry ◽

Metal Ion ◽

Molecular Mechanisms ◽

Disease Onset ◽

Native Mass Spectrometry ◽

Mass Spectrometry Analysis ◽

Zinc Binding ◽

Lag Phase ◽

Intrinsically Disordered ◽

Tau Aggregation

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer’s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

Download Full-text

HDX and Native Mass Spectrometry Reveals the Different Structural Basis for Interaction of the Staphylococcal Pathogenicity Island Repressor Stl with Dimeric and Trimeric Phage dUTPases

Biomolecules ◽

10.3390/biom9090488 ◽

2019 ◽

Vol 9 (9) ◽

pp. 488 ◽

Cited By ~ 1

Author(s):

Kinga Nyíri ◽

Matthew J. Harris ◽

Judit Matejka ◽

Olivér Ozohanics ◽

Károly Vékey ◽

...

Keyword(s):

Mass Spectrometry ◽

Pathogenicity Island ◽

Native Mass Spectrometry ◽

Inhibitory Effect ◽

Structural Basis ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Peptide Segments ◽

Shed Light

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases.

Download Full-text

Membrane Protein–Lipid Interactions Probed Using Mass Spectrometry

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111508 ◽

2019 ◽

Vol 88 (1) ◽

pp. 85-111 ◽

Cited By ~ 35

Author(s):

Jani Reddy Bolla ◽

Mark T. Agasid ◽

Shahid Mehmood ◽

Carol V. Robinson

Keyword(s):

Mass Spectrometry ◽

Membrane Protein ◽

Lipid Bilayers ◽

Protein Interactions ◽

Native Mass Spectrometry ◽

Lipid Interactions ◽

X Ray Crystallography ◽

Native Ms ◽

Molecular Aspects ◽

Lipid Protein Interactions

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid–protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein–lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo–electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein–lipid interactions in the native environment.

Download Full-text