A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL

Article abstract of DOI:10.1016/j.bmcl.2008.12.015

The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines.

Full text of DOI:10.1016/j.bmcl.2008.12.015

Bioorganic & Medicinal Chemistry Letters 19 (2009) 811–813
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A small molecule inhibitor selective for a variant ATP-binding site
of the chaperonin GroEL
b
b
a,b,c
Eli Chapman a, , George W. Farr , Krystyna Furtak , Arthur L. Horwich
*
a The Scripps Research Institute, Molecular Biology, 10550 North Torrey Pines Road, Mb46, La Jolla, CA 92037, USA
b Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA
c Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bac-
terial cell assisting protein folding to the native state through actions requiring ATP binding and hydro-
lysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal
pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An
initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iter-
atively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclo-
pentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by
the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will
enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and
might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Sho-
kat and his colleagues may thus also be used to study large macromolecular machines.
Received 13 November 2008
Revised 2 December 2008
Accepted 3 December 2008
Available online 7 December 2008
Keywords:
Chemical genetics
Chaperone
Chaperonin
GroEL
ATPase inhibitor
Ó 2008 Elsevier Ltd. All rights reserved.
Chaperonins are megadalton-sized double ring protein com-
plexes, found in a variety of cellular compartments, that provide
essential kinetic assistance to protein folding through the con-
sumption of ATP. The bacterial chaperonin, GroEL, provides such
assistance through a reaction cycle involving two major states. In
one state, an open ring binds non-native polypeptide in its central
cavity via contacts with its hydrophobic wall, forestalling misfold-
ing and aggregation. In the second state, after GroEL cooperatively
binds ATP in the 7 subunits of the ring, the cochaperonin ‘lid’ pro-
tein GroES is recruited, and the non-native polypeptide is released
into the encapsulated cavity, the ‘Anfinsen cage’, where it attempts
to properly fold in an isolated, now hydrophilic environment
where aggregation cannot occur. After what is the longest step of
the reaction cycle (ꢀ10 s), ATP hydrolyzes in the GroES-bound ring,
gating ATP binding in the (unoccupied) opposite ring, the latter
step allosterically ejecting GroES and polypeptide into the bulk
solution. ATP binding and hydrolysis thus play a crucial role in
driving the GroEL/GroES reaction cycle.1 While a mutant, D398K,
that blocks ATP hydrolysis has been identified, arresting the ma-
chine in a folding-active state,2 to date there have been no mutants
isolated that block the step of ATP binding. Such inhibition would
allow analyses of the step of folding activation, for example
addressing how many subunits need to bind ATP in order to bind
GroES or trigger folding of polypeptide. Inhibition of ATP binding
would seem to be readily accomplished by small molecule occupa-
tion of the ATP-binding site. To this end, the chemical biological
tools developed by Shokat and coworkers3 were adapted to gener-
ate a small molecule inhibitor tuned specifically to a GroEL variant
with an amino acid substitution in the ATP-binding pocket.
First, residues in the ATP-binding pocket were selected for sub-
stitution, employing the X-ray crystallographic model of an asym-
metric GroEL/GroES/ADPÁAlF3 complex (PDB 1svt).4 Eleven
residues were selected, each was altered to cysteine, and several
to glycine and alanine as well. Mutation of bulky residues, for
example, I493, N479, or M488, was particularly attractive, because
it would create a ‘hole’ in the binding pocket that could specifically
accommodate a compound that would not be recognized by the
wild-type binding site.5 Substitution with cysteine comprised an
attempt to generate a covalent link with an electrophile-bearing
small molecule. Function of the variants was tested in vivo by
transforming a GroEL-deficient strain of Escherichia coli with the
respective mutant groE operons and inspecting for rescue.6 With
the exception of double mutants and the I493G variant, all of the
substitutions were tolerated (see Supplemental Table 1). The res-
cuing variants were then over-produced and purified for biochem-
ical study.
An initial collection of ꢀ120 molecules,7 based on histidine and
pyrazolo-pyrimidine scaffolds, was screened for inhibition of wild-
type and variant GroEL ATPase activity, as measured using a mala-
chite green assay in 96-well plate format. From this, a number of
inhibitors obtained, all from the pyrazolo-pyrimidine scaffold.
* Corresponding author. Tel.: +1 858 784 7386; fax: +1 858 784 7388.
E-mail address: echapman@scripps.edu (E. Chapman).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.015

