I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.
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Feb 2023
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Open Access
Abstract: Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.
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Sep 2022
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Jan
Bláha
,
Tereza
Skálová
,
Barbora
Kalousková
,
Ondřej
Skořepa
,
Denis
Cmunt
,
Valéria
Grobárová
,
Samuel
Pazicky
,
Edita
Poláchová
,
Celeste
Abreu
,
Jan
Stransky
,
Tomas
Koval
,
Jarmila
Duskova
,
Yuguang
Zhao
,
Karl
Harlos
,
Jindřich
Hašek
,
Jan
Dohnalek
,
Ondřej
Vaněk
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.
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Aug 2022
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Lucy C.
Walters
,
Daniel
Rozbesky
,
Karl
Harlos
,
Max
Quastel
,
Hong
Sun
,
Sebastian
Springer
,
Robert P.
Rambo
,
Fiyaz
Mohammed
,
E. Yvonne
Jones
,
Andrew J.
Mcmichael
,
Geraldine M.
Gillespie
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuguang
Zhao
,
William
Mahy
,
Nicky J.
Willis
,
Hannah L.
Woodward
,
David
Steadman
,
Elliott D.
Bayle
,
Benjamin N.
Atkinson
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Karl
Harlos
,
Fiona
Jeganathan
,
Stefan
Constantinou
,
Artur
Costa
,
Svend
Kjær
,
Magda
Bictash
,
Patricia C.
Salinas
,
Paul
Whiting
,
Jean-Paul
Vincent
,
Paul V.
Fish
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[16814]
Open Access
Abstract: The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.
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Jun 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Bicyclic azetidine compounds possess antimalarial activity via targeting of the cytoplasmic Plasmodium falciparum (Pf) protein translation enzyme phenylalanine-tRNA synthetase (cFRS). These drugs kill parasites both in vitro and in vivo, including the blood, liver, and transmission developmental stages. Here we present the co-crystal structure of PfcFRS with a potent inhibitor, the bicyclic azetidine BRD7929. Our studies reveal high-affinity binding of BRD7929 with PfcFRS along with exquisite specificity compared with the human enzyme, leading in turn to potent and selective inhibition of the parasite enzyme. Our co-crystal structure shows that BRD7929 binds in the active site in the α subunit of PfcFRS, where it occupies the amino acid site, an auxiliary site, and partially the ATP site. This structural snapshot of inhibitor-bound PfcFRS thus provides a platform for the structure-guided optimization of novel antimalarial compounds.
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Apr 2022
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I03-Macromolecular Crystallography
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Dapeng
Li
,
Simon
Brackenridge
,
Lucy C.
Walters
,
Olivia
Swanson
,
Karl
Harlos
,
Daniel
Rozbesky
,
Derek W.
Cain
,
Kevin
Wiehe
,
Richard M.
Scearce
,
Maggie
Barr
,
Zekun
Mu
,
Robert
Parks
,
Max
Quastel
,
Robert J.
Edwards
,
Yunfei
Wang
,
Wes
Rountree
,
Kevin O.
Saunders
,
Guido
Ferrari
,
Persephone
Borrow
,
E. Yvonne
Jones
,
S. Munir
Alam
,
Mihai L.
Azoitei
,
Geraldine M.
Gillespie
,
Andrew J.
Mcmichael
,
Barton F.
Haynes
Open Access
Abstract: The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.
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Mar 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 19946]
Open Access
Abstract: Transmission of the New World hemorrhagic fever arenaviruses Junín virus (JUNV) and Machupo virus (MACV) to humans is facilitated, in part, by the interaction between the arenavirus GP1 glycoprotein and the human transferrin receptor 1 (hTfR1). We utilize a mouse model of live-attenuated immunization with envelope exchange viruses to isolate neutralizing monoclonal antibodies (NAbs) specific to JUNV GP1 and MACV GP1. Structures of two NAbs, termed JUN1 and MAC1, demonstrate that they neutralize through disruption of hTfR1 recognition. JUN1 utilizes a binding mode common to all characterized infection- and vaccine-elicited JUNV-specific NAbs, which involves mimicking hTfR1 binding through the insertion of a tyrosine into the receptor-binding site. In contrast, MAC1 undergoes a tyrosine-mediated mode of antigen recognition distinct from that used by the reported anti-JUNV NAbs and the only other characterized anti-MACV NAb. These data reveal the varied modes of GP1-specific recognition among New World arenaviruses by the antibody-mediated immune response.
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Mar 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS) represent a promising therapeutic anti‐malarial target. Cladosporin, a tool compound was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with x‐ray crystallography reveals compounds to be of moderate potency. Co‐crystals of Cla‐B and Cla‐C with PfKRS reveals key atomic configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these piperidine ring scaffold inhibitors lays a framework for further structural editing and functional modifications of cladosporin scaffold to obtain a potent lead.
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May 2021
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I24-Microfocus Macromolecular Crystallography
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Manmohan
Sharma
,
Nipun
Malhotra
,
Manickam
Yogavel
,
Karl
Harlos
,
Bruno
Melillo
,
Eamon
Comer
,
Arthur
Gonse
,
Suhel
Parvez
,
Branko
Mitasev
,
Francis G.
Fang
,
Stuart L.
Schreiber
,
Amit
Sharma
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
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Jan 2021
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