Development of Pyridine-based Inhibitors for the Human Vaccinia-related Kinases 1 and 2

Article abstract of DOI:10.1021/acsmedchemlett.9b00082

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (26) displayed an IC₅₀ value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04). Differences in compound binding mode and substituent preferences between the two VRKs were identified by the structure-activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point for the design of more specific and potent inhibitors against each of the two VRKs.

Full text of DOI:10.1021/acsmedchemlett.9b00082

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Letter
Development of Pyridine-Based Inhibitors for
the Human Vaccinia-Related Kinases 1 and 2
Ricardo Augusto Massarico Serafim, Fernando H. de Souza Gama, Luiz Antonio Dutra,
Caio V. dos Reis, Stanley N. S. Vasconcelos, André da Silva Santiago, Jéssica E
Takarada, Fúlvia Di Pillo, Hatylas Azevedo, Alessandra Mascarello, Jonathan M Elkins,
Katlin B. Massirer, Opher Gileadi, Cristiano R.W. Guimarães, and Rafael M. Couñago
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00082 • Publication Date (Web): 19 Aug 2019
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Development of Pyridine-Based Inhibitors for the Human Vaccinia-
Related Kinases 1 and 2
Ricardo A. M. Serafim, 1,2,# Fernando H. de Souza Gama, 3,# Luiz A. Dutra, 1,2,# Caio V. dos Reis, 1,2
Stanley N. S. Vasconcelos, 1,2 André da Silva Santiago, 1,2 Jéssica E. Takarada, 1,2 Fúlvia Di Pillo, 4
Hatylas Azevedo, 3 Alessandra Mascarello, 3 Jonathan M. Elkins, 2,5 Katlin B. Massirer, 1,2 Opher
Gileadi, 5 Cristiano R. W. Guimarães, 3,* Rafael M. Couñago 1,2,*
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1 Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade
Estadual de Campinas (UNICAMP), Campinas, SP, 13083-875, Brazil
2 Structural Genomics Consortium, Departamento de Genética e Evolução, Instituto de Biologia, UNICAMP, Campinas, SP,
13083-886, Brazil
3 Aché Laboratórios Farmacêuticos S.A., Guarulhos, SP, Brazil
4 PhD Program in Genetics and Molecular Biology (PGBM), UNICAMP, Brazil
5 Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, UK.
Vaccinia-related kinases, aminopyridine, difluorophenol, kinase inhibitors, structure-based compound development.
ABSTRACT: Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell
division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their
therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and
elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (18)
displayed an IC50 value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04).
Differences in compound binding mode and substituent preferences between the two VRKs were identified by the series structure-
activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point
for the design of more specific and potent inhibitors against each of the two VRKs.
The human genome encodes three vaccinia-related kinases
(VRK1, VRK2, and VRK3).1 VRK3 has a degraded ATP-
binding site and is thought to be a pseudo-kinase,2 while VRK1
and VRK2 feature active kinase domains and have been
associated with various cellular processes, including cell
replication, chromatin remodeling, and response to DNA
damage.3–5 VRK2 has two isoforms that share identical kinase
domains but locate to distinct cellular compartments:
endoplasmic reticulum (VRK2A); and cytoplasm and nucleus
(VRK2B).1,6 VRK1 can be found in both nucleus and
cytoplasm.1 Genetic studies have associated VRK1 and VRK2
with a number of human diseases and conditions. VRK2 is
associated with neurological disorders, such as schizophrenia,
multiple sclerosis, major depressive disorder, and epilepsy.7–11
VRK1 overexpression is associated with increased cell division
and poor prognosis in a number of cancers.12,13 Despite these
advances, our understanding of the cellular roles of human
VRKs and their therapeutic potential is limited. Currently, there
are no potent and specific small molecule inhibitors of the
VRKs.
family also includes MAP4K4, whose phosphate-binding loop
(P-loop, also referred to as glycine-rich loop) can adopt an
unusual conformation in which this conserved structural motif
folds over the ligand. It has been suggested that this folded P-
loop conformation might be targeted to obtain potent and
selective kinase inhibitors.16 Co-crystal structures of VRK1 and
VRK2 bound to the p90 RSK (ribosomal S6 kinase) inhibitor
BI-D1870 (1, Figure 1A) revealed that both kinases can also
adopt the folded P-loop conformation, further suggesting that
MAP4K4 and these two VRKs have similar topography and
structural features in their ATP-binding sites.15 BI-D1870
displays a highly developed pteridine scaffold and recent
selectivity data revealed this compound to be quite
promiscuous.17 These observations motivated us to search for
MAP4K4 ligands known to induce
a folded P-loop
conformation and that could be used as starting points to
develop potent and selective inhibitors against VRK1 and
VRK2.