812
E. Chapman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 811–813
R4
NH
Because only modest variant selectivity and inhibition were ob-
served, a small collection of derivatives of this scaffold was
synthesized in an effort to increase selectivity and potency
(Scheme 1).8
R3
R3
R1
NH
N
NH
N
n
N
N
N
R2
Briefly, acid chlorides bearing R1 were added to the anion of
malononitrile and after extractive workup, the resulting product
was refluxed with dimethylsulfate (DMS) in 9:1 dioxane:H2O to
yield the vinyl ether derivatives after chromatographic purifica-
tion. These two steps could also be combined into one step by add-
ing DMS directly to the first reaction and heating to reflux followed
by extractive workup and chromatographic purification. Formation
of the pyrazolol ring was carried out by refluxing the vinyl ethers
with a hydrazine bearing R2 in ethanol followed by workup and
chromatography. Finally heating to 180 °C in formamide yielded
the pyrazolol-pyrimidine. Position R3 was introduced by reaction
with the corresponding acid chloride in pyridine. In the cases in
which R1 was a nitro containing aryl group, the molecule could
be further elaborated by catalytic hydrogenation of the nitro group
to yield the aniline followed by filtration and condensation with an
acid chloride containing R4 using three equivalents of pyridine in
chloroform.
N
N
n = 0, 1
N
R2
R1 = alkyl or aryl
R2 = methyl, phenyl, benzyl, 3-nitrophenyl,
4-nitrophenyl, or t-butyl
R3 = H, alkylcarboxamides, arylcarboxamides,
alkyl, or aryl
R4 = alkylcarboxamides or arylcarboxamides
O
HN
NH2
N
N
N
N
Positions R1–R4 (Fig. 1a) were varied giving a total of ꢀ100 com-
pounds to be tested against chaperonin ATPase activities. It was
discovered that substitutions at position R3 were not tolerated. Po-
sition R2 tolerated a methyl, ethyl, isopropyl or tert-butyl group,
but no aryl substitutions were accepted and the tert-butyl group
showed the best activity. At position R1, only aryl substitutions
were tolerated. While screening compounds bearing electrophiles
at positions R1 and R4, it was noted that despite only modest cova-
lent bond formation with one of the mutants (G32C, data not
shown), the incorporation of an acrylamide group or a chloroacet-
amide at R4 yielded increased potency versus the unsubstituted
amine. This guided the synthesis of a small collective of aryl and
alkyl substitutions at the R4 position, with larger alkyl substituents
showing greater potency up to a cyclohexyl group which showed
diminished efficacy.
EC3016
Figure 1. The variable positions used to generate the panel of small molecules used
in the present study is indicated in (a). A small molecule with excellent selectivity
for the I493C GroEL variant is shown in (b).
Inhibition of activity was seen for several of the mutant/molecule
pairs, but the most sensitive discovered was I493C, in combination
with EC3016 (Fig. 1b). This pair was further assessed in a collective
of standard GroEL assays, ATP hydrolysis (Fig. 2a) and the refolding
of two GroEL substrate proteins, bovine rhodanese and pig heart
malate dehydrogenase (MDH; Fig. 2b).
The combined chemical and genetic approach taken here to
generating a small molecule ligand that targets a specific geneti-
cally modified protein to effect selective activation or inhibition
of activity has been employed in a number of previous studies.
An early example was the use of FK1012 and FKBP to generate
chemical control of protein dimerization.10 The current work di-
rectly borrows from an approach originally taken with kinases,
where uniquely for a selected kinase, a specific ATP analogue
was identified that could fit into the genetically engineered ATP
pocket of the kinase to enable labeling of its specific substrates.11
Subsequently, inhibition of individual kinases was programmed
using similar approaches with genetically engineered kinase ATP
pockets and small molecule pyrazolol-pyrimidine inhibitors like
those employed here.12 Such combined chemical genetic ap-
proaches have been adapted to the molecular motors, kinesin13
and myosin,14 employing modified ATP pockets and N6-substituted
ATP or ADP analogues, enabling action of individual isoforms of
these machines to be recognized.15 The present study of the GroEL
chaperonin takes the same general approach as these earlier stud-
ies, that is, modifying the nucleotide pocket at the position adja-
cent to the N6 of ATP and using as a ligand a small molecule
pyrazolol-pyrimidine compound related to those employed by
Shokat and coworkers,12 to inhibit ATP binding by the GroEL ma-
chine. Here, instead of a single or, as in the case of some of the mo-
tors, two targeted sites, there are seven sites within a ring. Beyond
this initial study, the use of varying numbers of targetable variant
sites versus non-recognized wild-type sites, by virtue of forming
rings with mixed subunit composition, has already enabled inves-
tigation of the ATP requirements for activating the machine at the
level of a ring.16 The experiments presented here and in the recent
mixed ring study have been carried out in vitro, but they establish
the feasibility of using the combination of a small molecule ligand
The ATPase activity of wtGroEL and each of the mutants was
measured in the presence and absence of each of the pyrazolo-
pyrimidine derivatives in a single point, 96-well plate format.9
R1
N
N
R2
NH2
N
R1
N
N
R2
NC
OCH3
CN
O
N
c
a, b
d
R1
R1 Cl
H2N
CN
O
R3 NH
R1
N
N
R2
e
N
N
O
NO2
NH2
HN
R4
NH2
NH2
NH2
N
N
N
g
f
N
N
N
N
N
R2
N
N
N
N
R2
R2
Scheme 1. General synthesis of pyrazolo-pyrimidines used in the present study. (a)
NaH, malononitrile, THF, 0–23 °C; (b) (CH3)2SO4, NaHCO3, dioxane/H2O, reflux; (c)
R2NHNH2, EtOH, reflux; (d) formamide, 180 °C; (e) R3COCl, pyridine, 23 °C; (f) H2,
Pd/C, EtOH, 23 °C; (g) R4COCl, pyridine, CHCl3, 23 °C.

