I04-Macromolecular Crystallography
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Leire
Iralde-lorente
,
Giusy
Tassone
,
Letizia
Clementi
,
Lorenzo
Franci
,
Claire C
Munier
,
Ylenia
Cau
,
Mattia
Mori
,
Mario
Chiariello
,
Adriano
Angelucci
,
Matthew W. D.
Perry
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Maurizio
Botta
Abstract: The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukaemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukaemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumour cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.
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Mar 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Luís A. R.
Carvalho
,
Vanessa T.
Almeida
,
Jose A.
Brito
,
Kenneth M.
Lum
,
Tânia F.
Oliveira
,
Rita C.
Guedes
,
Lídia M.
Gonçalves
,
Susana D.
Lucas
,
Benjamin F.
Cravatt
,
Margarida
Archer
,
Rui
Moreira
Diamond Proposal Number(s):
[20161]
Abstract: 3-Oxo-β-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-β-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-β-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-β-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.
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Mar 2020
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I03-Macromolecular Crystallography
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Abstract: We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.
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Jan 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9537]
Abstract: Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity towards primase, possibly leading to less toxic and more effective therapeutic agents.
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Sep 2019
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B21-High Throughput SAXS
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Amaia
Gonzalez-magaña
,
Alain
Ibáñez De Opakua
,
Nekane
Merino
,
Hugo
Monteiro
,
Tammo
Diercks
,
Javier
Murciano-calles
,
Irene
Luque
,
Pau
Bernadó
,
Tiago
Cordeiro
,
Alfredo
De Biasio
,
Francisco J
Blanco
Diamond Proposal Number(s):
[14707]
Abstract: The Proliferating Cell Nuclear Antigen-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, binds to PCNA as non-ubiquitinated p15PAF does, but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by 8 disordered residues, form transient dimers due to the high local effective concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1for maintenance of DNA methylation during replication.
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Sep 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Jon A.
Read
,
Jonathan
Tart
,
Philip B.
Rawlins
,
Clare
Gregson
,
Karen
Jones
,
Ning
Gao
,
Xiahui
Zhu
,
Ron
Tomlinson
,
Erin
Code
,
Tony
Cheung
,
Huawei
Chen
,
Sameer P.
Kawatkar
,
Andy
Bloecher
,
Sharan
Bagal
,
Daniel H.
O’donovan
,
James
Robinson
Abstract: Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates chromatin state and gene expression by methylating histone H3 lysine 27. EZH2 is overexpressed or mutated in various hematological malignancies and solid cancers. Our previous efforts to identify inhibitors of PRC2 methyltransferase activity by high-throughput screening (HTS) resulted in large numbers of false positives and thus a significant hit deconvolution challenge. More recently, others have reported compounds that bind to another PRC2 core subunit, EED, and allosterically inhibit EZH2 activity. This mechanism is particularly appealing as it appears to retain potency in cell lines that have acquired resistance to orthosteric EZH2 inhibition. By designing a fluorescence polarization probe based on the reported EED binding compounds, we were able to quickly and cleanly re-triage our previously challenging HTS hit list and identify novel allosteric PRC2 inhibitors.
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Sep 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14692]
Abstract: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.
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Aug 2019
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I02-Macromolecular Crystallography
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David R.
Chisholm
,
Charles W. E.
Tomlinson
,
Garr-layy
Zhou
,
Claire
Holden
,
Valerie
Affleck
,
Rebecca
Lamb
,
Katherine
Newling
,
Peter
Ashton
,
Roy
Valentine
,
Christopher
Redfern
,
János
Erostyák
,
Geza
Makkai
,
Carrie A.
Ambler
,
Andrew
Whiting
,
Ehmke
Pohl
Abstract: Retinoids, such as all-trans-retinoic acid (ATRA), are endogenous signalling molecules derived from Vitamin A that influ-ence a variety of cellular processes through mediation of transcription events in the cell nucleus. Due to these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they con-trol. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluo-rescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quanti-fy their binding to their cellular targets, including Cellular Retinoid Binding Protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding (Kd = 34.0 ± 2.5 nM) and we further used X-ray crystallography to solve the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid-binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behaviour of the com-pounds in cultured human epithelial cells, highlighting a fascinating nuclear localisation, and used RNA sequencing to con-firm that the compounds regulate similar cellular processes to ATRA. We anticipate that the unique properties of these fluo-rescent retinoids can now be used to shed new light on the vital and highly complex retinoid signalling pathway.
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Feb 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Abstract: Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation, but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-C oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labelling studies on the SpMnSOD-catalysed oxidation of lignin model compound guiaiacylglycerol--guaiacyl ether indicated that the oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of approximately 1 molar equivalent of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn centre for hydroxyl radical generation.
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Sep 2018
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I04-Macromolecular Crystallography
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Abstract: Analog-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogs of pyrazolopyrimidine-based Src kinase inhibitors (e.g. PP1). This ‘bump-hole’ method has been utilized for at least 85 of ~520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS-kinase technology, we designed type II kinase inhibitors, ASDO2/6 (Analog-Sensitive ‘DFG-Out’ kinase inhibitors-2/6), that target the ‘DFG-out’ conformation of cysteine (Cys)-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase and Cyclin-dependent kinase-1 and in cells using M110C-GWL expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6-inhibition, but not AS-kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS-kinase inhibitors, have the potential to inhibit multiple AS-kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.
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Sep 2018
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