Fragment-Based Discovery of Dual JC Virus and BK Virus Helicase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00486

There are currently no treatments for life-threatening infections caused by human polyomaviruses JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of JCV large T antigen (apo) and its use to drive the structure-based design of dual JCV and BKV ATP-competitive inhibitors. The crystal structures obtained by soaking our early inhibitors into the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from 18 μM for the early 6-(2-methoxyphenyl)- and the 6-(2-ethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole hits 1a and 1b to 0.6 μM for triazolopyridine 12i. In addition, we were able to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the absence of marked cytotoxicity, thus confirming the usefulness of this approach.

Full text of DOI:10.1021/acs.jmedchem.6b00486

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Article
Fragment-based discovery of dual JC virus and BK virus helicase inhibitors
Dominique Bonafoux, Suganthini Nanthakumar, Upul K. Bandarage, Christine
Memmott, Derek Bates Lowe, Alex M. Aronov, Govinda Bhisetti Rao, Kenny Bonanno,
Joyce Coll, Joshua Leeman, Christopher A. Lepre, Fan Lu, Emanuele Perola, Rene
Rijnbrand, William P. Taylor, Dean Wilson, Yi Zhou, Jacque Zwahlen, and Ernst ter Haar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00486 • Publication Date (Web): 07 Jul 2016
Downloaded from http://pubs.acs.org on July 7, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Fragmentꢀbased discovery of dual JC virus and BK
virus helicase inhibitors
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Dominique Bonafoux,* Suganthini Nanthakumar, Upul K. Bandarage, Christine Memmott,
a
Derek Lowe, Alex M. Aronov, Govinda Bhisetti Rao, Kenneth C. Bonanno, Joyce Coll, Joshua
b
Leeman, Christopher A. Lepre, Fan Lu, Emanuele Perola, Rene Rijnbrand, William P. Taylor,
c
Dean Wilson, Yi Zhou, Jacque Zwahlen, Ernst ter Haar*
Vertex Pharmaceuticals, Incorporated 50 Northern Avenue, Boston, Massachusetts 02210,
United States
a
:
Current address. Biogen Idec, 225 Binney Street, Cambridge, MA 02142, United States
Current address. Arbutus Biopharma Incorporated, 3805 Old Easton Road, Doylestown,
b
:
Pennsylvania 18902, United States
c
:
Current address. Assembly Biosciences, 409 Illinois Street, San Francisco, CA 94158, United
States
KEYWORDS: JCV, BKV, helicase, ATPꢀcompetitive, fragment based drug design
ABSTRACT
There are currently no treatment for lifeꢀthreatening infections caused by human polyomaviruses
JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of
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JCV large T antigen (apo) and its use to drive the structureꢀbased design of dual JCV and BKV
ATPꢀcompetitive inhibitors. The crystal structures obtained by soaking our early inhibitors into
the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from
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18 ꢀM for the early 6ꢀ(2ꢀmethoxyphenyl)ꢀ and the 6ꢀ(2ꢀethoxyphenyl)ꢀ[1,2,4]triazolo[3,4ꢀ
b][1,3,4]thiadiazole hits 1a and 1b to 0.6 ꢀM for triazolopyridine 12i. In addition, we were able
to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the
absence of marked cytotoxicity, thus confirming the usefulness of this approach.
1.
INTRODUCTION
Human polyomaviruses JC (JCV) and BK (BKV) infect a large fraction of the world’s
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population (>50 %), leading to asymptomatic persistent infections in healthy individuals but
causing severe diseases in immunocompromised patients. JCV leads to progressive multifocal
3
leukoencephalopathy (PML), a rapidly progressing and fatal central nervous system condition,
while reactivation of BKV triggers polyomavirusꢀassociated nephropathy (PVN) in kidney
transplant patients and hemorrhagic cystitis (HC) in cancer patients undergoing a bone marrow
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,5
transplant. There are currently no antiviral therapies available to treat these lifeꢀthreatening
infections.
JCV and BKV are members of the polyomavirus family of small doubleꢀstranded DNA (dsDNA)
viruses. Polyomaviruses possess a viral genome that is divided into three main regions: two
coding and one noncoding. Early in the infection cycle, the Polyomaviridae encode the small
tumor antigen (tAg) and the large tumor antigen (LTAg). Later in the infection cycle, they
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encode for the viral capsid proteins VP1, VP2, and VP3, as well as the viral nonstructural protein
called agnoprotein. The LTAgs encoded by the early coding region are essential for viral DNA
replication. They contain a SF3 hexameric helicase domain that uses energy derived from ATP
hydrolysis to unwind duplex DNA. Because LTags are solely viral proteins with no human
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counterpart, they represent attractive targets for therapeutic intervention.