E. Chapman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 811–813
813
120
100
80
60
40
20
0
14
12
10
8
6
4
2
Figure 2. Biochemical properties of wtGroEL and I493C measured in the presence or absence of EC3016. (a) The amount of inorganic phosphate released 10 min after addition
of ATP as measured by malachite green complex formation. (b) The maximum amount of rhodanese, MDH, or DHFR activity recovered. Red bars are wtGroEL, green bars are
wt GroEL plus 50
l
M EC3016, blue bars are I493C, and yellow bars are I493C plus 50
l
M EC3016. For both panels, each point was repeated in triplicate and averaged and the
error bars indicate one standard deviation from the mean.
and a cell containing homooligomeric variant GroEL to address the
action of GroEL in vivo. For example, addition of a cell permeable
small molecule ligand that rapidly and specifically inactivates var-
iant GroEL by binding to its modified nucleotide pocket, blocking
ATP access during cell growth, should potentially be able to ad-
dress which proteins are the immediate substrates of the chapero-
nin by their selective misfolding and aggregation. General
physiological defects of GroEL-deficient cells may also be further
defined by such an experiment.
In conclusion, a small molecule inhibitor that acts selectively on
a genetically modified GroEL ATP-binding pocket has been gener-
ated through use of a combination of synthetic chemistry and
genetics.
References and notes
1. (a) Horwich, A. L.; Farr, G. W.; Fenton, W. A. Chem. Rev. 2006, 106, 1917; (b)
Horovitz, A.; Willison, K. R. Curr. Opin. Struct. Biol. 2005, 15, 646.
2. Rye, H. S.; Burston, S. G.; Fenton, W. A.; Beechem, J. M.; Xu, Z.; Sigler, P. B.;
Horwich, A. L. Nature 1997, 388, 792.
3. Bishop, A. C.; Buzko, O.; Shokat, K. M. Trends Cell Biol. 2001, 11, 167.
4. Chaudhry, C.; Farr, G. W.; Todd, M. J.; Rye, H. S.; Brunger, A. T.; Adams, P. D.;
Horwich, A. L.; Sigler, P. B. EMBO J. 2003, 22, 4877.
5. Bishop, A. C.; Ubersax, J. A.; Petsch, D. T.; Matheos, D. P.; Gray, N. S.; Blethrow,
J.; Shimizu, E.; Tsien, J. Z.; Schultz, P. G.; Rose, M. D.; Wood, J. L.; Morgan, D. O.;
Shokat, K. M. Nature 2000, 407, 395.
6. Horwich, A. L.; Low, K. B.; Fenton, W. A.; Hirshfield, I. N.; Furtak, K. Cell 1993, 74,
909.
7. (a) Bishop, A. C. Ph.D. Thesis, Princeton University, November 2000.; (b)
Witucki. L. W. Ph.D. Thesis, Princeton University, November 2000.
8. Hanefeld, U.; Rees, C. W.; White, A. J. P.; Williams, D. J. J. Chem. Soc., Perkins
Trans. 1996, 1, 1545.
Acknowledgments
9. (a)Lanzetta,P.A.;Alvarez,L.J.;Reinach,P.S.;Candia,O.A.Anal.Biochem.1979,100,
95; (b) Henkel, R. D.; VandeBerg, J. L.; Walsh, R. A. Anal. Biochem. 1988, 169, 312.
10. Spencer, D. M.; Wandless, T. J.; Schreiber, S. L.; Crabtree, G. R. Science 1993, 262,
1019.
11. Shah, K.; Liu, Y.; Deirmengian, C.; Shokat, K. M. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 3565.
The authors wish to acknowledge Professor Kevan Shokat, Uni-
versity of California at San Francisco, for providing the initial li-
brary and invaluable consultation and Professor Chi-Huey Wong
for providing chemical synthesis space and advice.
12. Bishop, A. C.; Shah, K.; Liu, Y.; Witucki, L.; Kung, C.; Shokat, K. M. Curr. Biol.
1998, 8, 257.
13. Kapoor, T. M.; Mitchison, T. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9106.
14. Gillespie, P. G.; Gillespie, S. K.; Mercer, J. A.; Shah, K.; Shokat, K. M. J. Biol. Chem.
1999, 274, 31373.
15. Holt, J. R.; Gillespie, S. K.; Provance, D. W.; Shah, K.; Shokat, K. M.; Corey, D. P.;
Mercer, J. A.; Gillespie, P. G. Cell 2002, 108, 371.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.015.
16. Chapman, E.; Farr, G. W.; Fenton, W. A.; Johnson, S. M.; Horwich, A. L. Proc. Natl.
Acad. Sci. U.S.A. 2008. doi:10.1073/pnas.0810657105.