Structure-activity analysis and cell-based optimization of human galactokinase inhibitors

Article abstract of DOI:10.1021/ml200131j

Classic galactosemia is a rare human disease associated with the accumulation of a toxic level of galactose-1-phosphate (gal-1P) caused by the inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity. To reduce the toxic level of ga

Full text of DOI:10.1021/ml200131j

LETTER
pubs.acs.org/acsmedchemlett
StructureÀActivity Analysis and Cell-Based Optimization of Human
Galactokinase Inhibitors
Sina I. Odejinmi,† Rafael G. Rascon,† Manshu Tang,† Hariprasad Vankayalapati,‡ and Kent Lai*,†
†Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah 84132,
United States
‡Center for Investigational Therapeutics, Huntsman Cancer Institute, The University of Utah, Salt Lake City, Utah 84112, United States
S Supporting Information
b
ABSTRACT: Classic galactosemia is a rare human disease associated with
the accumulation of a toxic level of galactose-1-phosphate (gal-1P) caused
by the inherited deficiency of galactose-1-phosphate uridyltransferase
(GALT) activity. To reduce the toxic level of gal-1P in patients, we have
identified, via high-throughput screening, over 200 small molecule GALK
inhibitors. We selected a 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile
scaffold for further structureÀactivity relationships characterization, lead
optimization with regards to potency, and efficacy to reduce gal-1P
accumulation in patient cells.
KEYWORDS: Galactokinase, galactose-1-phosphate, dihydrothiazinone,
GHMP kinases, galactosemia
lassic galactosemia (CG) is an inherited metabolic condition
caused by deficiency of galactose-1-phosphate uridyltrans-
therapy, one must elucidate the pathogenic mechanisms of the
disease and identify useful therapeutic targets. Although the
precise pathophysiology of CG remains unexplained, decades
of clinical observations,14,15 confirmed recently by prospective
outcome study,16 showed that patients with galactokinase
(GALK) deficiency rarely manifest the chronic complications
seen in GALT-deficient patients. Note that GALK-deficient
patients do NOT accumulate 10À20-fold increases of gal-1P
over their lifetime as do GALT-deficient patients even when on
galactose-restricted diets. Others and we confirmed these clinical
findings in a Saccharomyces cerevisiae (baker's yeast) model for
CG. While a GALT-deficient mutant yeast was sensitive to
galactose in growth medium, disruption of GALK function in
this mutant reversed its susceptibility to galactose toxicity.17À19
These findings support the pathological role of gal-1P in GALT
deficiency in humans and yeast models for CG but also raise the
question about the origin of gal-1P, the enzymatic product of
GALK on galactose, in a galactosemic patient who refrains from
dairy products. It has been found that galactose moieties con-
verted to gal-1P can also come from nondairy sources, for
example, galactose-containing fruits and vegetables amounting
to as much as 30 mg/day.20 However, galactose moieties can also
be produced endogenously from UDP-glucose via the UDP-4-
galactose epimerase (GALE) reaction, as well as from the natural
turnover of glycolipids and glycoproteins. In fact, using isotopic
labeling, Berry et al. elegantly demonstrated that a 50 kg adult
male could produce up to 1.2 g of galactose per day, which is
C
ferase (GALT, EC 2.7.7.12) activity.1 GALT is the second
enzyme in the evolutionarily conserved Leloir pathway of
galactose metabolism and facilitates the simultaneous conversion
of uridine diphosphoglucose (UDP-glucose) and galactose-
1-phosphate (gal-1P) to uridine diphosphogalactose (UDP-
galactose) and glucose-1-phosphate.2 Consequently, GALT de-
ficiency leads to the accumulation of gal-1P and a deficiency of
UDP-galactose in patient cells.3,4 If untreated, CG can result in a
lethal disease in the affected newborn.5 Ever since most states in
the United States included this disorder in the newborn screen-
ing panel, neonatal morbidity and mortality have decreased
considerably. The current mainstay of treatment is the with-
drawal of (ga-)lactose from the diet.5 However, it has become
clear that despite optimal dietary management, chronic compli-
cations such as IQ deficits, ataxia, speech dyspraxia, premature
ovarian insufficiency, and decreased bone mineralization persist
in many affected adults.6,7 In the past few years alone, at least six
groups of investigators reported that the health-related quality of
life consequences of galactosemic patients and their parents were
worse than generally thought.8À13 Such an outcry of concerns for
a relatively rare disease suggested that the stressful conditions
suffered by the patients and their family members have been
underestimated for too long, and swift actions are required to
improve the current situation. Moreover, little can we deny that
the failure of galactose-restricted diet to prevent secondary
complications has slowly eroded the early success of newborn
screening of this genetic disorder, and the medical community is
yearning for a more effective therapy. To develop a more effective
Received: March 11, 2011
Accepted: July 4, 2011
Published: July 04, 2011
r
2011 American Chemical Society
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Figure 1. Lead compounds selected for SAR studies.