As a result, early
proof of concept screens for the modulation of LTags ATPase activity have recently been
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reported for polyomaviruses BKV,
JCV and SV40. To the best of our knowledge, these
efforts have not been pursued past the stage of hit identification.
2.
RESULTS AND DISCUSSION
We report herein the first structureꢀbased approach to the rational design of dual JCV and BKV
helicase inhibitors. The helicase ATPꢀbinding pockets of JCV and BKV are highly conserved,
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with only four divergent residues (I395/L396; A552/I553; S559/Q560 and C560/N561), and we
hypothesized that dual JCV/BKV inhibitors could be identified. As a result, we simultaneously
initiated a high throughput screen of approximately 350,000 compounds using a JCV DNAꢀ
unwinding assay as primary readout and a broad crystallization effort towards the elucidation of
the structure of the JCV LTAg helicase.
Crystallization of the JCV LTAg. The JCV LTAg is a 688 residue protein consisting of a
DNAꢀJ domain at the aminoꢀterminus, an origin binding domain and a helicase domain. In order
to unwind dsDNA, the LTAg assembles into a donutꢀshaped hexamer that allows one DNA
strand to pass through the small central hole, while the other strand is guided along the outside of
the hexamer. The separation of the two DNA strands occurs by steric occlusion as the enzyme
pulls the DNA strand through the central hole. This separation requires ATP hydrolysis and is
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believed to occur with a rotary mechanism where each of the six LTAg molecules cycles through
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an ATPꢀ, ADPꢀbound and nucleotide free states.
The JCV helicase domain stretches from residue ~260 to 630 and contains three
subdomains. We expressed and purified the helicase domain containing residues 261 to 628
from E. coli. Initial crystals were obtained with 25ꢀ30% Peg400, 0.1 M Li SO , 0.1M HEPES
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(pH 7.0), 0.2 M NaCl. The diffraction pattern showed high mosaicity and low resolution
preventing us from solving the structure. fter analyzing the crystal contacts from the previously
A
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published SV40 LTAg, we decided to introduce the following eleven mutations in the JCV
LTAg: E280D, D295N, N299A, Q301A, Q302A, K304A, K305A, E307A, K308A, K309A,
R624A. These mutated residues are high entropy residues located at the surface of the protein
and away from the ATP binding site.
The introduction of these nine mutations resulted in better diffracting crystals that allowed us to
solve the crystal structure using 2.2Å data. The protein crystals belonged to space group P6,
with one molecule in the asymmetric unit. The donutꢀshaped helicase complex was assembled
by applying the 6ꢀfold symmetry of the space group, forcing all six molecules to be in an
identical conformation (Figure 1).
12, 13
The JCV helicase sequence is 75% identical with SV40 LTAg
and is structurally very
similar. The RMSD between the two molecules is 0.692 (with 322 Cα superimposed). At the
Nꢀterminus is a Znꢀbinding domain (residues 267 to 331), followed by domains 2 and 3. The Znꢀ
binding domain is made from four helices (Figure 1b α5). The Znꢀion is held in place by C303,
C306, H314 and H318. The Zn binding domain at the aminoꢀterminus is connected with domain
3
through a long helical linker. Although Znꢀfingers are often involved in DNA binding, in the
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case of JCV (and SV40) the Zn ion is distant from the central channel and its purpose is
structural support to accommodate homoꢀhexamerization with other JCV LTAgs. Domain 2 is
inserted in between Domain 3 and has the typical AAA+ fold of a 5 stranded betaꢀsheet
surrounded by four alphaꢀhelices. Helices 10ꢀ12 are on one side of the betaꢀsheet while helix α9
is on the other side. Between betaꢀstrand 1 and helix 9 is the Walker A motif (or Pꢀloop) a
conserved motif involved in ATP’s triphosphate binding. Domain 2 also has a betaꢀhairpin
inserted after betaꢀstrand 3, betaꢀstrands 3’ and 3”, with the conserved H514 and K513
responsible for pulling the DNA strand through the pore of the hexamer. Domain 3 contains 7
alphaꢀhelices. Helices α6 through α8 are followed by Domain 2, followed by alphaꢀhelices α13
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through α16.
