RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: Design, biochemical activity, and structural information

Article abstract of DOI:10.1021/jm900597q

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antivira

Full text of DOI:10.1021/jm900597q

J. Med. Chem. 2009, 52, 5781–5784 5781
DOI: 10.1021/jm900597q
RNase H Active Site Inhibitors of Human
Immunodeficiency Virus Type 1 Reverse
Transcriptase: Design, Biochemical Activity, and
Structural Information†
Thorsten A. Kirschberg,* Mini Balakrishnan,
Neil H. Squires, Tiffany Barnes, Katherine M. Brendza,
Xiaowu Chen, Eugene J. Eisenberg, Weili Jin, Nilima Kutty,
Stephanie Leavitt, Albert Liclican, Qi Liu, Xiaohong Liu,
John Mak, Jason K. Perry, Michael Wang, William
J. Watkins, and Eric B. Lansdon
Departments of Medicinal Chemistry, Biology, Drug Metabolism,
and Structural Chemistry, Gilead Sciences, 333 Lakeside Drive,
Foster City, California 94404
Received May 7, 2009
Figure 1. Electronic analysis of inhibitor classes in the context of
dual metal chelation.
Abstract: Pyrimidinol carboxylic acids were designed as inhibitors
of HIV-1 RNase H function. These molecules can coordinate to two
divalent metal ions in the RNase H active site. Inhibition of
enzymatic activity was measured in a biochemical assay, but no
antiviral effect was observed. Binding was demonstrated via a solid
state structure of the isolated p15-Ec domain of HIV-1 RT showing
inhibitor and two Mn(II) ions bound to the RNase H active site.
More recently, the effect of NNRTIs on the efficiency and
specificity of RNase H-mediated cleavage events (likely
through allosteric effects) has become a novel avenue for
research.5 Recent crystallographic studies of the RNase H
protein from Bacillus halodurans have elucidated the metal
requirements and ligand sphere events in catalysis of endo-
nucleolytic phosphodiester hydrolysis.6 This information, to-
gether with results obtained with mixtures of divalent metals in
assays of HIV-1 RT RNase H function, supports a bimetallic
mode of action involving Mg(II).7 The enzymatic active site
contains four carboxylate residues, creating an environment
capable of stabilizing two Mg ions in close proximity. Chelators
of these two ions in the enzyme active site are therefore regarded
as potential inhibitors of the function of HIV-1 RNase H.
Three different scaffold types have been reported as RNase
H active site metal chelators and are depicted in Figure 1.8 R,γ-
Diketo acids have often served as starting points for the design
and optimization of inhibitors against enzymes reliant on a
two-metal mechanism of action for endonucleolytic phospho-
diester hydrolysis such as influenza endonuclease, HIV-1 in-
tegrase, and FLAP endonuclease.9 The structure of a
moderately potent R,γ-diketo acid (1), the result of a study
targeting the RNase H function of HIV-1 RT, is depicted in
Figure 1.10 N-Hydroxyimides have been reported as potent
inhibitors of HIV-1 RNase H function, acting via active site
metal chelation. These compounds were first described as
inhibitors of influenza endonuclease, but their high potency
in biochemical assays of HIV-1 RNase H inhibition make these
structures valuable leads against this target.11 Selected hydro-
xytropolones and especially the natural product β-thujaplicinol
have been reported as potent inhibitors of HIV-1 RNase H.12
This class has been explored for inhibition of multiple HIV-1
enzymes including HIV-1 integrase and HIV-1 RT polymerase,
albeit with low to moderate selectivities and potencies.13
Earlier, hydroxytropolones were shown to have inhibitory
properties toward inositol monophosphatase. An inhibitory
mechanism invoking dual metal chelation was described.14
An overarching connection among the three inhibitor
classes emerges upon analysis of different resonance and
The inhibition of enzyme-mediated processes associated
with the life cycle of the human immunodeficiency virus type
1 (HIV-1a) has led to major advancements in the treatment of
patients suffering from AIDS. Nonetheless, the hallmark of
this human pathogen is the ease with which it is capable of
adapting to selection pressures via genetic mutation. Hence,
the search for new members of established drug classes as well
asthediscoveryofagentstoinhibit HIVvia novelmechanisms
of action continue to be important research goals worldwide.1
One of the three virally encoded enzymes is HIV-1 reverse
transcriptase (HIV-1 RT). This enzyme is responsible for the
reverse transcription of single-stranded viral RNA into dou-
ble-stranded DNA, utilizing RNA- and DNA-dependent
polymerization activities. Additionally, the enzyme catalyzes
the hydrolysis of RNA phosphodiester bonds in the RNA/
DNA hybrids produced during RNA-dependent DNA synth-
esis.2 These two functions are linked to two distinct and
spatially well-resolved active sites within the same protein.
Exploitation of binding at or near the polymerase active site
has proven fruitful for the development of nucleoside reverse
transcriptase inhibitors and non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs), respectively. On the other hand,
inhibition of the RNase H function of HIV-1 RT has proven
to be more difficult.3 Although inhibitors of biochemical
activity have been described as early as 1990, no approved
drugs or clinical development candidates have emerged.4
†The atomic coordinates and structure factors have been deposited in
the Protein Data Bank for p15-Ec in complex with compound 9; PDB
code 3HYF.
*To whom correspondence should be addressed. Phone: 650.522.
5133. Fax: 650.522.5899. E-mail: tkirschberg@gilead.com.
a Abbreviations: HIV-1, human immunodeficiency virus type 1; RT,
reverse transcriptase; NNRTI, non-nucleoside reverse transcriptase
inhibitor; SAR, structure-activity relationship.
r
2009 American Chemical Society
Published on Web 09/10/2009
pubs.acs.org/jmc

