scholarly journals Identification of the first highly selective inhibitor of human lactate dehydrogenase B

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachio Shibata ◽  
Satoshi Sogabe ◽  
Masanori Miwa ◽  
Takuya Fujimoto ◽  
Nobuyuki Takakura ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Assay ◽  
Cancer Metabolism ◽  
Selective Inhibitor ◽  
Cancer Cell Proliferation ◽  
Allosteric Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Promising Target ◽  
Catalytic Active Site

AbstractLactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

scholarly journals Identification of the First Highly Selective Inhibitor of Human Lactate Dehydrogenase B

2020 ◽  
Author(s):  
sachio shibata ◽  
Satoshi Sogabe ◽  
Masanori Miwa ◽  
Takuya Fujimoto ◽  
Nobuyuki Takakura ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Assay ◽  
Cancer Metabolism ◽  
Selective Inhibitor ◽  
Cancer Cell Proliferation ◽  
Allosteric Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Promising Target ◽  
Catalytic Active Site

Abstract Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+ and has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To our knowledge, selective inhibitors for LDHB have yet to be reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. The screening campaign identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. It exhibited uncompetitive LDHB inhibition (IC50 = 42 nM). X-ray crystallography identified its binding with the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerization interface of the two dimers is critical for enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

scholarly journals Identification of the First Highly Selective inhibitor of Human Lactate Dehydrogenase B

2021 ◽  
Author(s):  
Sachio Shibata ◽  
Satoshi Sogabe ◽  
Masanori Miwa ◽  
Takuya Fujimoto ◽  
Nobuyuki Takakura ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Assay ◽  
Cancer Metabolism ◽  
Selective Inhibitor ◽  
Cancer Cell Proliferation ◽  
Allosteric Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Promising Target ◽  
Catalytic Active Site

Abstract Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+ and has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have yet to be reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. The screening campaign identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography identified its binding with the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers was critical for enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

scholarly journals Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

2018 ◽  
Vol 19 (11) ◽  
pp. 3664 ◽  
Author(s):  
Ali Munawar ◽  
Steven Beelen ◽  
Ahmad Munawar ◽  
Eveline Lescrinier ◽  
Sergei Strelkov
Keyword(s):  
Zika Virus ◽  
Dissociation Constants ◽  
Global Public Health ◽  
Human Pathogens ◽  
Allosteric Site ◽  
Replication Complex ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Atomic Precision

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015–2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

scholarly journals New insights into the catalytic active-site structure of multicopper oxidases

2014 ◽  
Vol 70 (3) ◽  
pp. 772-779 ◽  
Author(s):  
Hirofumi Komori ◽  
Ryosuke Sugiyama ◽  
Kunishige Kataoka ◽  
Kentaro Miyazaki ◽  
Yoshiki Higuchi ◽  
...  
Keyword(s):  
Electron Density ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Reduction Process ◽  
Multicopper Oxidases ◽  
Site Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Catalytic Active Site ◽  
Hydrated Electrons

Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO fromEscherichia coliand laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

Anilinoquinazoline Inhibitors of Fructose 1,6-Bisphosphatase Bind at a Novel Allosteric Site:  Synthesis, In Vitro Characterization, and X-ray Crystallography

2002 ◽  
Vol 45 (18) ◽  
pp. 3865-3877 ◽  
Author(s):  
Stephen W. Wright ◽  
Anthony A. Carlo ◽  
Maynard D. Carty ◽  
Dennis E. Danley ◽  
David L. Hageman ◽  
...  
Keyword(s):  
Allosteric Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
In Vitro Characterization ◽  
Fructose 1,6 Bisphosphatase

scholarly journals Allosteric modulation of caspase 3 through mutagenesis

2012 ◽  
Vol 32 (4) ◽  
pp. 401-411 ◽  
Author(s):  
Jad Walters ◽  
Joshua L. Schipper ◽  
Paul Swartz ◽  
Carla Mattos ◽  
A. Clay Clark
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Caspase 3 ◽  
Structural Defects ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Dimer Interface ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dynamics Simulations

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

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