I24-Microfocus Macromolecular Crystallography
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Lídia Dos Passos
Lima
,
Dev
Sriranganadane
,
Daiane Laise
Da Silva
,
Natália C.
Drebes Dörr
,
Enzo Breviglieri Sichi
Mello
,
Caio Vinicius
Dos Reis
,
Rogério Ferreira
Lourenço
,
José Felipe Teixeira Da Silva
Santos
,
Anita
Salmazo
,
Brenno Wendler
Miranda
,
Katlin B.
Massirer
,
Rafael M.
Counago
,
Cristina E.
Alvarez-Martinez
Open Access
Abstract: Serine/threonine (Ser/Thr) kinases of the Hanks-type family are widespread in bacteria, playing key roles in signal transduction. The transmembrane Ser/Thr kinase PknS (XAC4127) from the phytopathogenic bacterium Xanthomonas citri is required for the expression of a type VI secretion system, which confers resistance to predation by the soil amoeba Dictyostelium discoideum. PknS exerts its function via activation of the cognate ECF-type alternative sigma factor EcfK, ultimately triggering the expression of type VI secretion system (T6SS) genes. In this study, we characterize PknS, demonstrating its ability to undergo autophosphorylation both in vitro and within X. citri cells. Structural analysis of the PknS kinase domain revealed the conservation of the canonical fold characteristic of Hanks-type kinases. PknS directly phosphorylates EcfK at five Ser/Thr residues located in two distinct regions of the sigma factor: the conserved σ2 domain (residue T51) and a nonconserved linker connecting domains σ2 and σ4 (residues T104, T106, S108, and S110). The conserved residue T51, previously shown to be essential for sigma factor activity in an EcfK homolog, corresponds to a site that directly interacts with the RNA polymerase β′ subunit. Site-directed mutagenesis analyses further revealed that the conserved residue T106 is also critical for EcfK function. Structural studies indicated that, in addition to T51, phosphorylation at T106 activates EcfK by promoting its interaction with a positively charged pocket within the RNA polymerase β′ subunit. Collectively, our findings describe a previously unknown signal transduction pathway involving a Hanks-type kinase and a sigma factor, providing new insights into the mechanisms of sigma factor activation via phosphorylation in bacteria.
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Jan 2026
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I03-Macromolecular Crystallography
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Han Wee
Ong
,
Xuan
Yang
,
Jeffery L.
Smith
,
Sharon
Taft-Benz
,
Stefanie
Howell
,
Rebekah J.
Dickmander
,
Tammy M.
Havener
,
Marcia K.
Sanders
,
Jason W.
Brown
,
Rafael M.
Counago
,
Edcon
Chang
,
Andreas
Kramer
,
Nathaniel J.
Moorman
,
Mark
Heise
,
Alison D.
Axtman
,
David H.
Drewry
,
Timothy M.
Willson
Diamond Proposal Number(s):
[36057]
Open Access
Abstract: The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
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Fernando H.
De Souza Gama
,
Luiz A.
Dutra
,
Michael
Hawgood
,
Caio Vinícius
Dos Reis
,
Ricardo A. M.
Serafim
,
Marcos A.
Ferreira
,
Bruno V. M.
Teodoro
,
Jéssica Emi
Takarada
,
André S.
Santiago
,
Dimitrios-Ilias
Balourdas
,
Ann-Sofie
Nilsson
,
Bruno
Urien
,
Vitor M.
Almeida
,
Carina
Gileadi
,
Priscila Z.
Ramos
,
Anita
Salmazo
,
Stanley N. S.
Vasconcelos
,
Micael R.
Cunha
,
Susanne
Mueller
,
Stefan
Knapp
,
Katlin B.
Massirer
,
Jonathan M.
Elkins
,
Opher
Gileadi
,
Alessandra
Mascarello
,
Bennie B. L. G.
Lemmens
,
Cristiano R. W.
Guimarães
,
Hatylas
Azevedo
,
Rafael M.
Counago
Abstract: Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.
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May 2024
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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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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[16171]
Open Access
Abstract: Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.
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Oct 2020
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I24-Microfocus Macromolecular Crystallography
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Gerson S.
Profeta
,
Caio V.
Dos Reis
,
André Da S.
Santiago
,
Paulo H. C.
Godoi
,
Angela M.
Fala
,
Carrow I.
Wells
,
Roger
Sartori
,
Anita P. T.
Salmazo
,
Priscila Z.
Ramos
,
Katlin B.
Massirer
,
Jonathan M.
Elkins
,
David H.
Drewry
,
Opher
Gileadi
,
Rafael M.
Counago
Diamond Proposal Number(s):
[16171]
Open Access
Abstract: Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.
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Nov 2019
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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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