I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.
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Sep 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Abstract: α-mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297, and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer associated N-glycosylation.
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Jul 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[306, 7864]
Abstract: Solvent organization is a key but underexploited contributor to the thermodynamics of protein–ligand recognition, with implications for ligand discovery, drug resistance and protein engineering. Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavourable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP’s enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand or both. This suggests that solvent structure is an evolutionary con-straint on protein sequence that contributes to ligand affinity and selectivity.
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Sep 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zoltan
Szlávik
,
Levente
Ondi
,
Márton
Csékei
,
Attila
Paczal
,
Zoltán B.
Szabó
,
Gábor
Radics
,
James
Murray
,
James
Davidson
,
Ijen
Chen
,
Ben
Davis
,
Roderick E.
Hubbard
,
Christopher
Pedder
,
Pawel
Dokurno
,
Allan
Surgenor
,
Julia
Smith
,
Alan
Robertson
,
Gaetane
Letoumelin-braizat
,
Nicolas
Cauquil
,
Marion
Zarka
,
Didier
Demarles
,
Francoise
Perron-sierra
,
Audrey
Claperon
,
Frederic
Colland
,
Olivier
Geneste
,
András
Kotschy
Diamond Proposal Number(s):
[17182, 1194, 2103]
Abstract: Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using nuclear magnetic resonance and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compounds, and cellular activity was brought on scale by offsetting the negative charge of the anchoring carboxylate group. The obtained compounds described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compound, the chemical series leading to the identification of our more advanced compounds S63845 and S64315.
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Jul 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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James B.
Murray
,
James
Davidson
,
Ijen
Chen
,
Ben
Davis
,
Pawel
Dokurno
,
Christopher J.
Graham
,
Richard
Harris
,
Allan
Jordan
,
Natalia
Matassova
,
Christopher
Pedder
,
Stuart
Ray
,
Stephen D.
Roughley
,
Julia
Smith
,
Claire
Walmsley
,
Yikang
Wang
,
Neil
Whitehead
,
Douglas S.
Williamson
,
Patrick
Casara
,
Thierry
Le Diguarher
,
John
Hickman
,
Jerome
Stark
,
András
Kotschy
,
Olivier
Geneste
,
Roderick E.
Hubbard
Diamond Proposal Number(s):
[671, 1194, 17182]
Abstract: We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.
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May 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Douglas S.
Williamson
,
Garrick P.
Smith
,
Pamela
Acheson-dossang
,
Simon T.
Bedford
,
Victoria
Chell
,
I-jen
Chen
,
Justus C. A.
Daechsel
,
Zoe
Daniels
,
Laurent
David
,
Pawel
Dokurno
,
Morten
Hentzer
,
Martin C.
Herzig
,
Roderick E.
Hubbard
,
Jonathan D.
Moore
,
James B.
Murray
,
Samantha
Newland
,
Stuart C.
Ray
,
Terry
Shaw
,
Allan E.
Surgenor
,
Lindsey
Terry
,
Kenneth
Thirstrup
,
Yikang
Wang
,
Kenneth V.
Christensen
Diamond Proposal Number(s):
[5791, 12428, 2103, 14641, 5067]
Abstract: Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]-pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-pt. mut. structure). Compound 22 was shown to be potent, moderately selective, orally available and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 nM and 5 nM, respectively.
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Oct 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Open Access
Abstract: Modulation of enzyme activity is a powerful means of probing cellular function and can be exploited for diverse applications. Here, we explore a method of enzyme activation where covalent tethering of a small molecule to an enzyme can increase catalytic activity (kcat/KM) up to 35-fold. Using a bacterial glycoside hydrolase, BtGH84, we demonstrate how small molecule “fragments”, identified as activators in free solution, can be covalently tethered to the protein using Michael-addition chemistry. We show how tethering generates a constitutively-activated enzyme-fragment conjugate, which displays both improved catalytic efficiency and increased susceptibility to certain inhibitor classes. Structure guided modifications of the tethered fragment demonstrate how specific interactions between the fragment and the enzyme influence the extent of activation. This work suggests that a similar approach may be used to modulate the activity of enzymes such as to improve catalytic efficiency or increase inhibitor susceptibility.
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Sep 2017
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I04-Macromolecular Crystallography
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Open Access
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.
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Nov 2015
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221]
Open Access
Abstract: Fragment-based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small-molecule enzyme activators. Herein, we describe the discovery and characterization of small-molecule activators of a glycoside hydrolase (a bacterial O-GlcNAc hydrolase). A ligand-observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90?% increase in kcat/KM?values (kcat=catalytic rate constant; KM=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM?and Vmax?values (Vmax=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in?vivo, and could also be used for glycoside hydrolase activation in many industrial processes.
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Dec 2014
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Fragment-based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small-molecule enzyme activators. Herein, we describe the discovery and characterization of small-molecule activators of a glycoside hydrolase (a bacterial O-GlcNAc hydrolase). A ligand-observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in kcat/KM values (kcat=catalytic rate constant; KM=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM and Vmax values (Vmax=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.
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Dec 2014
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