scholarly journals Identification of an inhibitory pocket in falcilysin bound by chloroquine provides a new avenue for malaria drug development

2021 ◽  
Author(s):  
Grennady Wirjanata ◽  
Jerzy Dziekan ◽  
Jianqing Lin ◽  
Abbas El Sahili ◽  
Nur Elyza Binte Zulkifli ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Drug Candidate ◽  
Biological Pathway ◽  
Peptide Substrate ◽  
Protein Targets ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Cavity ◽  
Peptide Substrate Binding ◽  
Large Peptide

Despite their widespread use, our understanding of how malaria drugs work remains limited. This includes chloroquine (CQ), the most successful antimalarial ever deployed. Here, we used MS-CETSA and dose-response transcriptional profiling to elucidate protein targets and mechanism of action (MOA) of CQ, as well as MK-4815, a malaria drug candidate with a proposed MOA similar to CQ. We identified falcilysin (FLN) as the target of both compounds and found that hemoglobin digestion was the key biological pathway affected, with distinct MOA profiles between CQ-sensitive and CQ-resistant parasites. We showed that CQ and MK-4815 inhibit FLN proteolytic activity, and using X-ray crystallography, that they occupy a hydrophobic pocket situated within the large peptide substrate binding cavity of FLN. As a key protein in the MOA of CQ, FLN now constitute an interesting target for the development of novel anti-malarial drugs with improved resistance profiles.

scholarly journals Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

2021 ◽  
Author(s):  
vincent Chaptal ◽  
Veronica Zampieri ◽  
Benjamin Wiseman ◽  
Cedric Orelle ◽  
Juliette Martin ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Drug Transport ◽  
Rhodamine 6G ◽  
Drug Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Outer Leaflet ◽  
Molecular Features ◽  
Binding Cavity ◽  
Dynamics Simulations

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved two ATP-Mg2+-bound outward-facing (OF) conformations of the Bacillus subtilis (homodimeric) BmrA, one by X-ray crystallography without drug, and another by single-particle cryo-EM with rhodamine 6G (R6G). Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5[prime]-6[prime] of the other. R6G induces a rearrangement of TM1-2, highlighting a flexibility that was confirmed by H/D exchange and molecular dynamics simulations. The latter also shows a fast post-release occlusion of the cavity driven by hydrophobicity. Altogether, these data support a new swing mechanism for drug transport.

scholarly journals Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

2021 ◽  
Author(s):  
Mathieu Botte ◽  
Dongchun Ni ◽  
Stephan Schenck ◽  
Iwan Zimmermann ◽  
Mohamed Chami ◽  
...  
Keyword(s):  
Structural Biology ◽  
Transient State ◽  
Gram Negative Bacteria ◽  
Protein Targets ◽  
X Ray ◽  
Environmental Threats ◽  
X Ray Crystallography ◽  
Structural Framework ◽  
Bacterial Outer Membrane ◽  
Insight Into

Lipopolysaccharides (LPS) are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 Angstrom resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.

Structure of the Molecular Receptor 1,4,7,10-Tetrakis[(S)-2-hydroxy-2-phenylethyl]-1,4,7,10-tetraazacyclododecane: A Combined X-Ray Crystallographic and Theoretical Study Producing an Assessment of the Crystal Packing Energy

2006 ◽  
Vol 59 (2) ◽  
pp. 123
Author(s):  
Christopher B. Smith ◽  
Mark A. Buntine ◽  
Stephen F. Lincoln ◽  
Max R. Taylor ◽  
Kevin P. Wainwright
Keyword(s):  
Gas Phase ◽  
Crystal Packing ◽  
Minimum Energy ◽  
Basis Set ◽  
X Ray ◽  
Hartree Fock ◽  
X Ray Crystallography ◽  
Phenyl Rings ◽  
Binding Cavity ◽  
Aromatic Anions