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Figure 1. a) Structure of the JCV LTAg monomer; b) Structure of the JCV LTAg hexamer along
the 6ꢀfold axis
Inhibitor design. Following the highꢀthroughput screen, the hits underwent a rigorous triage that
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included i) removal of all hits with low binding efficiency (BEI < 15) ; ii) mechanistic
evaluation to identify ATPꢀcompetitive inhibitors that lacked DNAꢀbinding ability. This process
allowed us to identify two closely related, equipotent, ATPꢀcompetitive triazolothiadiazole
inhibitors of JCV DNA unwinding activity, the 6ꢀ(2ꢀmethoxyphenyl)ꢀ and the 6ꢀ(2ꢀ
ethoxyphenyl)ꢀ[1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole 1a and 1b (Figure 2, Table 1).
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1a : R = Me, JCV helicase IC50 : 18 ꢀM
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b : R = Et, JCV helicase IC : 19.4 ꢀM
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Figure 2. Fragment hits 1a,b
To follow up on the hits, we purchased commercially available 5a and 5b, and initiated the
synthesis of additional closely related analogs.
Initially, analogous triazolothiadiazoles were synthesized as shown in Scheme 1. The 4ꢀaminoꢀ
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Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol 4, obtained through the neat condensation of diaminothiourea with
formic acid, was condensed with benzoic acids, under microwave conditions to give products 5cꢀ
m.
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Scheme 1. Condensation of carboxylic acids with the 4ꢀaminoꢀ4Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol
Conditions: (a) reflux, neat, overnight, 42%; (b) POCl , 110ꢀ120˚C, 30ꢀ90 min, microwave, or
3
(c) T P, DIPEA, EtOAc, 140˚C, 20ꢀ120 min, microwave
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To probe the space for potential interactions with the helicase, while keeping an emphasis on
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introducing sp carbon rich chains, additional ether analogs were synthesized via alkylation of
5m with alkylchlorides or alkylbromides (5n-q) (Scheme 2).
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Scheme 2: Ether synthesis through alkylation of phenol 5m
Conditions: (a) K CO CH CN, 100ꢀ130˚C, 30 min, microwave
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Table 1. Biochemical activity of early triazolo[3,4ꢀb][1,3,4]thiadiazoles (TT) (5aꢀ5q).
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Compound
JCV helicase IC50 (ꢀM)
BKV helicase IC (ꢀM)
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Minimum Significant Ratio (MSR) = 3.2; i.e., compound that have a difference in IC of at
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least a factor of 3.2 are considered significantly different.
This effort rapidly established the importance of the ether at the 2ꢀposition of the phenyl ring
since the 2ꢀhydroxyphenyl analog 5m, the 2ꢀaminoꢀ and 2ꢀdimethylaminoꢀanalog 5a and 5e, as
well as the 2ꢀmethyl and 2ꢀcyclopropyl analogs 5b and 5c lost all activity against both enzymes
compared to the original hits 1a and 1b. The direct linkage of the phenyl ring to the thiadiazole
was also found to be of importance since the benzylic analog of 1a (5l) was inactive.
Even though a wide variety of ethers were introduced at the 2ꢀposition of the phenyl ring,
improving upon the activity of 1a,b proved to be difficult. Only the phenoxyꢀanalog 5d
displayed slightly improved IC50s on both JCV and BKV around 5 ꢀM. The THP ether analogs
5
n and 5o were also well tolerated whereas switching from THP (5o) to morpholine (5p) resulted
in a tenꢀfold decrease in potency.
Our first Xꢀray crystal structure of a triazolo[3,4ꢀb][1,3,4]thiazole in JCV was obtained by
soaking 5d into apo JCV crystals (Figure 3). This structure demonstrated that 5d makes a single
interaction with the backbone NH of K551 in the pseudoꢀhinge region while the phenoxy group
is engaged in πꢀstacking with R555, possibly accounting for the slight improvement in potency
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compared to methoxy and ethoxy hits 1a,b from 18 ꢀM to 4.5 ꢀM. It is worth noting that 5d
interacts solely with one of the JCV monomers that constitute the ATP pocket and does not
engage any of the residues of the neighboring JCV monomer, as shown in Figure 3.
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shown in green and the other in blue.
Based on the crystal structure of 5d in JCV, we identified the closest residues, within 4 Å, that
would be available for additional interactions with our scaffold. These residues are i) the
carbonyls of S431 and K551 in the hinge region as well as the side chain OH of T435 and the
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side chain amino group of Gln571 from the first JCV monomer, ii) the terminal amino group
K419 from the second JCV monomer.
In order to optimally target K551 and S431 in the pseudoꢀhinge region, we morphed our 5,5ꢀ
membered ring triazolothiadiazole core into a 6,5ꢀmembered ring thiazolopyridine core. The two
regioisomeric thiazolopyridines corresponding to original hit 1, the thiazolo[4,5-c]pyridine (12a),
and the thiazolo[5,4-c]pyridine (15) were synthesized according to Schemes 3 and 4.