5782 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Kirschberg et al.
Table 1. Biochemical RNase H Inhibitory Potency of the Compoundsa
HIV-1 RNase H
IC50 [μM]
hu RNase H
IC50 [μM]
compd
1
6.5/13.2 (3.2)
0.12/0.15 (0.6-1)
0.60/0.72 (0.21)
5.9/6.1
nd
nd
2
3
nd
nd
Figure 2. Modified scaffolds derived from literature leads.
4
tautomeric forms of the core scaffolds, as depicted in Figure 1.
Limited SAR on the N-hydroxyimide class has revealed the
absolute requirement for one enolizable proton within the six-
membered imide ring structure.7 Consistent with this, an iso-
quinoline N-oxide is proposed as the biologically relevant
tautomer (20). A similar distribution of electron density around
the triad of coordinating oxygen atoms is found in the 6-π
aromatic resonance structure of the hydroxytropolone (30).
From both these species, a single deprotonation yields a doubly
negatively charged triad of oxygen atoms. The R,γ-diketo acid
system can be analyzed in a similar way: at physiological pH,
the carboxylic acid is deprotonated, as in structure 10. The
acidity of the 1,3-dicarbonyl system is anticipated to be suffi-
cient to generate small quantities of dianions under these
conditions. Supporting evidence for the biological relevance
of the dianion is found in studies of influenza endonuclease
inhibition by R,γ-diketoacids, where reduction of the pH of the
assay to below the second pKa of the diketoacid sharply
decreased the inhibitory activity.15 In all of these cases, there-
fore, a doubly anionic species may be necessary for effective
coordination of a putative inhibitor to both Mg ions in the
structural environment of the enzyme active sites.
5
22.4/23.9
11.4/14.5
2.1/2.9
nd
6
>100/>100
>100/>100
>100/>100
>100/>100
78/70
7
8
9.6/10.7
1.2/1.7
9
10
11
12
0.9/1.4
0.17/0.18
70.0/74.0
48/49
nd
a Inhibitory activities were determined via a fluorescence-based
RNase H assay (ref 21) using either HIV-1 RT or human RNase H enzyme.
Numbers in parentheses for compounds 1-3 are IC50s reported in the original
publications and are cited for comparison. All compounds had >95% purity
with exception of 1 (92.4%). nd = not determined.
Figure 3. Simple pyrimidinol carboxylic acids with C2 variants.
Scheme 1. General Access to Pyrimidinol Carboxylic Acidsa
While this analysis allows for the creation of a unifying
metal-chelating pharmacophore model for HIV RNase H,
scaffolds 1-3 all present intrinsic problems as starting points
for inhibitor design, mainly with respect to their stability
under aqueous conditions.16 For example, compound 2 de-
composes to uncharacterized more polar species in aqueous
solution at pH 7.4 at room temperature over hours
(unpublished results). The instability problem was solved via
the introduction of a nitrogen atom to yield the bicyclic
aromatic N-hydroxy quinazolinedione 4 (Figure 2).17 In the
hydroxytropolone series, the chelating scaffold was modified
via attachment of an aromatic ring, yielding compound 5,
with a geometrically altered oxygen triad (Figure 2). This
