B21-High Throughput SAXS
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Beatrice
Mercorelli
,
Alessandro
Bazzacco
,
Michela
Eleuteri
,
Samuele
Di Cristofano
,
Jenny
Desantis
,
Alessandro
Paciaroni
,
Maria Grazia
Ortore
,
Sara
Tuci
,
Francesco
Spinozzi
,
Domenico
Raimondo
,
Laura
Goracci
,
Gabriele
Cruciani
,
Arianna
Loregian
Diamond Proposal Number(s):
[29982]
Open Access
Abstract: SARS-CoV-2 Main protease (Mpro) is the most explored coronavirus antiviral target, being most antivirals approved or under development protease inhibitors. Mpro is active as a dimer and the molecular details of its maturation are poorly understood. Some compounds that crystallize at the dimerization interface rather than at the catalytic pocket have been proposed as allosteric inhibitors. Here, we characterize a series of novel compounds starting from a scaffold identified by an in silico screening for Mpro catalytic pocket. Several compounds showed anti-SARS-CoV-2 activity in infected cells, but they did not inhibit Mpro in vitro. Time-of-addition studies pointed to a stage compatible with Mpro targeting. Molecular modelling studies suggested that compounds 1 and 11 bind Mpro similarly to the allosteric inhibitor AT7519. Small-angle X-ray scattering studies revealed that 1 and 11 strongly shift Mpro equilibrium to the monomeric form, while the allosteric inhibitor pelitinib and the catalytic inhibitors nirmatrelvir and GC376 stabilize the dimer. Compounds 1 and 11 inhibited Mpro proteolytic activity in SARS-CoV-2 infected cells acting as allosteric inhibitors that stabilize the monomeric form. In conclusion, we validated an allosteric site in Mpro that could be exploited for the development of effective anti-SARS-CoV-2 antivirals targeting Mpro with a novel mechanism.
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Aug 2025
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Krios III-Titan Krios III at Diamond
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Zhen
Hou
,
Yao
Shen
,
Stanley
Fronik
,
Juan
Shen
,
Jiong
Shi
,
Jialu
Xu
,
Long
Chen
,
Nathan
Hardenbrook
,
Alan N.
Engelman
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.
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Jul 2025
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Madeline E.
Kavanagh
,
Kirsty J.
Mclean
,
Sophie H.
Gilbert
,
Cecilia N.
Amadi
,
Matthew
Snee
,
Richard B.
Tunnicliffe
,
Kriti
Arora
,
Helena I. M.
Boshoff
,
Alexander
Fanourakis
,
Maria Jose
Rebollo-Lopez
,
Fatima
Ortega
,
Colin W.
Levy
,
Andrew W.
Munro
,
David
Leys
,
Chris
Abell
,
Anthony G.
Coyne
Diamond Proposal Number(s):
[8997, 17773, 24447]
Open Access
Abstract: Tuberculosis is the deadliest infectious disease in history and new drugs are urgently required to combat multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Here, we exploit the relience of Mtb on host-derived cholesterol to develop a novel class of antitubercular compounds that target Mtb CYP125 and CYP142; the enzymes that catalyze the first step of cholesterol metabolism. A combination of fragment screening and structure-based drug design was used to identify a hit compound and guide synthetic optimization of a dual CYP125/142 ligand 5m (KD 40–160 nM), which potently inhibits enzyme activity in vitro (KI < 100 nM), and the growth of Mtb in extracellular (MIC99 0.4–1.5 μM) and intracellular assays (IC50 1.7 μM). The structural data and lead compounds reported here will help study Mtb cholesterol metabolism and guide the development of novel antibiotics to combat MDR Mtb.
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Jul 2025
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I04-Macromolecular Crystallography
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Lindsay B.
Tulloch
,
Hugh
Tawell
,
Annie E.
Taylor
,
Marta Lopes
Lima
,
Alice
Dawson
,
Sandra
Carvalho
,
Richard J.
Wall
,
Victoriano
Corpas-Lopez
,
Gourav
Dey
,
Jack
Duggan
,
Luma Godoy
Magalhaes
,
Leah S.
Torrie
,
Laura
Frame
,
David
Robinson
,
Stephen
Patterson
,
Michele
Tinti
,
George W.
Weaver
,
William J.
Robinson
,
Monica
Cal
,
Marcel
Kaiser
,
Pascal
Mäser
,
Peter
Sjö
,
Benjamin
Perry
,
John M.
Kelly
,
Amanda Fortes
Francisco
,
Avninder S.