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59
60
As expected from the binding mode of 5d in JCV, thiazolopyridines 12a-b were found to inhibit
JCV’s DNA unwinding ability in a comparable manner to triazolothiadiazoles 1a-b, with an IC50
of 5.0 and 9.1 uM respectively while 12b’s regioisomeric thiazolopyridine 19 was inactive in
these assays with IC50s > 80 ꢀM. Furthermore, thiazolo[4,5-c]pyridines 12a-f were found to be
equipotent to their triazolothiadiazole analogs 1a-b, 5d, 5j, 5m, , and 5o, allowing us to transfer
existing triazolothiadiazole SAR to the new series (Table 2).
Scheme 3. Synthesis of thiazolo[4,5-c]pyridines (12a-12f)
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Conditions: (a) tꢀBuCOCl, Et N, DCM 0˚C to RT, 78%; (b) nꢀBuLi, tetraisopropylthiuram
3
disulfide, THF, ꢀ78˚C to RT, 74%; (c) NaOH, MeOH , RT , 1 hr, 88%; (d) RCOCl, Et N, DCM,
3
RT, 10 min, 62%; (e) aq HCl (5M), 80˚C, 2 hrs, 45%; (f) 4ꢀ(2ꢀbromoethyl)tetrahydropyran,
K CO , CH CN, 80˚C, 3 hrs, 87%
2
3
3
Scheme 4. Synthesis of the 2ꢀ(2ꢀethoxyphenyl)thiazolo[4,5ꢀc]pyridine (15).
Conditions: (a) 2ꢀEthoxybenzoyl chloride, K CO , THF, RT, overnight, 11 %; (b) Lawesson’s
2
3
reagent, toluene, reflux, overnight, 43%
Table 2. Biochemical activity of early thiazolo[4,5ꢀc]pyridines
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R
JCV helicase IC50 (ꢀM) BKV helicase IC (ꢀM)
50
12a
5.0
10.7
10
11
12
13
14
15
16
17
18
19
20
21
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60
1
2b
9.1
8.0
12c
10.5
8.7
37
10.2
15
12d
1
2e
53
12f
9.6
11
Probing for interactions with K551 and S431 required the introduction of an amino group at
position 6 (20, 21 Scheme 5) and/or position 4 (26, Scheme 5) of the thiazolopyridine core.
Interestingly, the presence of the amino group was tolerated at position 6 (20) and detrimental at
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6
position 4 (30). However, the additional hydrogenꢀbond donor at position 6 did not lead to an
increase in activity as 20 was found to be equipotent with its unsubstituted analog 12a. This is
all the more surprising, as subsequent monomethylation of the 6ꢀamino group in 20 led to a
complete loss of activity (21, Table 3).
0
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9
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Scheme 5: Synthesis of the 2ꢀ(2ꢀmethoxyphenyl)thiazolo[5,4ꢀc] pyridinꢀ6ꢀamine (20) and 2ꢀ(2ꢀ
methoxyphenyl)ꢀNꢀmethylthiazolo[5,4ꢀc]pyridinꢀ6ꢀamine (21).
Conditions: (a) Thiocarbonyl dichloride, Na CO ,DCM, RT, 4 days, 57%; (b) 1ꢀbromoꢀ2ꢀ
2
3
methoxybenzene, nꢀBuLi, THF, DMF, ꢀ78˚C, 20 min (assumed quantitative); (c) Na CO , DMF,
2
3
120˚C, 3hrs, 26%; for 20 (d) tertꢀbutyl carbamate, NaOtꢀBu, XPhos palladacycle, dioxane,
20˚C, 20 min, microwave; (e) TFA, THF, 41 %; for 21 (d) methylamine, NaOtꢀBu, BrettPhos
1
palladacycle, dioxane, 135˚C, 30 min, microwave, 42 %
Scheme 6: Synthesis of the 2ꢀ(2ꢀmethoxyphenyl)thiazolo[5,4ꢀc]pyridinꢀ4ꢀamine (26)
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Conditions: (a) Thiocarbonyl dichloride, Na CO , CSCl , DCM, RT, 24 h, 51%; (b) 1ꢀbromoꢀ2ꢀ
2
3
2
methoxybenzene, nꢀBuLi, THF, ꢀ78˚C, 20 min; (c) Na CO , 120˚C, 12 hr ,40%; (d) tertꢀbutyl
2
3
carbamate, NaOtꢀBu, XPhos palladacycle, dioxane, 135˚C, 30 min, microwave; (e) TFA, THF,
4%
4
Table 3: Biochemical activity of thiazolo[5,4ꢀc]pyridinamines
Inhibitor
JCV helicase IC50 (ꢀM)
BKV helicase IC (ꢀM)
50
2
2
0
1
4.3
>80
>80
8.7
>80
>80
26
st
As the addition of a donorꢀacceptor motif targeting the hinge region of the 1 JCV monomer did
not result in increased activity, our efforts shifted towards T435 and K419, the neighboring
residues of the second JCV monomer (Figure 3). In an attempt to engage these residues, we
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6
initially evaluated the replacement of the 2ꢀphenoxyphenyl group with a 2ꢀphenoxypyridine or 2ꢀ
phenoxypyrimidine groups.