2-aminopyridines are well-known kinase inhibitor scaffolds
and such compounds (exemplified by 2, Figure 1A) have been
shown to promote a folded P-loop conformation in MAP4K4.18
Additionally, this scaffold is small and versatile, allowing the
attachment of different substituents on both sides of the
aminopyridine core by a short synthetic route. The co-crystal
Previously, our analysis of the Published Kinase Inhibitor Set
(PKIS)14 revealed that VRK1 shared a similar activity profile to
a small number of kinases, including members of the kinase
family Ste20, such as MINK1 and TNIK.15 The Ste20 kinase
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structures of VRK1 and VRK2 bound to 1 (BI-D1870) also
polarities and electronic proprieties, but none of these changes
resulted in an improved ΔTm over the difluorophenol moiety.
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revealed that the ligand difluorophenol moiety promoted
favorable polar interactions to structurally-conserved residues
within the VRK ATP-binding site.15 Thus, our design strategy
to develop new small molecule inhibitors for VRK1 and VRK2
was to generate hybrid ligands by fusing the aminopyridine core
with the difluorophenol group.
We next explored the ability of various phenyl derivatives in
R2 to optimize the compound binding profile, while maintaining
a difluorophenol group in R1 (Supplementary Table S1). Our
results confirmed that polar groups in R2 are important for
binding the VRKs and indicated that, overall, these groups were
better tolerated in the meta position (10 vs. 11; 12 vs. 13; 14 vs.
15 and 16), although the addition of a carboxylic acid group in
this position was detrimental to binding (17). For VRK2-KD,
the best compound had a para-sulfonamide group on the R2
phenyl ring (18), although a benzothiazole fused ring (19) was
also tolerated in this position. VRK1-FL tolerated both of these
groups in R2 equally well. Nevertheless, for VRK1, we could
not find a R2 substituent that increased Tm-shifts compared to
the difluorophenol moiety (5). For VRK2-KD, further attempts
to modify the difluorophenol in R1 while maintaining the
sulfonamide group in R2 were unsuccessful (20, 21 and 22).
(A)
F
O
N
N
OH
F
Cl
N
NH2
N
N
9
N
N
H
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H2N
N
1
2
BI-D1870
(B)
R1
Br
Y
R1
X
R2
Y
I
Br
Y
R1 B(OH)2
R2 B(OH)2
Pd(dppf)Cl2 (10 mol%)
Cs2CO3 (2.5 eq)
EtOH:H2O (5:1)
Pd(dppf)Cl2 (10 mol%)
Cs2CO3 (2.5 eq)
EtOH:H2O (5:1)
X
N
X
N
N
MW, 85 °C, 1 h
MW, 85 °C, 1 h
Table 1 - Structures and values for DSF and enzyme
inhibition assays for key compounds
X, Y = H or NH2 or CH3
Figure 1: (A) Chemical structure of 1 (BI-D1870) and the
MAP4K4 inhibitor 2. (B) Synthetic route to aminopyridine
derivatives.
DSF ΔTm (°C)
IC50 / 95% CIb
(nM)VRK1
#
1
Structure
VRK1
VRK2
Synthesis of aminopyridine derivatives was performed using
a rapid and efficient two-step Suzuki-Miyaura coupling (Figure
1B) with yields ranging between 18% and 89%. Each step was
performed by a microwave-assisted reaction in one hour.
Details of the procedure are available in the Supporting
Information.