modification however resulted in a significant drop in potency
(Table 1). The enzyme-mediated synthesis of this revised
scaffold did not allow for a convenient exploration of
SAR.18 Tautomeric structures of the stabilized scaffolds A,
B, and C, emphasizing the pharmacophore model for dual
metal chelation, are depicted in Figure 1, and potencies of key
examples are reported in Table 1.
Pyrimidinol carboxylic acid derivatives were recently re-
ported as stable substitutes for R,γ-diketo acids and R,γ-
diketo amides.19 Hence, this scaffold was selected to study
the inhibition of HIV-1 RNase H, for it had already been
shown that customization of dual metal chelation via mod-
ification of the acidity of the central hydroxyl group was
feasible.20 The synthesis of compounds in this series utilized
the published four-step route outlined in Scheme 1.
Anticipating that the carboxylate and both phenolic hydro-
xyl groups would be important for dual metal chelation,
investigations focused on variations of the 2-substituent.
A simple first set of analogues is depicted in Figure 3, with
corresponding biochemical data in Table 1. All compounds
presented here were evaluated in a biochemical RNase H
assay as described in the literature.21
a (a) NH2OH (2ꢀ), MeOH, 50 °C, 14 h; (b) DMAD (1.05ꢀ) CHCl3,
60 °C; (c) m-xylenes, 130 °C 4 h; (d) NaOH, MeOH, H2O, then HCl aq.
These compounds proved informative for establishing in-
itial SAR within this series. A 2-phenyl substituted analogue
(6) displayed moderate inhibitory activity in the RNase H
cleavage assay with an IC50 value of 12.9 μM (average of two
experiments). The introduction of a methylene linker pro-
vided the most active compound (7) in this initial set (IC50
2.5 μM), contrasting with the SAR for inhibition of HCV
polymerase, which showed a clear preference for a phenyl or
thiophene group at C2 optimally substituted at the ortho
position.20 This observation is suggestive of a secondary
binding interaction of the side chain that could be exploited
for affinity and selectivity. Hence, further exploration focused
on benzyl variants of 7. Examples of key inhibitors are
depicted in Figure 4. Disubstituted analogues 9 and 10 proved
a little more potent when compared to 7. The most active
inhibitor within this series was compound 11 (IC50 0.18 μM),
which is more than 10-fold more potent than the initial lead 7.
Consistent with the dual metal chelating pharmacophore
model, only marginal inhibitory activity was observed for
compound 12, the methyl ester of compound 7. All pyrimidi-
nol carboxylic acids exhibited minimal to no inhibitory activ-
ity against the human RNase H enzyme (Table 1) consistent
with good selectivity for the viral enzyme.
Time of addition experiments were studied with compound 9
to support the mechanistic metal dependence of the inhibitor.
The assay was performed at a high enzyme to substrate ratio to
capture a large burst amplitude upon initiation of the reaction
(Figure 5A). Preincubation of the enzyme with inhibitor and
Mg before substrate addition significantly quenched the initial

