I02-Macromolecular Crystallography
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Alfredo
Picado
,
Apirat
Chaikuad
,
Carrow I.
Wells
,
Safal
Shrestha
,
William J.
Zuercher
,
Julie E.
Pickett
,
Frank E.
Kwarcinski
,
Parvathi
Sinha
,
Chandi S.
De Silva
,
Reena
Zutshi
,
Shubin
Liu
,
Natarajan
Kannan
,
Stefan
Knapp
,
David H.
Drewry
,
Timothy M.
Willson
Abstract: STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.
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Nov 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Yousuke
Yamada
,
Hajime
Takashima
,
David Lee
Walmsley
,
Fumihito
Ushiyama
,
Yohei
Matsuda
,
Harumi
Kanazawa
,
Toru
Yamaguchi-sasaki
,
Nozomi
Tanaka-yamamoto
,
Junya
Yamagishi
,
Risa
Kurimoto-tsuruta
,
Yuya
Ogata
,
Norikazu
Ohtake
,
Hayley
Angove
,
Lisa
Baker
,
Richard
Harris
,
Alba
Macias
,
Alan
Robertson
,
Allan
Surgenor
,
Hayato
Watanabe
,
Koichiro
Nakano
,
Masashi
Mima
,
Kunihiko
Iwamoto
,
Atsushi
Okada
,
Iichiro
Takata
,
Kosuke
Hitaka
,
Akihiro
Tanaka
,
Kiyoko
Fujita
,
Hiroyuki
Sugiyama
,
Roderick E.
Hubbard
Diamond Proposal Number(s):
[12428]
Abstract: UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 μg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.
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Nov 2020
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I02-Macromolecular Crystallography
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Zoltan
Szlavik
,
Marton
Csekei
,
Attila
Paczal
,
Zoltan B.
Szabo
,
Szabolcs
Sipos
,
Gabor
Radics
,
Agnes
Proszenyak
,
Balazs
Balint
,
James
Murray
,
James
Davidson
,
Ijen
Chen
,
Pawel
Dokurno
,
Allan E
Surgenor
,
Zoe Marie
Daniels
,
Roderick E.
Hubbard
,
Gaëtane
Le Toumelin-braizat
,
Audrey
Claperon
,
Gaëlle
Lysiak-auvity
,
Anne-marie
Girard
,
Alain
Bruno
,
Maia
Chanrion
,
Frédéric
Colland
,
Ana-leticia
Maragno
,
Didier
Demarles
,
Olivier
Geneste
,
Andras
Kotschy
Diamond Proposal Number(s):
[2103]
Abstract: Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.
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Nov 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Christopher
Douse
,
Iva A.
Tchasovnikarova
,
Richard T.
Timms
,
Anna V.
Protasio
,
Marta
Seczynska
,
Daniil M.
Prigozhin
,
Anna
Albecka
,
James
Wagstaff
,
James C.
Williamson
,
Stefan M. V.
Freund
,
Paul J.
Lehner
,
Yorgo
Modis
Diamond Proposal Number(s):
[11235]
Open Access
Abstract: The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.
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Oct 2020
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I02-Macromolecular Crystallography
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Weimei
Sun
,
Xuqing
Zhang
,
Maxwell D.
Cummings
,
Kamal
Albarazanji
,
Jiejun
Wu
,
Mina
Wang
,
Richard
Alexander
,
Bin
Zhu
,
Yuemei
Zhang
,
James
Leonard
,
James
Lanter
,
James
Lenhard
Abstract: Inhibition of the serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of metabolic diseases. Camostat has been used clinically for treating chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (kinact/KI) of 1.5 x 104 M-1s-1. High-resolution LC-MS showed addition of 161.6 Da to EP following reaction with camostat, consistent with insertion of the carboxyphenylguanidine moiety of camostat. Covalent inhibition of EP by camostat is reversible, with an enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19Å, showing modification of the catalytic serine of EP by a close analog of camostat leading to formation of the carboxyphenylguanidine acyl enzyme identical to that expected for reaction with camostat. Of particular note, minor structural modifications of camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and body weight, with concomitant improved blood glucose levels in obese and diabetic ob/ob mice. Thus, the structure-activity relationship (SAR) needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of metabolic diseases, and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy.
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Oct 2020
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I02-Macromolecular Crystallography
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Gary
Tresadern
,
Ingrid
Velter
,
Andrés A.
Trabanco
,
Frans
Van Den Keybus
,
Gregor J.
Macdonald
,
Marijke V. F.
Somers
,
Greet
Vanhoof
,
Philip M.
Leonard
,
Marieke B. A. C.
Lamers
,
Yves E. M.
Van Roosbroeck
,
Peter J. J. A.
Buijnsters
Diamond Proposal Number(s):
[5603]
Abstract: We describe the hit-to-lead exploration of a [1,2,4]triazolo[1,5-a]pyrimidine phosphodiesterase 2A (PDE2A) inhibitor arising from high-throughput screening. X-ray crystallography enabled structure-guided design, leading to the identification of preferred substructural components. Further rounds of optimization used relative binding free-energy calculations to prioritize different substituents from the large accessible chemical space. The free-energy perturbation (FEP) calculations were performed for 265 putative PDE2A inhibitors, and 100 compounds were synthesized representing a relatively large prospective application providing unexpectedly active molecules with IC50′s from 2340 to 0.89 nM. Lead compound 46 originating from the FEP calculations showed PDE2A inhibition IC50 of 1.3 ± 0.39 nM, ∼100-fold selectivity versus other PDE enzymes, clean cytochrome P450 profile, in vivo target occupancy, and promise for further lead optimization.
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Oct 2020
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I02-Macromolecular Crystallography
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Petra
Dzianová
,
Seiya
Asai
,
Martina
Chrudinová
,
Lucie
Kosinová
,
Pavlo
Potalitsyn
,
Pavel
Šácha
,
Romana
Hadravová
,
Irena
Selicharová
,
Jan
Kříž
,
Johan P.
Turkenburg
,
Andrzej Marek
Brzozowski
,
Jiří
Jiráček
,
Lenka
Žáková
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.
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Oct 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7641, 9475, 13467]
Abstract: Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, this data provides important new insights to the factors determining stability, substrate recognition and the structural mechanism of hydrolysis in this industrially important group of enzymes.
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Oct 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Sander
Herfst
,
Jie
Zhang
,
Mathilde
Richard
,
Ryan
Mcbride
,
Pascal
Lexmond
,
Theo M.
Bestebroer
,
Monique I. J.
Spronken
,
Dennis
De Meulder
,
Judith M.
Van Den Brand
,
Miruna E.
Rosu
,
Stephen R.
Martin
,
Steven J.
Gamblin
,
Xiaoli
Xiong
,
Wenjie
Peng
,
Rogier
Bodewes
,
Erhard
Van Der Vries
,
Albert D. M. E.
Osterhaus
,
James C.
Paulson
,
John J.
Skehel
,
Ron A. M.
Fouchier
Diamond Proposal Number(s):
[9826, 13775]
Abstract: In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.
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Oct 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14631, 17180, 20015]
Open Access
Abstract: The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor ‘example 172’ (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.
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Oct 2020
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