I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Benjamin Nicholas
Atkinson
,
David
Steadman
,
Yuguang
Zhao
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis Reinholds
Ruza
,
Fiona
Jeganathan
,
Georgie
Lines
,
Sarah
Frew
,
Amy
Monaghan
,
Svend
Kjaer
,
Magda
Bictash
,
Yvonne
Jones
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 14744]
Open Access
Abstract: NOTUM is a carboxylesterase that has been shown to act by mediating the O-depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in in vitro disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide 3 as binding in the palmitoleate pocket with modest inhibition activity (IC50 33 μM). Optimization of hit 3 by SAR studies guided by SBDD identified indazole 38 (IC50 0.032 μM) and isoquinoline 45 (IC50 0.085 μM) as potent inhibitors of NOTUM. The binding of 45 to NOTUM was rationalized through an X-ray cocrystal structure determination which showed a flipped binding orientation compared to 3. However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.
|
Apr 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Faraz
Shaikh
,
Yuguang
Zhao
,
Luis
Alvarez
,
Maria
Iliopoulou
,
Christopher Thomas
Lohans
,
Christopher J.
Schofield
,
Sergi
Padilla-parra
,
Shirley W. I.
Siu
,
Elizabeth
Fry
,
Jingshan
Ren
,
David I.
Stuart
Diamond Proposal Number(s):
[10627]
Abstract: Potent Ebolavirus (EBOV) inhibitors will help to curtail outbreaks such as that which occurred in 2014-16 in West Africa. EBOV has on its surface a single glycoprotein (GP) critical for viral entry and membrane fusion. Recent high resolution complexes of EBOV GP with a variety of approved drugs revealed that binding to a common cavity prevented fusion of the virus and endosomal membranes, inhibiting virus infection. We performed docking experiments, screening a database of natural compounds to identify those likely to bind at this site. Using both inhibition assays of HIV-1-derived pseudovirus cell entry and structural analyses of the complexes of the compounds with GP we show here that two of these compounds attach in the common binding cavity, out of eight tested. In both cases two molecules bind in the cavity. The two compounds are chemically similar but the tighter binder has an additional chlorine atom that forms good halogen bonds to the protein and achieves an IC50 of 50 nM, making it the most potent GP-binding EBOV inhibitor yet identified, validating our screening approach for the discovery of novel anti-viral compounds.
|
Feb 2019
|
|
|
|
Abstract: Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease—a disease endemic especially in the Asia-Pacific region1. Scavenger receptor class B member 2 (SCARB2) is the major receptor of EV71, as well as several other enteroviruses responsible for hand, foot and mouth disease, and plays a key role in cell entry2. The isolated structures of EV71 and SCARB2 are known3,4,5,6, but how they interact to initiate infection is not. Here, we report the EV71–SCARB2 complex structure determined at 3.4 Å resolution using cryo-electron microscopy. This reveals that SCARB2 binds EV71 on the southern rim of the canyon, rather than across the canyon, as predicted3,7,8. Helices 152–163 (α5) and 183–193 (α7) of SCARB2 and the viral protein 1 (VP1) GH and VP2 EF loops of EV71 dominate the interaction, suggesting an allosteric mechanism by which receptor binding might facilitate the low-pH uncoating of the virus in the endosome/lysosome. Remarkably, many residues within the binding footprint are not conserved across SCARB2-dependent enteroviruses; however, a conserved proline and glycine seem to be key residues. Thus, although the virus maintains antigenic variability even within the receptor-binding footprint, the identification of binding ‘hot spots’ may facilitate the design of receptor mimic therapeutics less likely to quickly generate resistance.
|
Dec 2018
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8423]
Abstract: LYPD6 is a Wnt signaling enhancer that promotes phosphorylation of the Wnt co‐receptor LRP6 (low‐density lipoprotein receptor‐related protein 6). It also binds the nicotinic acetylcholine receptor (nAChR). We report here the 1.25 Å resolution structure of the LYPD6 extracellular Ly6/uPAR (LU) domain and map its interaction with LRP6 by mutagenesis and surface plasmon resonance (SPR). The LYPD6LU structure reveals a “tri‐fingered protein domain” fold with the middle fingertip of the bearing a “NxI” motif, a tripeptide motif associated with LRP5/6 binding by Wnt inhibitors. Of the Ly6 protein family members, only LYPD6 has an NxI motif. Since mutations in the LYPD6 NxI motif abolish or severely reduce interaction with LRP6, our results indicate its key role in the interaction of LYPD6 with LRP6.
