I24-Microfocus Macromolecular Crystallography
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Indran
Mathavan
,
Lawrence J.
Liu
,
Sean W.
Robinson
,
Nelly
El-Sakkary
,
Adam Jo J.
Elatico
,
Darwin
Gomez
,
Ricky
Nellas
,
Raymond J.
Owens
,
William
Zuercher
,
Iva
Navratilova
,
Conor R.
Caffrey
,
Konstantinos
Beis
Diamond Proposal Number(s):
[12579]
Open Access
Abstract: Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivoS. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.
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Oct 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Ricardo A. M.
Serafim
,
Fiona J.
Sorrell
,
Benedict-Tilman
Berger
,
Ross J.
Collins
,
Stanley N. S.
Vasconcelos
,
Katlin B.
Massirer
,
Stefan
Knapp
,
James
Bennett
,
Oleg
Fedorov
,
Hitesh
Patel
,
William J.
Zuercher
,
Jonathan M.
Elkins
Diamond Proposal Number(s):
[15433]
Abstract: SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.
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Aug 2021
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I03-Macromolecular Crystallography
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Benjamin J.
Eduful
,
Sean N.
O'Byrne
,
Louisa
Temme
,
Christopher R. M.
Asquith
,
Yi
Liang
,
Alfredo
Picado
,
Joseph R.
Pilotte
,
Mohammad Anwar
Hossain
,
Carrow I.
Wells
,
William J.
Zuercher
,
Carolina M. C.
Catta-Preta
,
Priscila
Zonzini Ramos
,
André De S.
Santiago
,
Rafael M.
Counago
,
Christopher G.
Langendorf
,
Kévin
Nay
,
Jonathan S.
Oakhill
,
Thomas L.
Pulliam
,
Chenchu
Lin
,
Dominik
Awad
,
Timothy M.
Willson
,
Daniel E.
Frigo
,
John W.
Scott
,
David H.
Drewry
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.
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Jul 2021
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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
Diamond Proposal Number(s):
[442]
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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I03-Macromolecular Crystallography
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Carrow
Wells
,
Rafael M.
Counago
,
Juanita C.
Limas
,
Tuanny L.
Almeida
,
Jeanette Gowen
Cook
,
David H
Drewry
,
Jonathan M.
Elkins
,
Opher
Gileadi
,
Nirav R.
Kapadia
,
Alvaro
Lorente-Macias
,
Julie E.
Pickett
,
Alexander
Riemen
,
Roberta R.
Ruela-De-Sousa
,
Timothy M.
Willson
,
Cunyu
Zhang
,
William J
Zuercher
,
Reena
Zutshi
,
Alison D.
Axtman
Diamond Proposal Number(s):
[14664]
Abstract: Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.
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Oct 2019
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I02-Macromolecular Crystallography
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Megan J.
Agajanian
,
Matthew P.
Walker
,
Alison D.
Axtman
,
Roberta R.
Ruela-De-Sousa
,
D. Stephen
Serafin
,
Alex D.
Rabinowitz
,
David M.
Graham
,
Meagan B.
Ryan
,
Tigist
Tamir
,
Yuko
Nakamichi
,
Melissa V.
Gammons
,
James M.
Bennett
,
Rafael M.
Counago
,
David H.
Drewry
,
Jonathan M.
Elkins
,
Carina
Gileadi
,
Opher
Gileadi
,
Paulo H.
Godoi
,
Nirav
Kapadia
,
Susanne
Müller
,
André S.
Santiago
,
Fiona J.
Sorrell
,
Carrow I.
Wells
,
Oleg
Fedorov
,
Timothy M.
Willson
,
William J.
Zuercher
,
Michael B.
Major
Open Access
Abstract: β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
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Jan 2019
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I03-Macromolecular Crystallography
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Christopher
Asquith
,
Paulo
Godoi
,
Rafael M.
Counago
,
Tuomo
Laitinen
,
John W.
Scott
,
Christopher
Langendorf
,
Jonathan S.
Oakhill
,
David
Drewry
,
William
Zuercher
,
Panayiotis
Koutentis
,
Timothy
Willson
,
Andreas
Kalogirou
Diamond Proposal Number(s):
[14664]
Open Access
Abstract: We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.
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May 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Rafael M.
Counago
,
Charles K.
Allerston
,
Pavel
Savitsky
,
Hatylas
Azevedo
,
Paulo H.
Godoi
,
Carrow I.
Wells
,
Alessandra
Mascarello
,
Fernando H.
De Souza Gama
,
Katlin B.
Massirer
,
William J.
Zuercher
,
Cristiano R. W.
Guimarães
,
Opher
Gileadi
Diamond Proposal Number(s):
[12988, 10619]
Open Access
Abstract: The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.
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Dec 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Jonathan M
Elkins
,
Vita
Fedele
,
Marta
Szklarz
,
Kamal R
Abdul Azeez
,
Eidarus
Salah
,
Jowita
Mikolajczyk
,
Sergei
Romanov
,
Nikolai
Sepetov
,
Xi-Ping
Huang
,
Bryan L
Roth
,
Ayman
Al Haj Zen
,
Denis
Fourches
,
Eugene
Muratov
,
Alex
Tropsha
,
Joel
Morris
,
Beverly A
Teicher
,
Mark
Kunkel
,
David H
Drewry
,
William J
Zuercher
,
Stefan
Knapp
Abstract: Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein–coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.
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Oct 2015
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