F
O
N
N
OH
F
N
33 /
24-44
5.5a
4.3a
N
N
H
F
F
HO
F
OH
F
260 /
202-332
To test our hybridization hypothesis, we prepared prototype
compound 5, bearing difluorophenol moieties in both, the 3-
and 5- positions of the 2-aminopyridine core (Table 1). We
5
3.2 ± 0.3 2.2 ± 0.2
2.1 ± 0.7 3.2 ± 0.0
2.2 ± 0.2 3.0 ± 0.0
H2N
N
F
HO
F
SO2NH2
employed
a thermal-shift assay (Differential Scanning
18
19
NDc
Fluorimetry, DSF) to identify aminopyridine analogues that
could bind to the full-length VRK1 (residues Met1-Lys396,
VRK1-FL) and the kinase domain of VRK2 (residues Pro14-
His335, VRK2-KD). DSF is a robust method to estimate
binding affinities and has been used successfully to triage large
compound libraries.14,19,20 For these experiments, we considered
positive hits those compounds inducing changes in VRK1-FL
and VRK2-KD melting temperatures, ΔTm, larger than 2.0ꢀ°C.
DSF analysis indicated that 5 induced thermal shifts above this
threshold for both VRK1 and VRK2 (Table 1, Supplementary
Figure S1).
H2N
N
F
HO
F
N
S
674 /
530-860
H2N
F
N
N
F
HO
F
OH
F
150 /
120-188
26
3.5 ± 0.3 1.1 ± 0.2
a values from Couñago et al. 2017; b confidence interval; c not
determined
Encouraged by these results, we prepared a first series of 2-
aminopyridine analogues to explore the relevance of the
difluorophenol moiety to the thermal stabilization of the two
VRKs (Table 1, Supplementary Table S1). For both proteins,
simultaneously replacing the difluorophenol moiety with
phenol groups in R1 and R2 (6) resulted in a drop of the observed
ΔTm, suggesting that increasing the phenol acidic character or
altering the electronic properties of the aromatic ring was
important for binding.21,22 VRK1-FL seemed to tolerate the
substitution of a difluorophenol by a phenol on either R1 or R2
position equally well, whereas maintaining a difluorophenol
group in R1 proved to be more important for VRK2-KD binding
(8 and 7). Not unexpectedly, removal of the HB (hydrogen
bond) capacity in R2, whilst maintaining the difluorophenol
group in R1, still resulted in a ΔTm drop for both VRKs (9). We
attempted to improve compound binding by replacing the
difluorophenol in R1 with a variety of groups of distinct sizes,
Although robust, DSF provides only limited information on
compound binding to a target protein. We then developed an
enzyme inhibition assay (Supplementary Figure S2) and
determined the potency (IC50) of key compounds against
VRK1-FL (Table 1, Figure 2, Supplementary Table S2). In
these assays, our prototype compound 5 had an IC50 value of
260 nM, ~8-fold higher than the one observed for 1 (BI-D1870,
33 nM). Estimated IC50 values were in good agreement with our
DSF results. For example, 19, with a ΔTm of 2.2 °C, had an IC50
of 674 nM against VRK1-FL (Supplementary Figure S3). More
importantly, our enzymatic assays confirmed these compounds
as inhibitors of VRK1.
Unfortunately, we did not succeed in developing a similar
enzymatic assay for VRK2. Thus, we employed Isothermal
Titration Calorimetry (ITC) to estimate binding potency (KD) of
selected compounds to VRK2-KD, including 18, our top
compound from the DSF assay (Figure 2, Supplementary
Figure S4). Our ITC data confirmed binding of these
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compounds to VRK2-KD, and revealed these interactions to be
mostly enthalpy-driven (|ΔH| > |TΔS|) (Supplementary Table
S3). As expected, 18 had the strongest binding to VRK2 (KD
~400 nM). At the moment, we cannot offer an explanation for
the discrepancy between ITC and DSF results for 13 and 19.
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Figure 2: Biochemical, biophysical and selectivity profile characterization of key compounds. (A) IC50 determination curves for 1, 5 and 26
against VRK1-FL. Individual data points are the mean ± SD of three technical replicates. Estimated IC50 values are shown in parenthesis.