Triazolothiazoles 5r-s were obtained from the 2ꢀphenoxypyridineꢀ3ꢀcarboxylic acid and the 4ꢀ
phenoxypyrimidineꢀ5ꢀcarboxylic acid respectively, using the reaction conditions depicted in
Scheme 1 (5r conditions (a;c); 5s conditions (a;b)). Triazolothiazole 5t was synthesized from the
0
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6ꢀ(4ꢀchloropyridinꢀ3ꢀyl)ꢀ[1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole as shown in Scheme 7. Finally,
the thiazolopyridine analog of 5r (12g) was obtained in 41% yield through the acid promoted
intramolecular cyclization of intermediate 30 as depicted in Scheme 8.
Scheme 7 : Synthesis of 6ꢀ(4ꢀphenoxypyridinꢀ3ꢀyl)ꢀ[1,2,4]triazolo[3,4ꢀb][1,3,4]thiadiazole 5t.
Conditions: (a) POCl , 100˚C, 35 min, microwave, 43%; b) phenol, K CO , DMF, 130˚C, 60
3
2
3
min, microwave, 3%
Scheme 8: Synthesis of the 2ꢀ(2ꢀphenoxypyridinꢀ3ꢀyl)thiazolo[5,4ꢀc]pyridine (12g).
Conditions: (a) HATU, DIPEA, DMF; b) 6M HCl, 80˚C, 5hrs, 41%.
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Table 4: Activity of phenoxypyridines and phenoxypyrimidines.
R
Inhibitor
JCV helicase IC50 (ꢀM)
BKV helicase IC (ꢀM)
50
10
11
12
13
14
15
16
17
18
19
20
21
22
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53
54
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57
58
59
60
5
r
8.8
11.0
>80
7.3
5s
>80
5.5
1.4
5
t
12g
1.3
The introduction of a 2ꢀphenoxypyridinyl group was well tolerated in the thiazolotriazole
scaffold (5r, 5s), but did not lead to an increase in activity compared to 2ꢀphenoxyphenyl analog
5d. The corresponding 2ꢀphenoxypyrimidinyl analog 5s lost activity. In contrast, 12g showed a
1
0ꢀfold increase in activity on both viruses, with IC50s of 1.4 ꢀM (JCV) and 1.3 ꢀM (BKV)
compared to 2ꢀphenoxyanalog 12c with IC50s of 10.5 ꢀM (JCV) and 10.2 ꢀM (BKV). This
increase in activity could be attributed to the formation of a new productive interaction between
the phenoxypyridine nitrogen of 12g and K 419 of the second JCV monomer as shown in the
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6
crystal structure obtained by soaking 12g into JCV apo (Figure 4a). It is worth noting that K419
in JCV corresponds to K420 in BKV, a key residue of the ATP pocket found to be critical for
8
viral replication using an in vitro directed mutagenesis approach.
0
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9
0
As we continued to look for additional interactions with the second JCV monomer, we
discovered that another lysine, K420, could effectively form a hydrogen bond with our
thiazolopyridines when an acetamide was introduced at the pꢀposition of the phenoxy group of
12c (12h, Scheme 9). In this case, K420 was found to produce a productive interaction with the
acetamide carbonyl of 12h located at 2.8 Å, leading to a 10ꢀfold increase in potency with IC50
values of 0.9 ꢀM and 1.6 ꢀM on JCV and BKV respectively (Figure 4b).
Scheme 9: Synthesis of the Nꢀ(4ꢀ(2ꢀ(thiazolo[5,4ꢀc]pyridinꢀ2ꢀyl)phenoxy)phenyl)acetamide
(12h) and the Nꢀ(4ꢀ((3ꢀ(thiazolo[5,4ꢀc]pyridinꢀ2ꢀyl)pyridinꢀ2ꢀyl)oxy)phenyl)acetamide (12i)
Conditions: (a) Et N, CH Cl , RT, 11ꢀ18 hrs; (b) 6M HCl, RT or 80˚C, 30 min; (c) Nꢀ(4ꢀ
3
2
2
hydroxyphenyl)acetamide,CS CO , or K CO , DMSO, 150˚C, 4 hrs to overnight
2
3
2
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Since the introduction of a 2ꢀphenoxypyridine (12g) and that of an acetamide at the pꢀposition of
the phenoxy group (12h) both led to a profound increase in activity on both viruses, these two
key structural features were combined within a single compound, thiazolopyridine 12i (Scheme
9).