|
Aug 2018
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10627]
Abstract: A large number of Food and Drug Administration (FDA) approved drugs have been found to inhibit cell entry of Ebola virus (EBOV). However, since these drugs have various primary pharmacological targets their mechanisms of action against EBOV remain largely unknown. We have previously shown that six FDA approved drugs inhibit EBOV infection by interacting with and destabilizing the viral glycoprotein (GP). Here we show that the antidepressants imipramine and clomipramine, and antipsychotic drug thioridazine also directly interact with EBOV GP, and determine the mode of interaction by crystallographic analysis of the complexes. The compounds bind within the same pocket as observed for other, chemically divergent, complexes but with different binding modes. These details should be of value for the development of potent EBOV inhibitors.
|
May 2018
|
|
I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10627]
Abstract: Here we show that four chemically divergent approved drugs reported to inhibit Ebolavirus infection, benztropine, bepridil, paroxetine and sertraline, directly interact with the Ebolavirus glycoprotein. Binding of these drugs destabilise the protein, suggesting that this may be the mechanism of inhibition, as reported for the anticancer drug toremifene and the painkiller ibuprofen, which bind in the same large cavity on the glycoprotein. Crystal structures show that the position of binding and the mode of interaction within the pocket vary significantly between these compounds. The binding constants (Kd) determined by thermal shift assay correlate with the protein-inhibitor interactions as well as with the antiviral activities determined by virus cell entry assays, supporting the hypothesis that these drugs inhibit viral entry by binding the glycoprotein and destabilising the pre-fusion conformation. Details of the protein–inhibitor interactions of these complexes and their relation with binding affinity may facilitate the design of more potent inhibitors.
|
Dec 2017
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10627]
Abstract: The role of B cells and posttranslational modifications in pathogenesis of organ-specific immune diseases is increasingly envisioned but remains poorly understood, particularly in human disorders. In celiac disease, transglutaminase 2–modified (TG2-modified; deamidated) gluten peptides drive disease-specific T cell and B cell responses, and antibodies to deamidated gluten peptides are excellent diagnostic markers. Here, we substantiate by high-throughput sequencing of IGHV genes that antibodies to a disease-specific, deamidated, and immunodominant B cell epitope of gluten (PLQPEQPFP) have biased and stereotyped usage of IGHV3-23 and IGHV3-15 gene segments with modest somatic mutations. X-ray crystal structures of 2 prototype IGHV3-15/IGKV4-1 and IGHV3-23/IGLV4-69 antibodies reveal peptide interaction mainly via germline-encoded residues. In-depth mutational analysis showed restricted selection and substitution patterns at positions involved in antigen binding. While the IGHV3-15/IGKV4-1 antibody interacts with Glu5 and Gln6, the IGHV3-23/IGLV4-69 antibody interacts with Gln3, Pro4, Pro7, and Phe8 — residues involved in substrate recognition by TG2. Hence, both antibodies, despite different interaction with the epitope, recognize signatures of TG2 processing that facilitates B cell presentation of deamidated gluten peptides to T cells, thereby providing a molecular framework for the generation of these clinically important antibodies. The study provides essential insight into the pathogenic mechanism of celiac disease.
|
Sep 2017
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10627]
Abstract: Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme’s GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side-chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP’s catalytic cycle and reveals significant active site plasticity that may underlie its substrate permissiveness.
|
Mar 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[8423, 10627]
Open Access
Abstract: Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.
|
Sep 2016
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
Data acquisition
Detectors
Diagnostics
Health Physics
|
Diamond Proposal Number(s):
[10627]
Abstract: Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion(1-7). GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered(8-10). Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein-drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 degrees C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.
|
Jul 2016
|
|