many times the amount of exogenous galactose potentially
present in galactose-restricted diets.21 Therefore, endogenous
synthesis of galactose is likely to undermine the efficacy of dietary
management as a standard therapy. Because endogenous galac-
tose production is not amenable to dietary manipulation, there is a
need for innovative, nondietary therapy. Because gal-1P, an enzy-
matic product of GALK, is a major culprit for the complications
seen in CG patients and GALK deficiency is more manageable than
GALT deficiency, a few investigators have advocated the inhibition
of human GALK as an innovative approach to treat CG.16,22
Previously, we initiated a campaign to identify small molecule
inhibitors for the human GALK enzyme.23,24 We hypothesized
that GALK deficiency induced pharmacologically in GALT-defi-
cient patients would significantly reduce gal-1P accumulation and
prevent the chronic outcomes. Lastly, as for the uniqueness of the
therapeutic target, human GALK is unique because even though it
phosphorylates galactose, it does not belong to the same family of
other sugar kinases such as glucokinase (E.C. 2.7.1.2) or hexokinase
(E.C. 2.7.1.1). Instead, it belongs to the superfamily of small
molecule kinases, also known as the GHMP (Galactose, Homo-
serine, Mevalonic acid, Phosphomevalonic acid) kinases family.22,25
In our previous work, we identified over 150 small molecules
inhibitors of human GALK via high-throughput screening (HTS).23
We selected 34 compounds for further characterization.24 Although
their IC50 values were determined as 200 nM to 33 μM, their
selectivity among the individual GHMP kinases varied, and some
were shown to be toxic to cells.24 Thus, these first-generation
compounds will require further optimization for therapeutic use.
In this study, we chose 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbo-
nitrile (compound 1) (Figure 1) for structureÀactivity relationship
(SAR) studies because it shares a similar aromatic core and
functionality array with many of the positive hits.24 The IC50 of com-
pound 1 in the in vitro galactokinase inhibitory assay was 12 μM;
therefore, our major objective was to improve its potency. We began
by exploring nine commercially available compounds with structural
similarities to compound 1 (Figure 1). These compounds carried
structural modifications in the A ring or the C ring with none in
the central 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile core.
In the in vitro GALK inhibitory assays, only compounds 2, 3, and
4 possessed inhibitory activities against GALK, while the remaining
compounds were not active below 50 μM.
On the basis of these results, we rationalized that the presence
of hydroxyl functional group(s) is necessary on the aryl ring A,
since the active compounds have either a hydroxyl or a carboxylic
acid group. However, not all of the compounds with hydroxyl
groups on the A ring displayed inhibitory activity as is the case
with compound 7. This compound shares the same structural
similarity to compound 1 except that it has a thiobutyl group
instead of the aromatic C ring. Therefore, the aromatic C ring is
deemed necessary for activity since it was absent in compound 7.