10
11
12
13
14
15
16
17
18
19
20
21
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57
58
59
60
To our surprise, 12i was found to be equipotent to 12h with IC50s of 0.6 ꢀM of both JCV and
BKV. This finding can be rationalized with the crystal structures obtained with 12g and 12h
(Figure 4). In these structures, significant movements were observed for both K419 and K420.
As K420 moves in 2.5Å to bind to the acetamide carbonyl of 12h, K419 moves 3.4Å away from
the pyridine nitrogen of 12g, only allowing for a single productive interaction to be formed
between our inhibitors and the second JCV monomer.
The selectivity of two of our inhibitors 5d and 12h was evaluated against a subset of 66 kinases
TM
of the Eurofin Kinaseprofiler panel. Both compounds displayed an exquisite selectivity with
1
6
no inhibition greater than 35% at 10 ꢀM.
While improving the activity by increasing the number of interactions between the inhibitor and
the residues of the ATP pocket proved to be challenging, we believed that the level of activity
achieved with some of our most potent analogs would allow us to demonstrate a measurable
effect on viral replication in a cellular assay. We therefore developed a branched DNA (bDNA)
assay that measured the production levels of the late viral protein and major capsid protein VP1.
The data summarized in Table 5, showed that the triazolopyridine were generally better tolerated
than their thiazolotriazoles analogs with cell viability windows (CC /EC ) > 5 with the single
50
50
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exception of phenoxy analogs 5d and 12c. In spite of their lack of general cytotoxicity, the
antiviral activity of the thiazolopyrines did not correlate to their intrinsic ability to inhibit DNA
unwinding. This is clearly demonstrated with the most efficacious thiazolopyridine (12d) with an
EC50 of 0.26 ꢀM in the bDNA assay and an IC50 of 8.7 ꢀM in the unwinding assay. This
phenomenon can potentially be explained by the fact that the bDNA assay monitors late capsid
protein VP1 and therefore provides a read on the entire viral life cycle that not only involves
DNAꢀunwinding, but a variety of host proteins. An orthogonal assay with a readout that would
be more proximal to the DNAꢀunwinding step could potentially provide a better alignment. The
antiviral activity of the thiazolopyridines may also be the result of a mechanism of action far
more complex than DNA unwinding inhibition and possibly involving more than one target. The
success of such a multiꢀtarget inhibitory profile has been demonstrated for Herpes virus
helicase(SF1)ꢀprimase inhibitor amenamevir (ASP2151) which was found to be a more potent
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
6
7
8
9
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3
4
5
6
7
8
9
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2
3
4
5
6
7
8
9
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2
3
4
5
6
7
8
9
0
1
7
primase inhibitor than DNA unwinding inhibitor.
Table 5. Antiꢀviral activity in Vero cells
(TT) vs
(TP)
a
Inhibitor Core
R
JCV bDNA
JCV bDNA
CC /EC
50 50
EC (ꢀM)
CC (ꢀM)
5
0
50
1a
TT
>60
>80
ND
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1
2a
TP
TT
TP
TT
TP
TT
TP
TT
1.5
44
>50
>65
>33
ND
>6
1b
10
11
12
13
14
15
16
17
18
19
20
21
22
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26
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41
42
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50
51
52
53
54
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57
58
59
60
12b
7.4
34
>50
>50
>50
>50
>50
>50
5
d
ND
ND
ND
>166
ND
12c
36
5
j
>16
0.26
15
12d
5
o
1
2f
TP
7.7
>50
>6
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5
r
TT
TP
TP
76
3.8
48
>50
>50
>50
ND
> 13
ND
12g
0
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12h
1
2i
TP
40
>50
ND
a
15
Minimum Significant Ratio (MSR) = 2.6; i.e., compound that have a difference in EC of at
5
0
least a factor of 2.6 are considered significantly different
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Figure 4. Crystal structure of (a) 12g and (b) 12h bound to JCV LTAg
CONCLUSION
In summary, we have described our path towards the structureꢀbased design of dual JCV and
BKV ATPꢀcompetitive inhibitors. To our knowledge, this is the first report of a structure
based approach targeting a hexameric helicase of the SF3 family. We reported the first
crystal structure of the hexameric helicase of JCV LTag (apo) which was used for soaking
our fragment inhibitors and for driving fragment design. In agreement with our hypothesis,
we demonstrated that the activity of our ATPꢀcompetitive inhibitors of DNAꢀunwinding
tracked well between the two viruses. Based on structural information, we rapidly increased
the activity of our scaffolds 30ꢀfold from 18 ꢀM for the original hits 1a,b to 0.6 ꢀM for
triazolopyridine 12i. Capitalizing further on our structural findings, was however hindered by
the complexity of the ATP pocket in combination with the mobility of two of its key
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5
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5
5
6
residues, K419 and K420, thus only allowing for the formation of two productive
interactions between the inhibitor and the helicase. In spite of this limitation, it is
encouraging to note that we were able to demonstrate measurable antiviral activity for our
thiazolopyridines in the absence of marked cytotoxicity.