(B,C) ITC measurements for top compounds in the DSF assay for VRK2-KD (18) and VRK1-FL (26). Values for binding energies are shown
in kJ/mol. (D) Selectivity profile of 1 (BI-D1870), 5 and 26 against a representative panel of 48 human kinases at 1 µM concentration. Colors
denote % residual kinase activity: green - residual activity >80%; yellow - residual activity < 40% and < 80%; red - residual activity < 40%.
Residual activity values are shown in Supplementary Table S5. Numbers in parenthesis report the selectivity score S(50%) for each
compound. S(50%) was obtained by dividing the number of protein kinases having % residual activity < 50%, by the total number of tested
protein kinases. Distribution of selected kinases across the human kinome is shown in Supplementary Figure S7. The VRKs were not part
of the panel.
To better understand the molecular basis for ligand binding,
we obtained co-crystals of VRK1-KD (residues Arg3-Glu364)
bound to 5 and VRK2-KD bound to 18 (Figure 3). Both
structures were solved by molecular replacement
(Supplementary Table S4). Ligand-bound VRK1-KD and
VRK2-KD adopted an active conformation, in which conserved
residues from structurally-conserved elements of the kinase
domain (Glu83 and Lys71 within αC-helix and the ATP-
binding site, respectively - VRK1 numbering) are brought into
close proximity and the side chain of Asp197 (VRK1
numbering) from the conserved DFG motif (DYG in the VRKs)
facilitated HBs with main chain carbonyl and amide,
respectively, from VRK1-KD hinge residue Phe134.
The co-crystal structures helped us rationalize the relevance
of the difluorophenol moiety for binding. Regardless of
compound binding pose, this group facilitated an HB network
with polar side chains from structurally-conserved residues
within the kinase domain of VRK1 (Lys71 and Glu83) and
VRK2 Lys61 and Glu73). The difluorophenol group
participating in these contacts displayed distinct dihedral angles
to the 2-amino core depending on its attachment position: ~45°
in R1 and ~9° in R2. In VRK1, these different orientations of the
difluorophenol group were accommodated by a corresponding
movement of the side chain from residue Met131, which
occupies the gatekeeper position in this protein. Consequently,
the difluorophenol group fitted tightly between the αC-helix
and the gatekeeper residue in both poses. These observations
might explain why we could not find substituents that improved
binding over the difluorophenol group.
points towards the ATP-binding site (Figure
Supplementary Figure S5A,B).
3 and
As expected, 5 and 18 were found in the ATP-binding sites
of VRK1 and VRK2, respectively (Figure 3A,B). The binding
pose for 18 showed the compound 2-amino moiety pointed
towards the back of VRK2 ATP-binding site. The compound 2-
amino group and its pyridine N atom established one hydrogen
bond each to the carbonyl and amide groups of VRK2 hinge
residues Glu122 and Leu124, respectively. In VRK1-KD
crystals, the ligand could be observed in three out of the four
protein molecules in the asymmetric unit and, surprisingly, was
found in two different poses. The first of these was equivalent
to the one observed for 18 bound to VRK2-KD. In the second
binding mode, the 2-amino group of 5 pointed towards the
solvent and, together with the pyridine nitrogen atom,
The VRK2-KD co-crystal structure also revealed that the 18
sulfonamide group pointed away from the protein ATP-binding
site and was mostly solvent-exposed. A similar observation was
made for the difluorophenol group in 5 that did not interact with
VRK1-KD αC-helix (Supplementary Figure S5D-F). Our DSF
results also indicated that placement of polar groups in the
meta-position resulted in slight increases of ΔTm, specially for
VRK2-KD (10 vs. 11, for example). At this position, polar
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groups from the ligand might be able to engage polar groups
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P-loop did not fold over 18. In our VRK2 co-crystal, the P-loop
was found rotated towards the protein αC-helix by ~6 Å
(Supplementary Figure S5C). Consequently, equivalent
aromatic residues within the P-loop of VRK1 (Phe48) and
VRK2 (Phe40) occupied different positions in each of the
proteins’ ATP-binding site.
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from VRK2-KD P-loop.