Another observation in our limited SAR studies revealed that
compound 3 having 2-hydroxy aryl substituent in the A ring was
active, whereas compound 10 with the same thiomethyl sub-
stituent at the sixth position having 2,4-dichloro substituents in
the A ring was inactive. This clearly suggests that electronic-
withdrawing hydrophobic groups at the 2- and 4-positions may
not be tolerated in the A ring for activity. Compound 9, however,
has a spiroketal carboxyl group instead of an aryl carboxyl group
that exhibited no activity. Similarly, compounds 5, 6, and 8 that
do not have aromatic A ring did not display any inhibitory
activity. This further underscores the preference of an aromatic
ring as the A ring adjacent to the 4-oxo-3,4-dihydro-2H-1,3-
thiazine-5-carbonitrile core.
Through the above SAR studies, the IC50 of compound 2 with
two aryl hydroxyl groups from this selection was determined to
be in the low micromolar range (1.4 ( 0.7 μM) for GALK in
contrast to the original compound 1, which has an IC50 of 12 μM.
To modify compound 2 for further optimization of potency, it
was considered necessary to identify the functional groups that
are needed for potency. As a result, new compounds with different
positions of the hydroxyl groups in the A ring were designed
and synthesized by a procedure described by Yokoyama26,27 where
many commercially available aldehydes such as aldehyde 11 were
treated with mercaptoacrylamide 1227 in the presence of catalytic
HCl in MeOH under refluxing conditions and afforded solid
compound 2 in 61% yield (Scheme 1).
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The IC50 values of these compounds were subsequently
determined and used to assess the efficacy of the positions of
the aryl hydroxyl groups in the newly designed inhibitors
(Table 1). Results showed that compound 14 with the meta-
hydroxyl group exhibited a very high IC50 of 50 μM. When the
hydroxyl group was changed to the para position for compound
15, the potency of this compound dramatically improved to an
IC50 of 1.8 μM. When this aryl hydroxyl group was substituted
with ÀOCH3 functionality in compound 16, the GALK inhibi-
tory activity was abolished (IC50 > 50 μM).
Scheme 1. General Synthesis of 4-Oxo-3,4-dihydro-2H-1,3-
thiazine-5-carbonitrile by Refluxing with Acidic MeOH
Similarly, in compound 17, the substitution for benzo[d]-
[1,3]dioxole group caused an increase in the IC50 (23 μM). The
thiazinone compounds with 2,4- and 2,5-aryl hydroxyl substitu-
tions were assayed for inhibitory activities, and compound 19
with the 2,5-dihydroxy functional group was more potent than
the 2,4-dihydroxy thiazione derivative 18.
It was previously realized through computational modeling
studies that the aromatic C ring of the 4-oxo-3,4-dihydro-2H-1,3-
thiazine-5-carbonitrile core mimics the adenine moiety of ATP
Table 1. IC50 Values of Synthesized Thiazinones Derivatives of Compound 1a
a Experimental conditions for IC50 determination are provided in the Supporting Information. Standard deviations were calculated from three replicate
measurements.
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Figure 2. Mode of binding of compound 25 in complex with GALK. The active site ATP pocket is shown with critical residues that are in contact with
central 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile. The endocyclic amideÀArg228 interactions are depicted as dashed yellow lines. Similarly, the
dihydroxy-substituted aryl ring is positioned to γ-phosphate binding site and positioned within the distance to Mg2+. The Tyr109 is also within the
distance for wedge-face ππ interactions with morpholine-substituted aryl ring.
Scheme 2. Synthesis of Substituted Morpholine Derivatives of Compounds 2, 15, and 19
within the active site of human GALK crystal structure as shown
in Figure 2. Reference protein coordinates used for structure-
based lead optimization were taken from the X-ray structure of
the human GALK in complex with the nonhydrolyzable ATP
analog, AMP-PNP (PDB: 1WUU),28 and were used in all
computational experiments using the ICM suite. The active site
for the GALK protein was defined as being within 8 Å of AMP-
PNP in the X-ray cocrystal structure. Energy grids representing
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Table 2. IC50 Values of Synthesized Morpholine Derivatives
25, 27, and 29a
Therefore, we proceeded to determine the intracellular inhibi-
tory activity of morpholine derivative 25 against GALK by
measuring the reduction of gal-1P accumulation. This compound
inhibited GALK in patient fibroblast cells and lowered cellular
gal-1P by 16% at 50 μM.
