Small molecule inhibition of SAMHD1 dNTPase by tetramer destabilization

Article abstract of DOI:10.1021/ja5035717

SAMHD1 is a GTP-activated nonspecific dNTP triphosphohydrolase that depletes dNTP pools in resting CD4+ T cells and macrophages and effectively restricts infection by HIV-1. We have designed a nonsubstrate dUTP analogue with a methylene bridge connecting the α phosphate and 5′ carbon that potently inhibits SAMHD1. Although pppCH₂dU shows apparent competitive inhibition, it acts by a surprising allosteric mechanism that destabilizes active enzyme tetramer.

Full text of DOI:10.1021/ja5035717

Communication
pubs.acs.org/JACS
Open Access on 07/01/2015
Small Molecule Inhibition of SAMHD1 dNTPase by Tetramer
Destabilization
Kyle J. Seamon,† Erik C. Hansen,† Anastasia P. Kadina,‡ Boris A. Kashemirov,‡ Charles E. McKenna,*,‡
Namandje N. Bumpus,† and James T. Stivers*,†
́
†Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, Maryland 21205-2185, United States
‡Department of Chemistry, Dana and David Dornsife College of Arts, Letters and Sciences, University of Southern California, 810
Downey Way, Los Angeles, California 90089-0744, United States
S
* Supporting Information
nucleoside products were determined using mass spectrometry
(Figure S1). Exclusive incorporation of 18O into the
ABSTRACT: SAMHD1 is a GTP-activated nonspecific
dNTP triphosphohydrolase that depletes dNTP pools in
resting CD4+ T cells and macrophages and effectively
restricts infection by HIV-1. We have designed a
nonsubstrate dUTP analogue with a methylene bridge
connecting the α phosphate and 5′ carbon that potently
inhibits SAMHD1. Although pppCH2dU shows apparent
competitive inhibition, it acts by a surprising allosteric
mechanism that destabilizes active enzyme tetramer.
triphosphate product confirms that nucleophilic attack occurs
at the α phosphate and that the leaving group is the sugar 5′
alcohol. It follows that if the 5′ oxygen is replaced by a
nonhydrolyzable moiety9,10 (such as a methylene group),
enzymatic cleavage of dNTP would be prevented. We therefore
synthesized a 5′ methylene dUTP (pppCH 2dU, 1) by the
route outlined in Scheme 1.
Scheme 1. Synthesis of pppCH2dU (1)
AMHD1 is a homotetrameric human enzyme with
S
nonspecific dNTP triphosphohydrolase activity that serves
to deplete the dNTP substrates of reverse transcriptase in
myeloid lineage target cells of HIV-1 such as resting T cells,
macrophages, and dendritic cells, resulting in viral restriction.1,2
Paradoxically, SAMHD1-related suppression of HIV infection
in dendritic cells may prevent their activation of CD4+ T cells
through the IFN1-toll receptor pathway, thereby preventing the
stimulation of a strong adaptive immune response by the
host.3,4 Thus, inhibitors of SAMHD1 could serve as useful tool
compounds to explore host adaptive immune responses in
these immune cells.5
SAMHD1 shows a complex activation mechanism. The
enzyme displays ordered essential activation by binding of GTP
to a guanine nucleotide-specific activator site (A1) on each
monomer which induces dimerization,6 followed by binding of
any dNTP to a second nonspecific activator site on each
subunit (A2) as well as the substrate sites.7 Thus, the active
tetramer contains four occupied pairs of activator sites (A1A2)4
and four catalytic sites.7,8 This mechanism of activation, which
involves coupling of nucleotide binding energy to drive
formation of the active tetramer, presents multiple sites for
the rational design of small molecule modulators of the enzyme
activity, including subunit interfaces. In this report we describe
the synthesis and characterization of a dUTP-derived inhibitor
of SAMHD1 that acts by the surprising mechanism of
preventing tetramer association.