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5783
burst of catalysis (Figure 5C). When the Mg ions were omitted
in this preincubation period (Figure 5B), enzymatic activity
more closely resembled the inhibitor free conditions, rendering
strong support for a metal dependent mechanism of action for
this inhibitor class with the physiologically relevant Mg. The
results are in agreement with data from similar experiments
using β-thujaplicinol.22
Compound 9 was successfully crystallized in the active site
of the truncated HIV-1 RNase H domain (p15-Ec) from a
solution containing Mn(II) ions as a surrogate for Mg(II). In
agreement with early structural work, two Mn(II) ions are
observed.23 The inhibitor is coordinated to both metal ions,
and the carboxylate is coordinated to the metal ion that is
mechanistically associated with the leaving group of the
hydrolysis step (i.e., the 30 hydroxyl group of the sugar),
corresponding to metal B in the B. halodurans structure.24 In
that work, the authors propose that the primary catalytic role
of metal B is to lower the activation barrier to the transition
state by destabilizing the enzyme-substrate ground state.
Metal A, by contrast, serves to activate a molecule of water
for nucleophilic attack. In the present structure, this metal ion
is coordinated by the two phenolic oxygen atoms of compound
9. The coordination geometry of metal A is octahedral, with
the remaining ligands derived from active site carboxylates and
two apical water molecules. This geometry is the most com-
mon in solid-state structures of Mg(II) coordinated by oxygen
ligands and is presumably energetically preferred.25 The li-
gands for metal B are less symmetrically disposed, with
geometry akin to that seen in the B. halodurans structure,
where the perturbation is proposed as the basis for the
destabilization of the enzyme-substrate ground state. In
addition to the chelation of the inhibitor to the metal ions, a
binding interaction is apparent between the C2 aromatic
substituent and the imidazole of histidine 539, consistent with
the improved potency observed with the methylene linker. The
distance from the center of the phenyl ring to the edge of the
˚
imidazole is 3.8 A and is best characterized as an edge-on π
interaction. The side chain orientation of histidine 539 depicted
Figure 4. Structures of pyrimidinol carboxylic acid derivatives.
in Figure 6 is consistent with the short distance between the Nε
˚
of the histidine and the outer phenolic oxygen (2.7 A) of the
ligand as well as the water network in the crystal structure.
It should be emphasized that the structure was obtained
with Mn(II) ions in the active site and that an analogous
binding modefor inhibitionofthe enzymecanonly beinferred
under the conditions of the biochemical assay, where only Mg
is present. The structure discussed here is consistent with data
from another metal chelating scaffold with respect to the
nature and stoichiometry of metals and inhibitors in isolated
HIV-1 RNase H domains.26
None of the pyrimidinol carboxylic acids discussed herein
displayed activity in a multicycle MT-4 HIV infectivity assay at
concentrations up to 100 μM. One or more reasons might
account for this lack of antiviral activity: insufficient intrinsic
potency, low cell permeability, and high protein binding, for
example, may all play a role. Modulation of physicoche-
mical properties, particularly those affecting binding to the
bovine serum albumin in the medium of the assay, was key to
achieving high cellular potency for a series of structurally
Figure 5. Time of addition experiment. HIV-1 RT was preincu-
bated in DMSO with Mg (A), or 9 without Mg (B), or 9 with Mg (C).
Enzymatic reactions were initiated by combining with substrate
solutions. All experiments used otherwise identical conditions (60
nM enzyme, 100 nM substrate, 5 mM MgCl2, 25 μM of 9, 2 min
preincubation). There is a 10 s delay between addition of the
substrate and recording of the first time point. No hydrolysis was
observed without enzyme (D).
˚
Figure 6. Crystal structure of inhibitor 9 bound in the active site of RNase H at 1.7 A resolution. (A) Composite omit map contoured at 1.5σ
showing 9 (blue) and both Mn atoms with the two tightly coordinated water molecules to metal A (orange). (B) Depiction of the complex
˚
highlighting the metal coordination. Interatomic distances are indicated in A. Key amino acids are labeled (HIV-1 RT numbering).

5784 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Kirschberg et al.
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complex wherein the coordination geometry of metal B is
similar to that responsible for the elevation of the ground-state
energy during catalytic phosphodiester hydrolysis might be
intrinsically too low for useful levels of inhibition to be observed.
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