Additionally, morpholine derivative 27 was also well tolerated
in cells at up to 100 μM and reduced cellular gal-1P by 16% at
100 μM without gross cell lethality, while morpholine derivative
29 was proved to be lethal to cells at 100 μM or higher. However,
at 50 μM, this compound was well-tolerated. Unlike compound
25, neither 27 nor 29 was able to inhibit GALK or lowered gal-1P
at 50 μM.
Unknown cellular side reactions with 4-oxo-3,4-dihydro-2H-
1,3-thiazine-5-carbonitrile scaffold might have limited the inhi-
bitory potential of these morpholine derivatives to further lower
the cellular gal-1P below 16% at 50 or 100 μM. On the basis of
this observation, we have considered the design and synthesis of
new 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile scaffold
containing compounds that might be much less prone to cellular
side reactions, and our ongoing efforts will be reported in the
near future.
a Experimental conditions for IC50 determination are provided in the
Supporting Information. Standard deviations were calculated from three
replicate measurements.
In summary, we have conducted limited SAR studies and
optimization on a selected 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-
carbonitrile chemotype identified previously as a GALK inhibitor
and was initially toxic. The new series of compounds showed
improved in vitro potency, improved cell tolerance, and efficacy
to partially inhibit GALK in cell-based assays. Because the
accumulation of gal-1P plays a pathological role in the disorder
CG, the discovery of the oxo-3,4-dihydro-2H-1,3-thiazine-5-
carbonitrile scaffold substituted with morpholine is first in the
class of new compounds that can be explored as therapeutic
agents to lower cellular gal-1P in patient cells.
the active site (van der Waals, hydrogen bonding, electrostatics,
and hydrophobic interactions) were calculated with 0.5 Å grid
spacing, and docking experiments were performed using the
defined AMP-PNP binding pocket with the application of our
docking workflow.
The meta position to the thiobenzyl group of the C ring could
be modified with a morpholine group to further stabilize the
binding of the molecule to the adenine binding site. Computa-
tional studies suggested that this group was well tolerated within
the 4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile scaffold and its
binding mode within the ATP site of GALK. Hence, morpholine
derivatives of compounds 2, 15, and 19 were synthesized with the
aim to further improve the potency (Scheme 2). We chose the
4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile scaffold 2, 15, and
19 (Table 1) to prepare new morpholine derivatives 25, 27, and 29
(Scheme 2) because we had previously determined that the
compounds 2, 15, and 19 had IC50 values of 1.4, 1.8, and 0.7 μM,
respectively (Table 1), which were among the lowest IC50 values in
our in vitro GALK assays. As a result, the preparation of their
morpholine derivatives was pursued by the condensation of acryla-
mide 23 with aldehydes 24, 26, and 28 (Scheme 2).
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
spectral data for the test compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: kent.lai@hsc.utah.edu.
The synthesis of morpholine 25 (Scheme 2) began with the
transformation of benzylic alcohol 20 to bromide 21 by the
addition of NBS/DMS cocktails.29 A slow addition of bromide
21 to the solution of bis(ammoniothio)-2-cyanoacrylamide 22
selectively afforded acrylamide 23 in 55% from alcohol 20. The
synthesis of morpholine 25 was completed by the treatment of
acrylamide 23 with aldehyde 24 under acid-catalyzed conditions
in MeOH with a fair yield of 19%. We determined the IC50 values
of the morpholine derivatives, and the results are shown in
Table 2.
Initially, the intracellular inhibition of GALK and the subse-
quent lowering of gal-1P for compound 2 with an IC50 value of
1.4 μM could not be determined in patient cells because of its
toxicity. In the subsequent cell-based studies, compound 2 in its
protected form as diacetate 13 (Scheme 1) continued to be toxic
to the human fibroblast cells at either 50 μM or higher concen-
trations and resulted in total cell lethality within hours. However,
compound 25 with an IC50 value of 1.2 μM (Table 2) was
tolerated at 50 μM by the cells without visible signs of lethality.
Funding Sources
This work was supported by U.S. National Institutes of Health
Grants 5R01HD054744-05 and 3R01HD054744-04S1.
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