Regardless of the ligand binding pose, the P-loop of VRK1 was
found to be folded over 5. This conformation was likely
stabilized by hydrophobic interactions observed between P-
loop residue Phe48 and 5 three-ring system. By contrast, VRK2
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Figure 3: Co-crystal structures revealed distinct compound binding modes. (A, B) VRK1-KD bound to 5. The compound adopts two
different binding modes with different VRK1 molecules in the crystal. (C) VRK2-KD bound to 18. Black dashed lines represent potential
hydrogen bonds. Bottom panels show electron density maps (2Fo-Fc) contoured at 1.0 σ.
The two binding modes observed for 5 in VRK1 suggested
that the 2-amino moiety had no binding preference for either of
the hinge carbonyl groups it can interact with (Figure 3A,B).
This led us to hypothesize that these two interactions were
either equally productive or equally weak in the binding
process. To address these hypotheses, we synthesized the
following analogues: (i) 23, with two amino groups that could
interact with both hinge carbonyl groups simultaneously; (ii)
24, with a 2-amino and a space-filling 6-methyl group; (iii) 25,
with the 2-amino group removed; and (iv) 26 with the 2-amino
group substituted by a 2-methyl group (Table 1, Supplementary
Table S1).
Further, the increase in ΔTm observed for 26 (2-methyl
pyridine core) over 25 (pyridine core) for VRK1 might arise
from favorable electron-donating properties or conformational
effects conferred to the pyridine ring system by the presence of
an adjacent methyl group.23,24 Alternatively, the lack of a
substituent group in the 2-position may incur a free energy
penalty associated with 25 inability to fill in a cavity in the
protein binding pocket.
Enzyme inhibition assays further confirmed 26 as a VRK1
inhibitor with an IC50 value of ~150 nM, a ~2-fold increase in
potency over prototype compound 5 (Figure 1A, Table1). ITC
experiments determined that binding of 26 to VRK1-FL was
enthalpy-driven and had a KD of 190 nM (Figure 1C,
Supplementary Table S3).
DSF assays revealed that none of these new analogs had
improved ΔTm values for VRK2-KD (Table 1, Supplementary
Table S1). These results suggested that the HB between the
hinge carbonyl group and the 2-aminopyridine core is a
productive interaction for VRK2. Likewise, for VRK1-FL,
compounds 23, 24 and 25 did not improve ΔTm values over
those observed for 5. Poor results observed for 23 and 24 might
be explained by clashes between one of the two substituents in
these compounds (at the 2- or 6-position in the pyridine core)
and main chain atoms from residues within the kinase hinge
region.
The 2-amino-pyridine moiety is a common feature of many
kinase inhibitors, and often plays a role in binding to the kinase
hinge region (Supplementary Figure S6).25 We were surprised
that changes in hinge-binding groups within the pyridine core
had such marked impact on compound binding to each of the
VRKs, as these proteins have highly similar ATP-binding sites.
Motivated by these findings, we assessed the activity of 1, 5 and
26 against a representative panel of 48 human kinases at 1 µM
concentration (Figure 2D, Supplementary Figure S7). Our
results showed that introduction of the methyl group in the
pyridine scaffold markedly improved selectivity of 26 over
prototype 5 - S(50%) scores of 0.04 and 0.25, respectively. Our
selectivity data also confirmed previous reports that 1 (BI-
D1870) is fairly promiscuous17 - S(50%) score of 0.19.
By contrast, 26 and 5 were equipotent in the DSF assay,
supporting the hypothesis that the 2-amino moiety contributed
little to the binding of 5 to VRK1. Thus, both our DSF results
and crystal structures suggested that VRK1, but not VRK2, was
capable of binding equally well to the 2-aminopyridine core in
two different conformations.
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analyzed ITC experiments for VRK1-FL. C.V.R., A.S.S., and
To ascertain that 18 and 26 represented tractable molecules
for development into chemical probes suitable for biological
studies, we performed cell viability assays based on the
metabolic conversion of a tetrazolium dye (MTT, see
Supporting Information) to its insoluble formazan by human
(HeLa) cells. Our results indicated that there was no noticeable
effect on cell viability after a 24-hour treatment with 3.2 µM of
18 and only a slight decrease with 3.2 µM of 26; and > 60% of
cells remained viable after the compound concentration was
raised to 12.5 µM of 18 and 25 µM of 26 (Supplementary Figure
S8).