X-Ray crystallography demonstrates that the guest molecule binding cavity within the molecular receptor ligand 1,4,7,10-tetrakis[(S)-2-hydroxy-2-phenylethyl]-1,4,7,10-tetraazacyclododecane, (S)-thpec12, is a poorly defined conical region stabilized by three O–H···O hydrogen bonds and a single O–H···N hydrogen bond. Two similar, but crystallographically independent, molecules exist within the unit cell. Ab initio calculations, using Gaussian 03 (LanL2DZ basis set at the Hartree–Fock level of theory), predict that these have steric energies of 97.73 and 97.06 kJ mol−1, respectively, above that of the minimum energy (gas phase) conformer of the same hydrogen-bonding configuration, which is believed to be the structure of global minimum energy. The mean of these energies (97.4 kJ mol−1) represents a best estimate of the crystal packing energy for (S)-thpec12, some of which is seen to be expended in rotating the phenyl rings away from the positions favoured in the gas phase. The ability of the CdII complex of (S)-thpec12 to act as a molecular receptor for aromatic anions is demonstrated by the isolation of two inclusion compounds in which p-nitrophenolate and anthraquinone-2-sulfonate are retained.

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.

Tele-Substitution Reactions in the Synthesis of a Promising Class of Antimalarials

2020 ◽  
Author(s):  
Marat Korsik ◽  
Edwin Tse ◽  
David Smith ◽  
William Lewis ◽  
Peter J. Rutledge ◽  
...  
Keyword(s):  
Heterocyclic Ring ◽  
Ring System ◽  
Substitution Reactions ◽  
Laboratory Notebook ◽  
Polar Solvents ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Core ◽  
Nmr Data

<p></p><p>We have discovered and studied a <i>tele</i>substitution reaction in a biologically important heterocyclic ring system. Conditions that favour the <i>tele</i>-substitution pathway were identified: the use of increased equivalents of the nucleophile or decreased equivalents of base, or the use of softer nucleophiles, less polar solvents and larger halogens on the electrophile. Using results from X-ray crystallography and isotope labelling experiments a mechanism for this unusual transformation is proposed. We focused on this triazolopyrazine as it is the core structure of the <i>in vivo </i>active anti-plasmodium compounds of Series 4 of the Open Source Malaria consortium.</p> <p> </p> <p>Archive of the electronic laboratory notebook with the description of all conducted experiments and raw NMR data could be accessed via following link <a href="https://ses.library.usyd.edu.au/handle/2123/21890">https://ses.library.usyd.edu.au/handle/2123/21890</a> . For navigation between entries of laboratory notebook please use file "Strings for compounds in the article.pdf" that works as a reference between article codes and notebook codes, also this file contain SMILES for these compounds. </p><br><p></p>

Highly Electron-Deficient Pyridinium-Nitrones for Rapid and Tunable Inverse-Electron-Demand Strain-Promoted Alkyne-Nitrone Cycloaddition to Bicyclo[6.1.0]nonyne

2019 ◽  
Author(s):  
Praveen Gunawardene ◽  
Wilson Luo ◽  
Alexander M. Polgar ◽  
John F. Corrigan ◽  
Mark Workentin
Keyword(s):  
Density Functional Theory ◽  
Reaction Kinetics ◽  
Density Functional ◽  
Functional Theory ◽  
X Ray ◽  
Inverse Electron Demand ◽  
X Ray Crystallography ◽  
Electron Demand

<div> <div> <p>Highly accelerated inverse-electron-demand strain-promoted alkyne-nitrone cycloaddition (IED SPANC) between a sta- ble cyclooctyne (bicyclo[6.1.0]nonyne (BCN)) and nitrones delocalized into a Cα-pyridinium functionality is reported, with the most electron-deficient “pyridinium-nitrone” displaying among the most rapid cycloadditions to BCN that is currently reported. Density functional theory (DFT) and X-ray crystallography are explored to rationalize the effects of N- and Cα-substituent modifications at the nitrone on IED SPANC reaction kinetics and the overall rapid reactivity of pyridinium-delocalized nitrones.</p> </div> </div>

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