I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
|
May 2020
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14043]
Abstract: The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
|
Jan 2020
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Irina
Kufareva
,
Benoit
Bestgen
,
Paul
Brear
,
Renaud
Prudent
,
Béatrice
Laudet
,
Virginie
Moucadel
,
Mohamed
Ettaoussi
,
Celine F.
Sautel
,
Isabelle
Krimm
,
Matthias
Engel
,
Odile
Filhol
,
Marc
Le Borgne
,
Thierry
Lomberget
,
Claude
Cochet
,
Ruben
Abagyan
Diamond Proposal Number(s):
[18548]
Abstract: CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.
|
Nov 2019
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14043]
Abstract: Unlike many other well-characterized bacteria, the opportunistic human pathogen Pseudomonas aeruginosa relies exclusively on the Entner–Doudoroff pathway (EDP) for glycolysis. Pyruvate kinase (PK) is the main “pacemaker” of the EDP, and its activity is also relevant for P. aeruginosa virulence. Two distinct isozymes of bacterial PK have been recognized, PykA and PykF. Here, using growth and expression analyses of relevant PK mutants, we show that PykA is the dominant isoform in P. aeruginosa. Enzyme kinetics assays revealed that PykA displays potent K-type allosteric activation by glucose 6-phosphate and by intermediates from the pentose phosphate pathway. Unexpectedly, the X-ray structure of PykA at 2.4 Å resolution revealed that glucose 6-phosphate binds in a pocket that is distinct from the binding site reported for this metabolite in the PK from Mycobacterium tuberculosis (the only other available bacterial PK structure containing bound glucose 6-phosphate). We propose a mechanism by which glucose 6-phosphate binding at the allosteric site communicates with the PykA active site. Taken together, our findings indicate remarkable evolutionary plasticity in the mechanism(s) by which PK senses and responds to allosteric signals.
|
Sep 2019
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18548]
Abstract: With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a non-conserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations (MIC).
|
Sep 2019
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18548]
Open Access
Abstract: The discovery of new Protein–Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, CAM7117, prevents the formation of the holoenzyme assembly in vitro, slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. CAM7117 could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.
|
Apr 2019
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[9537, 14043]
Abstract: The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa, expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa. We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle–glyoxylate shunt branch-point in P. aeruginosa has a complex enzymology which is profoundly different to that in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.
|
Jul 2018
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9537]
Abstract: Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC50 of 44 μM and a molecular weight of only 257 gmol−1 has been identified as the most promising compound. Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.
|
May 2018
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9537, 9007]
Open Access
Abstract: CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC50 = 7 μM, GI50 = 10.0 ± 3.6 μM), has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50).
|
Feb 2018
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9537, 9007]
Abstract: Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors.
|
Apr 2017
|
|