Briefly, intermediate 3 was prepared by protection of the 3′-
and 5′-hydroxyl groups of deoxyuridine (dU) with tert-
butyldimethylsilyl chloride (TBDMSCl)11,12 followed by
selective removal of the 5′-silyl group from the product 2
with trichloroacetic acid.12,13 Adapting the procedure of
Schinazi,12 alcohol 3 was then oxidized with 2-iodoxybenzoic
acid, and the resulting aldehyde 4 was reacted with the sodium
salt of tetramethyl methylenebis(phosphonate) (TMMBP)
carbanion, leading to formation of the alkene 5, predominantly
as the trans-isomer. Compound 5 was hydrogenated in the
presence of Pd/C, affording the 3′-TBS protected dimethyl
ester of pCH2dU, 6.
As a mechanistic prelude to our studies, we first ascertained
whether SAMHD1 catalyzes attack of water at the α phosphate
or the 5′ carbon of the nucleotide sugar. Reactions of the self-
activating substrate dGTP were performed in 16O- and 18O-
substituted water, and the masses of the triphosphate and
Received: April 10, 2014
Published: July 1, 2014
© 2014 American Chemical Society
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The methyl- and t-butyldimethylsilyl-protecting groups were
removed by bromotrimethylsilane (BTMS),14,15 making
possible facile hydrolysis of the resulting trimethylsilyl
ester14,15 of 6 to pCH2dU, 7. It was previously reported16
that conventional BTMS deprotection of the diethyl ester of 7
resulted in significant anomerization of the nucleotide (β:α
anomer ratio 2:3). In contrast, we found that rapid microwave-
assisted BTMS silyldealkylation17 (MeCN, 60 °C, 7 min, MW),
followed by hydrolysis with H2O provided phosphonic acid 7
with minimal anomerization (β:α anomer ratio 8:1) (Figure
S14; the ratios are based on values obtained after conversion to
1). Although it was also possible to prepare 7 free of α-anomer
by HPLC purification, the 7 as obtained was more conveniently
first converted to the corresponding dUTP analogue 1 (via
coupling of morpholidate 8 with tributylammonium pyrophos-
phate (TBA/PPi) in DMSO),18,19 followed by isolation of the
pure β-anomer 1 by dual HPLC purification.17 The structures
of 1 and 5−7 were confirmed by 1H, 31P, and 13C NMR and by
HRMS.
We evaluated whether 1 was a substrate of SAMHD1 by
incubating 1 mM 1 with SAMHD1 in the presence and absence
of GTP activator (Figure S2). Compound 1 was found to be
completely unreactive even after 24 h incubation with
SAMHD1 regardless of whether GTP activator was present.
For comparison, we also evaluated the reactivity of dGTPαS
(Figure S2), which is known to occupy all activator and
substrate sites of SAMHD1 based on structural studies.8 Unlike
1, dGTPαS (racemic) was slowly hydrolyzed both in the
absence and presence of GTP activator (t1/2 ∼ 24 h), indicating
that this analogue is not only a slow substrate but also an
activator for its own hydrolysis. Compound 1 is the first
completely nonreactive substrate analogue of SAMHD1. The
inhibition mechanism of 1 was investigated using dUTP as the
substrate (Figure 1a). The entire data set was globally fitted to a
competitive inhibition model to obtain the inhibition constant
Ki = 80 6 μM. Thus, 1 appeared to follow simple competitive
inhibition with respect to dUTP with a Ki value that was a
surprising 20-fold lower than the Km for the substrate dUTP.6
Since the Ki of 1 was much less than the Km for dUTP and
dUTP acts as a coactivator by binding to the A2 sites,6 we
tested whether 1 might target the A2 site but fail to activate.