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EXPERIMENTAL SECTION
JCV large T antigen purification. JCV LTag (E264ꢀD628) N299A, Q301A, Q302A,
K304A, K305A, E307A, K308A, K309A, R624A was cloned in pET28b.1 plasmid and
expressed at 15˚C in E. coli for 28 hours. All purification steps were performed at 4˚C or on
ice. Cell paste was resuspended in 10 volumes of buffer A (50 mM HEPES [pH 7.8], 10%
glycerol, 400 mM NaCl, 5 mM βꢀmercaptoethanol, 5 mM imidazole, 0.2% Tweenꢀ20, 2.5
g/ml leupeptin, 2.0 ꢀg/ml Eꢀ64, 10 ꢀM DFP) containing benzonase™ nuclease (Novagen)
and lysed by one pass through a microfluidizer (Microfluidics Corporation, Newton, MA).
The lysate was clarified by centrifugation at 54,000 x g for 1 hour and the supernatant was
batch absorbed for 2 hours with nickelꢀnitrilotriacetic acid (NTA) agarose resin (Sigmaꢀ
Aldrich). The resin was poured into a gravity flow column, washed with 10 volumes of
buffer B (buffer A containing 10 mM imidazole) and eluted with 5 volumes of buffer C (50
mM HEPES [pH 7.8], 10% glycerol, 400 mM NaCl, 5 mM βꢀmercaptoethanol, 0.2% Tweenꢀ
20, 350 mM imidazole). Fractions containing JCV protein were pooled, concentrated and
loaded onto a HiPrep 26/60 Sephacryl Sꢀ300 HR column (GE Healthcare Life Sciences)
equilibrated in buffer D (25 mM HEPES [pH 7.2], 10% glycerol, 300 mM NaCl, 2 mM
dithiothreitol). Fractions containing JCV protein were pooled, concentrated and the
polyhistidine tag was removed by incubation with bovine thrombin (Calbiochem). After tag
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removal was complete, the protein was loaded onto a Superdex 200 10/300 gl column (GE
Healthcare Life Sciences) equilibrated in buffer E (25 mM HEPES [pH 7.5], 250 mM NaCl,
2
mM dithiothreitol). Fractions containing JCV were pooled and concentrated to 25ꢀ30
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mg/ml.
BKV large T antigen purification. BKV LTag (E265ꢀD629) was cloned in pET28b.1
plasmid and expressed at 15˚C in E. coli for 28 hours. All purification steps were performed
at 4˚C or on ice. Cell paste was resuspended in 10 volumes of buffer F (50 mM HEPES [pH
7.8], 10% glycerol, 500 mM NaCl, 5 mM βꢀmercaptoethanol, 20 mM imidazole, 0.2%
Tweenꢀ20, 2.5 g/ml leupeptin, 2.0 ꢀg/ml Eꢀ64, 10 ꢀM DFP) containing benzonase™
nuclease (Novagen) and lysed by one pass through a microfluidizer (Microfluidics
Corporation, Newton, MA). The lysate was clarified by centrifugation at 54,000 x g for 1
hour and the supernatant was batch absorbed overnight with nickelꢀnitrilotriacetic acid
(
NTA) agarose resin (Qiagen 304430). The resin was poured into a gravity flow column,
washed with 20 volumes of buffer G (50 mM HEPES [pH 7.8], 10% glycerol, 500 mM
NaCl, 5 mM βꢀmercaptoethanol, 20 mM imidazole) and eluted with 3 volumes of buffer H
(50 mM HEPES [pH 7.8], 10% glycerol, 400 mM NaCl, 5 mM βꢀmercaptoethanol, 350 mM
imidazole). The eluate was immediately diluted 3ꢀfold with buffer I (25 mM HEPES [pH 7.0],
10% Glycerol, 400 mM NaCl, 2 mM dithiothreitol) and incubated overnight with bovine
thrombin (Calbiochem) to remove the polyhsitidine tag. The cleaved protein was diluted with 5
volumes of buffer J (25 mM HEPES [pH 7.0], 10% Glycerol, 2 mM dithiothreitol) and loaded
onto a HiTrap™ SP FF column (GE Healthcare Life Sciences 17ꢀ5157ꢀ01) equilibrated in
buffer K (buffer J containing 70 mM NaCl). The column was eluted with a 30 column
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volume linear gradient from 70 mM NaCl to 500 mM NaCl in buffer J. Fractions containing
BKV protein were pooled and stored at minus 70˚C.