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L.A.D. designed, performed, and analyzed DSF experiments.
F.D.P. designed, performed and analyzed cell assays. H.A., A.M.,
J.M.E., K.B.M., O.G., C.R.W.G. and R.M.C. coordinated the
project. R.A.M.S. and R.M.C. wrote the manuscript. All authors
revised the manuscript. All authors have given approval to the
final version of the manuscript. # These authors contributed
equally.
Funding Sources
This work was supported by the Brazilian agencies FAPESP
(Fundação de Amparo à Pesquisa do Estado de São Paulo)
(2013/50724-5 and 2014/5087-0), Embrapii (Empresa Brasileira
de Pesquisa e Inovação Industrial), CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico) (465651/2014-3).
The SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck
KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario
Ministry of Economic Development and Innovation, Pfizer,
Takeda, and Wellcome [106169/ZZ14/Z]. R.A.M.S. and S.N.S.V.
received FAPESP postdoctoral fellowships (2016/25320-6 and
2018/09475-5, respectively). C.V.R and A.S.S. received CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)
postdoctoral fellowships (88887.146077/2017-00 and
88887.136342/2017-00, respectively).
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In conclusion, we report here the use of the pyridine scaffold
for the development of inhibitors against VRK1 and VRK2. Our
data revealed 26 to be a selective (S(50%) 0.04) and potent (IC50
~150 nM) inhibitor of VRK1. Compound 18 binds to VRK2 in
the mid-nM range (ITC KD 400 nM). Further characterization
of this compound is likely to confirm 18 as an inhibitor of the
VRK2 protein. Both compounds are not overtly toxic to human
cells and represent promising starting points for developing
more potent and selective inhibitors for VRK1 and VRK2.
Future optimization efforts will take advantage of the scaffold´s
good synthetic accessibility.
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website. The Supporting Information file contains:
Supplementary Tables S1-S5, Supplementary Figures S1-S8,
Supplementary Methods, and Supplementary References.
Notes
The authors F.H.S.G., H.A., A.M., C.R.W.G. are employed by
Aché Laboratórios Farmacêuticos. All other authors declare no
competing financial interest.
Accession Codes
The atomic coordinates and crystallographic structures factors for
VRK1-KD bound to 5 and for VRK2-KD bound to 18 have been
deposited in the Protein Data Bank (www.rcsb.org) with accession
codes: 6BU6 and 6NCG, respectively.
ACKNOWLEDGMENT
We thank the staff of the Proteomics section of the Life Sciences
Core Facility (LaCTAD), part of the University of Campinas
(UNICAMP), for their contributions to ITC analysis. We thank the
staff at the Northeastern Collaborative Access Team beamlines
(GU56413 and GU54127), which are funded by the National
Institute of General Medical Sciences from the National Institutes
of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C
beam line is funded by a NIH-ORIP HEI grant (S10 RR029205).
This research used resources of the Advanced Photon Source, a
U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National
Laboratory under Contract No. DE-AC02-06CH11357.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rafael.counago@unicamp.br;
cristiano.guimaraes@ache.com.br.
ORCID
Ricardo A. M. Serafim: 0000-0003-0614-1798
Fernando H. de Souza Gama: 0000-0002-3745-1046
Luiz A. Dutra: 0000-0002-3316-8526
Stanley N. S. Vasconcelos: 0000-0001-7289-5753
Caio V. dos Reis: 0000-0001-6271-7523
André da Silva Santiago: 0000-0002-3973-3370
Jessica E. Takarada: 0000-0001-7311-8665
Fulvia Di Pillo: 0000-0002-3496-0574
Hatylas Azevedo: 0000-0001-9233-0696
Alessandra Mascarello: 0000-0001-7672-4070
Jonathan M. Elkins: 0000-0003-2858-8929
Katlin B. Massirer: 0000-0001-6390-2560
Opher Gileadi: 0000-0001-6886-898X
ABBREVIATIONS
VRK, Vaccinia-related kinase; KD, kinase domain; FL, full-length;
HB, hydrogen bond; MTT, 3-(4,5-Dimetrylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide.
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