(The possibility that 1 binds antagonistically with GTP to the
A1 site is excluded because this site is highly specific for
guanine nucleotides and this type of inhibition would not
appear as competitive.)6 Our approach took advantage of the
previous finding that the A1 and A2 sites become inaccessible
to free ligands after formation of the tetramer, but that the
catalytic site remained accessible.6 Thus, we reasoned that
order-of-addition experiments where 1 was added before or
after dUTP and GTP might differentiate between inhibition
caused by binding of 1 to the A2 or catalytic site or both
(Figure 1b). To test for binding of 1 to the catalytic site, a
reaction of SAMHD1 with GTP and dUTP was initiated and
allowed to proceed for 2.5 min to ensure occupation of the A1
and A2 sites with GTP and dUTP and the sequestration of
these sites from free ligand. Then the reactions were spiked
with various concentrations of 1 and the subsequent rate of
dUTP consumption was followed over time (Figures 1b and
S3a). The Ki = 670 110 μM that was obtained under these
conditions was similar to the 1.5 mM Km for dUTP, suggesting
that when the A2 site was sequestered, 1 targeted the catalytic
site.6 In contrast, when GTP and 1 were preincubated with
SAMHD1 before addition of dUTP, a Ki = 52 5 μM was
Figure 1. Inhibition by 1. (a) Double-reciprocal plot of dUTP
hydrolysis in the presence of various fixed concentrations of 1 as
indicated. The reactions contain a saturating concentration of GTP
activator (5 mM). The entire data were fitted globally to a competitive
inhibition model to obtain the inhibition constant (Ki = 80 6 μM).
The two right panels show silver stained polyacrylamide gel images of
reactions (with and without 2 mM 1) that were quenched with the
cross-linking agent glutaradehyde. The monomer (M), dimer (D), and
tetramer (T) cross-linked forms of SAMHD1 are indicated. (b) Order
of addition experiments in which 1 is added before or after substrate
(see text). (c) DJK and corresponding cross-linking (DJXL)
experiment to evaluate the effects of 1 on the activity and oligomeric
state of SAMHD1 (see text). The various pre-jump conditions are
listed above the dotted lines and the post-jump reactions contained 1
mM dUTP in all cases. The post-jump delay times at which 50 mM
glutaraldehyde was added are indicated above the gel images and the
monomer (M), dimer (D), and tetramer (T) cross-linked forms are
indicated.
obtained, which is 30-fold lower than the Km for dUTP6
(Figures 1b and S3b). We conclude that 1 targets both the A2
and catalytic sites, but its potency largely arises from tight
binding to the A2 site.
Since 1 seemed to behave as a dUTP analogue, we wondered
why low concentrations of 1 that only populated the A2 site
gave rise to inhibition rather than activation. Our initial
hypothesis was that 1 might be antagonistic to formation of
active tetramer. This was confirmed by glutaraldehyde cross-
linking in the presence and absence of 1 under the conditions
of the inhibition experiment in Figure 1a.6 Separation of the
cross-linked forms by denaturing gel electrophoresis with
visualization by silver staining showed that 2 mM 1 completely
prevented tetramer formation in the presence of 1 mM dUTP
and 5 mM GTP (Figure 1a).
To further explore this surprising mechanism, we employed
our recently developed dilution-jump method that allows
investigation of the activity and stability of various oligomeric
forms of SAMHD1.6 This approach involved the formation of
complexes of SAMHD1 with activators and/or dNTPs at high
concentration (pre-jump condition) followed by rapid 100-fold
dilution into a solution with low activator and/or substrate
dNTP composition (post-jump condition). The post-jump
assay took two general forms: kinetic and structural. In the
structural mode the oligomeric state(s) that were present at the
time of dilution as well as their rate of decay in a given post-
jump condition, were probed using glutaraldehyde cross-linking
(DJXL). The dilution-jump kinetic (DJK) mode queried the
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time-dependent changes in catalytic activity after dilution into a
post-jump solution containing a radiolabeled substrate ([5-3H]
dUTP).
unusual inhibition mechanism that takes advantage of the
ordered essential activation mechanism and the ligand
specificities of the A1, A2, and catalytic sites.6,7 Antagonistic
binding of 1 to the A2 sites provides a reasonable explanation
for the unexpected long-term inhibition of tetramer formation
caused by 1 in the DJXL experiment (Figure 1c). That is, when
1 binds to the A2 sites in the pre-jump solution, it disrupts the
ordered-essential binding of GTP and dUTP, and since GTP is
absent in the post-jump, tetramer reassembly is not possible.