Bhambra
,
Susan
Wyllie
Diamond Proposal Number(s):
[26793]
Abstract: The protozoan parasite Trypanosoma cruzi causes Chagas disease, which is among the deadliest parasitic infections in Latin America. Current therapies are toxic and lack efficacy against the chronic stage of infection; thus, new drugs are urgently needed. Here, we describe a previously unidentified series of quinazoline compounds with potential against Trypanosoma cruzi and the related trypanosomatid parasites Trypanosoma brucei and Leishmania donovani. We demonstrated partial efficacy of a lead quinazoline compound in a mouse model of acute Chagas disease. Mechanism of action studies using several orthogonal approaches showed that this quinazoline compound series targeted the ATP-binding pocket of T. cruzi lysyl-tRNA synthetase 1 (KRS1). A high-resolution crystal structure of KRS1 bound to the drug indicated binding interactions that led to KRS1 inhibition. Our study identified KRS1 as a druggable target for treating T. cruzi infection in a mouse model. This quinazoline series shows potential for treating Chagas disease but will require further development to become a future treatment for this neglected disease.
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Jul 2025
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I03-Macromolecular Crystallography
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Nutpakal
Ketprasit
,
Chia-Wei
Tai
,
Vivek Kumar
Sharma
,
Yogavel
Manickam
,
Yogesh
Khandokar
,
Xi
Ye
,
Con
Dogovski
,
David H.
Hilko
,
Craig J.
Morton
,
Anne-Sophie C.
Braun
,
Michael G.
Leeming
,
Bagale
Siddharam
,
Gerald J.
Shami
,
Pushpangadan Indira
Pradeepkumar
,
Santosh
Panjikar
,
Sally-Ann
Poulsen
,
Michael D. W.
Griffin
,
Amit
Sharma
,
Leann
Tilley
,
Stanley C.
Xie
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5′-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial, dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple aminoacyl-tRNA synthetases (aaRSs), including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38885]
Open Access
Abstract: Cholesterol is an essential sterol in cell membranes that regulates organization and fluidity. This biomolecule has been identified as one of the critical factors in the internalization process of various viruses in human cells. Therefore, understanding these mechanisms is crucial for a deeper comprehension of viral pathogenicity in the search for practical therapeutic approaches against viral diseases. The biochemical and biophysical processes related to these diseases are highly complex. For this reason, studying model systems capable of mimicking the interaction of lipid membranes with cholesterol and proteins is fundamental. In this work, we propose to study the structural and elastic changes in mono-, bi-, and tridimensional lipid systems composed of dipalmitoylphosphatidylcholine (PC) with varying amounts of cholesterol in the presence and absence of the S protein (Spike) and its receptor-binding domain (RBD) from SARS-CoV-2. To characterize these systems, we used both experimental and theoretical approaches such as Langmuir trough, atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), electrochemical methods, and molecular dynamics (MD) simulations. With the interpretation of all results obtained in this work, it was possible to propose a structural model of the membrane in the presence of cholesterol and the interaction with the Spike protein and RBD. The behavior of the adsorption isotherm and SAXS data, together with the results provided by MD simulations, led us to conclude that cholesterol in PC monolayers promotes local alterations, inducing the formation of more rigid membrane regions. More importantly, cholesterol plays a crucial role in facilitating the allocation of SARS-CoV-2 proteins in lipid systems. This is especially true for the Spike protein, which displayed a non-ACE2 mediated stable binding to the lipid membrane with high internalization.
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Ester
Serrano
,
Tianxiao
Zhao
,
David R.
Mark
,
Mostafa
Soroor
,
Iris
Floria
,
Nicholas J.
Terrill
,
Nikil
Kapur
,
Arwen I. I.
Tyler
,
Mathew H.
Horrocks
,
Andrew J.
Roe
,
Olwyn
Byron
Diamond Proposal Number(s):
[28516]
Open Access
Abstract: Enterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors.
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Jun 2025
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2’ cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2’ cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike’s stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Antimicrobial resistance has emerged as a critical global public health threat, impacting human, animal and environmental health. An important mechanism of resistance is the production of β-lactamases, enzymes that hydrolyze the β-lactam ring, rendering β-lactam antibiotics ineffective. Metallo-β-lactamases (MBLs), which contain zinc ions in their active sites, are particularly challenging to counter as there are currently no inhibitors targeting these enzymes available on the market. Therefore, there is an urgent need for innovative drug discovery strategies to develop MBL-targeted therapies. New Delhi Metallo-β-Lactamase 1 (NDM-1) is the most widely disseminated MBL, with a global distribution in Enterobacterales. In this study, we used our library of fragment-sized chloroacetamides as a starting point to synthesize mercaptoacetamides as potential NDM-1 inhibitors. This resulted in a compound (14a) with an IC50 of 20 μM, which crystallography shows binds to NDM-1 in two different poses. Using this structure as a starting point for in silico design, we developed a series of larger thiol-based compounds designed to occupy more space in the active site and to utilize other novel zinc-binding groups. Although some showed minimal inhibition (which makes them valuable as decoys for metalloenzyme studies) one compound exhibited an IC50 of 14 μM, with crystallography indicating that an additional aromatic group, compared to 14a, interacts with hydrophobic residues on an NDM-1 active site loop. These data identify promising scaffolds for the further development of potent MBL inhibitors and show the utility of repurposing chemical libraries to target clinically important enzymes.
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Jun 2025
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