JCV DNA unwinding assay
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The assay was run in 50 mM HEPES pH 7.5, 100 mM NaCl, 7 mM MgCl , 0.002% Brijꢀ35,
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1 mM DTT, 2% DMSO, 360 nM of JCV LTAg, 15nM Cy5/BHQ3 dsDNA, 15 nM of
unlabeled dsDNA and 30u M ATP. The ATP was regenerated with the PK/LDH coupling
All assay components were assembled and incubated at room temp for 20 min to initiate the
reaction. 20 uL of the reaction mixture was added to assay ready plates and incubated 120ꢀ
180 minutes at room temperature. Fluorescence intensity was read throughout incubation and
slope of RFU v, then time analyzed.
BKV DNA unwinding assay
The assay was run in 50 mM HEPES pH 7.5, 100 mM NaCl, 7 mM MgCl , 0.002% Brijꢀ35,
2
1mM DTT, 2% DMSO, 540 nM BK LTAg, 15 nM Cy5/BHQ3 dsDNA, 15nM unlabeled
dsDNA and 30uM ATP. The ATP was regenerated with the PK/LDH coupling system.
All assay components were assembled and incubated at room temperature for 20 mn to
initiate the reaction. 20 uL of the reaction mixture were added to assay ready plates and
incubated 120ꢀ180 mn at room temperature. The fluorescence intensity was read throughout
incubation and slope of RFU v then time analyzed.
DNA binding assay.
10 mM DMSO stocks of compounds were added at 500 uM final concentration (5% DMSO)
to a solution of 4 uM salmon sperm DNA (Aldrich) dissolved in 50 mM potassium
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phosphate pH 7, 50 mM Na EDTA, D O. NMR experiments were performed at 295 K on a
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500 MHz Bruker Avance III NMR spectrometer equipped with a 5 mm triple resonance
1
inverse (TXI) cryoprobe. One dimensional H NMR spectra were collected with
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WATERGATE 3919 water suppression to identify insoluble or impure compounds.
Saturation transfer difference (STD) spectra were collected using a 2 sec train of 50 ms
Gaussian pulses for selective saturation at 5.8 ppm (H5’,5” region), with 32 interleaved sets
of 8 onꢀ and offꢀresonance scans. Compounds giving STD signals with signalꢀtoꢀnoise > 10
were classified as DNA binders. 500ꢀ ethidium bromide was used as a positive control.
DNA bDNA assay: JC virus VP1 specific DNA bDNA probes were requested by Vertex and
synthesized by Affymatrix, based on Affymatrix v2.0 kit. Cultured Vero cells were lysed by
adding 1/3 volume of lysis mixture from Quantigene v2.0 kit. 20 ꢁl of the completely mixed
lysate were mixed with 20 mM EDTA, 10 ꢁL 2.5N NaOH and 60 ꢁL dH O, incubated at
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2˚C for 30 min to denature the DNA and remove the RNA. 50 ꢁl of 2.0 M HEPES was
added, mixed, and then transferred 20 ꢁl of mixture into bDNA capture plates containing 80
l of JC VP1 specific DNA bDNA probes (50 ꢁl of lysis mixture, 28.7 ꢁl of H O, 1 ꢁl of
ꢁ
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blocking solution, and 0.3 ꢁl of probe stock). Purified DNA is applied to the assay once the
DNA was suspended into lysis buffer (1:2 diluted lysis mixture).
EC determination of Antiviral Agents. 7.5xE6 G144 Vero cells were plated in a Tꢀ150
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flask preꢀcoated with polyꢀOrinithine (0.005%) and Laminie (10 ꢁg/ml) in the growth
medium for 3 hr to overnight. The cells were infected with MAD4 virus at MOI = 1 TCID50
for 24 hr. For the BKV assay, 10xE6 Vero cells were plated in a Tꢀ150 flask in the growth
medium for overnight. The cells were infected with BKV virus at MOI = 0.1 TCID for 24
50
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