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57 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Standalone β-ketoreductase acts concomitantly with biosynthesis of the antimycin scaffold Asif Fazal , Glyn R. Hemsworth , Michael E. Webb , Ryan F. Seipke Acs Chemical Biology DOI: 10.1021/acschembio.1c00229 Diamond Proposal Number(s): [19248] Open Access Show Abstract ▼ Abstract: Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone β-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone β-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone β-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.	Jun 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Fragment screening reveals starting points for rational design of galactokinase 1 inhibitors to treat classic galactosemia Sabrina R. Mackinnon , Tobias Krojer , William R. Foster , Laura Diaz-Saez , Manshu Tang , Kilian V. M. Huber , Frank Von Delft , Kent Lai , Paul Brennan , Gustavo Arruda Bezerra , Wyatt W. Yue Acs Chemical Biology 152 DOI: 10.1021/acschembio.0c00498 Diamond Proposal Number(s): [18145] Show Abstract ▼ Abstract: Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP- competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.	Mar 2021
I24-Microfocus Macromolecular Crystallography	Fluorescence anisotropy-based tethering for discovery of protein–protein interaction stabilizers Eline Sijbesma , Bente A. Somsen , Galen P. Miley , Iris A. Leijten-Van De Gevel , Luc Brunsveld , Michelle R. Arkin , Christian Ottmann Acs Chemical Biology DOI: 10.1021/acschembio.0c00646 Open Access Show Abstract ▼ Abstract: Protein–protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools to fundamentally study PPIs and as starting points for potential new therapeutics. Yet, systematic approaches for their discovery are not widely available, and the design prerequisites of “molecular glues” are poorly understood. Covalent fragment-based screening can identify chemical starting points for these enhancers at specific sites in PPI interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach for a cysteine residue in the hub protein 14–3–3, an important regulator of phosphorylated client proteins. Here, we invert the strategy and report the development of a functional read-out for systematic identification of PPI enhancers based on fluorescence anisotropy (FA-tethering) with the reactive handle now on a client-derived peptide. Using the DNA-binding domain of the nuclear receptor Estrogen Related Receptor gamma (ERRγ), we target a native cysteine positioned at the 14–3–3 PPI interface and identify several fragments that form a disulfide bond to ERRγ and stabilize the complex up to 5-fold. Crystallography indicates that fragments bind in a pocket comprised of 14–3–3 and the ERRγ phosphopeptide. FA-tethering presents a streamlined methodology to discover molecular glues for protein complexes.	Nov 2020
I03-Macromolecular Crystallography	Pentavalent sialic acid conjugates block coxsackievirus A24 variant and human adenovirus type 37 – viruses that cause highly contagious eye infections Emil Johansson , Rémi Caraballo , Nitesh Mistry , Georg Zocher , Weixing Qian , C. David Andersson , Daniel L. Hurdiss , Naresh Chandra , Rebecca Thompson , Lars Frängsmyr , Thilo Stehle , Niklas Arnberg , Mikael Elofsson Acs Chemical Biology DOI: 10.1021/acschembio.0c00446 Open Access Show Abstract ▼ Abstract: Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection, and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.	Aug 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Substrate plasticity of a fungal peptide α-N-methyltransferase Haigang Song , Jūratė Fahrig-Kamarauskaitė , Emmanuel Matabaro , Hannelore Kaspar , Sally L. Shirran , Christina Zach , Amy Pace , Bozhidar-Adrian Stefanov , James Naismith , Markus Künzler Acs Chemical Biology DOI: 10.1021/acschembio.0c00237 Open Access Show Abstract ▼ Abstract: The methylation of amide nitrogen atoms can improve the stability, oral availability and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harbouring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline depending on its sequence context can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.	Jun 2020
I04-Macromolecular Crystallography	Identification of phosphate-containing compounds as new inhibitors of 14-3-3/c-Abl protein-protein interaction 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 Acs Chemical Biology DOI: 10.1021/acschembio.0c00039 Show Abstract ▼ 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.	Mar 2020
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	3-Oxo-β-sultam as a sulfonylating chemotype for inhibition of serine hydrolases and activity-based protein profiling 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 Acs Chemical Biology DOI: 10.1021/acschembio.0c00090 Diamond Proposal Number(s): [20161] Show Abstract ▼ 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.	Mar 2020
I03-Macromolecular Crystallography	Targeting cavity-creating p53 cancer mutations with small-molecule stabilizers: the Y220X paradigm Matthias Bauer , Andreas Krämer , Giovanni Settanni , Rhiannon N Jones , Xiaomin Ni , Raysa Khan Tareque , Alan R. Fersht , John Spencer , Andreas C. Joerger Acs Chemical Biology DOI: 10.1021/acschembio.9b00748 Open Access Show Abstract ▼ 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.	Jan 2020
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Structural basis for inhibition of human primase by arabinofuranosyl nucleoside analogues fludarabine and vidarabine Sandro Holzer , Neil J. Rzechorzek , Isobel R. Short , Michael Jenkyn-bedford , Luca Pellegrini , Mairi Kilkenny Acs Chemical Biology DOI: 10.1021/acschembio.9b00367 Diamond Proposal Number(s): [9537] Show Abstract ▼ 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.	Sep 2019
B21-High Throughput SAXS	Double monoubiquitination modifies the molecular recognition properties of p15 PAF promoting binding to the reader module of Dnmt1 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 Acs Chemical Biology DOI: 10.1021/acschembio.9b00679 Diamond Proposal Number(s): [14707] Show Abstract ▼ 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.	Sep 2019

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