These findings reveal an unexpected diversity of inhibition
mechanisms for dNTP analogues of SAMHD1. While the slow
substrate and inhibitor dGTPαS occupies all activator and
substrate sites of SAMHD1 and induces tetramerization (Figure
S4),6,8 1 interferes with the ordered activation mechanism and
prevents tetramerization. The antithetical inhibitory mecha-
nisms of these two analogues and the multiple ligand binding
pockets on SAMHD1 indicate that this enzyme is highly
amenable to rational design of inhibitors and activators that
could modulate its activity in cells. Such ligands could have
research or therapeutic utility given the emerging role of
SAMHD1 in autoimmune and chronic inflammatory diseases20
and its established role in restricting viral infection of antigen-
presenting cells.21
The DJXL experiment assessed the effect of 1 on the
oligomeric states of SAMHD1. Without 1 in the pre-jump
solution, the presence of [dUTP + GTP] resulted in the
efficient formation of tetramer (Figure 1c), while in the absence
of these nucleotides only the monomer and dimer forms of
SAMHD1 were present.6 The tetramer generated in the pre-
jump persisted for hours even in the absence of GTP activator
in the post-jump, confirming our previous findings that the
tetramer is long-lived.6 When 1 was included in the pre-jump
solution, tetramer formation was completely abrogated as
judged by glutaraldehyde cross-linking, and the resulting
monomer and dimer forms persisted in the post-jump reaction
for at least 1 h (Figure 1c).
The DJK method probed the effect of 1 on the post-jump
enzymatic activity. The pre-jump solution consisted of
SAMHD1, GTP activator, and dUTP substrate in the absence
or presence of 1. The post-jump solution contained [5-3H]
dUTP substrate but no GTP activator. In the absence of 1 in
the pre-jump, SAMHD1 showed a burst-phase for dUTP
hydrolysis in the post-jump that decayed exponentially and
eventually yielded a linear steady-state rate that persisted for
hours (Figure 1c).6 We have attributed the burst-decay period
(t1/2 ∼ 10 min) to the decay of SAMHD1 from a highly active
form generated in the pre-jump solution to a less active but
long-lived form that persists throughout the steady-state period
even in the absence of GTP activator.6 When 1 (5 mM) was
added to the pre-jump solution, the post-jump burst-decay
amplitude was almost abolished, and the subsequent linear
steady-state rate was reduced indefinitely even though the
diluted concentration of pppCH2dU in the post-jump was 1/20
that of the dUTP substrate (Figure 1c).
ASSOCIATED CONTENT
* Supporting Information
■
S
Mechanistic mass spectometry, synthetic details and spectro-
scopic data for 1, inertness of 1 in the presence of SAMHD1,
and kinetics for inhibition of SAMHD1 by 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jstivers@jhmi.edu; mckenna@usc.edu
■
Notes
In conclusion, although we initially viewed 1 as an excellent
substrate mimic, the above data require that 1 binds to
SAMHD1 in a manner that is disruptive to both tetramer
formation and catalytic activity (Figure 2). The data suggest an
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by US National Institute of Health
grants T32GM008763, T32GM080189, GM056834 (J.T.S.),
GM103853 (N.N.B.) and U19CA177547 (C.E.McK.).
C.E.McK. thanks Dr. Samuel Wilson for suggesting this
collaboration, UCR High Resolution Mass Spectrometry
Facility and Ron New for performing HRMS.
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