Hydan oin analogs inhibi he ully assembled ClpXP p o ease
wi hou a ec ing he indi idual pep idase and chape one
domains
Ch is ian Fe ze ,a Vadim S. Ko o ko a,b and S ephan A. Siebe a,†
P o eolysis media ed by ClpXP is a c ucial cellula p ocess linked o
bac e ial pa hogenesis. The de elopmen o speci ic inhibi o s has
la gely ocused on ClpP. Howe e , his ocus was challenged by a
ecen inding showing ha con o ma ional con ol by ClpX leads
o a ejec ion o ClpP binde s. Thus, we he e ollow up on a hi
molecule om a high h oughpu sc een pe o med agains he
whole ClpXP complex and demons a e ha s able inhibi ion wi h
high po ency is possible. Fu he in es iga ions e ealed ha he
small molecule binds o ClpP wi hou a ec ing i s ac i i y. Likewise,
he molecule does no inhibi ClpX and e ains he o e all
oligome ic s a e o ClpXP upon binding. S uc u e ac i i y
ela ionship s udies con i med s uc u al cons ain s in all h ee
pa s o he molecule sugges ing binding in o a de ined
s e eospeci ic pocke . O e all, he inhibi ion o ClpXP wi hou
a ec ing he indi idual componen s ep esen s a no el mechanism
wi h pe spec i es o u he op imiza ion o in si u applica ions.
Caseinoly ic p o ease P (ClpP) is a e adecame ic enzyme
belonging o he se ine hyd olase amily.1, 2 The enzyme is able
o diges small pep ides bu canno ac on la ge p o ein
subs a es by i sel . In he cell ClpP binds o cogna e chape ones
such as ClpX ia bo h apical si es o he e adecame ic ba el
o ming he p o eoly ically ac i e ClpXP complex.3 Wi hin his
complex ClpX ecognizes p o eins p one o deg ada ion, e.g.
du ing cellula s ess, un olds hem unde consump ion o ATP
and inally pushes he pep ide chain in o he ClpP ba el o
subsequen p o eoly ic diges . ClpXP ac i i y has been shown o
be c ucial o cell homeos asis in cance cells as well as o he
con ol o i ulence in pa hogenic bac e ia such as
S aphylococcus au eus.4, 5 As a consequence o hese unc ions
he gene ic dele ion as well as chemical inhibi ion led o posi i e
he apeu ic e ec s in ea ed leukemia as well as bac e ial cells,
espec i ely.5-7 Howe e , majo challenges in he de elopmen
o inhibi o s a e he selec i i y o compounds o ClpP o ClpX,
hei s abili y in plasma, esidence ime when bound o he
enzyme as well as con o ma ional con ol o ClpX o e ClpP
esul ing in ejec ion o compounds.8, 9 One o he easons o
hese limi a ions was he p e ious ocus on solely ClpP du ing
inhibi o de elopmen and jus ecen mechanis ic insigh s
un a eled he impo ance o he whole ClpXP complex as mo e
sui able a ge o mimic cellula ac i i y. In o de o o e come
he limi a ions o p e ious co alen inhibi o s10 such as be a-
lac ones, phenyl es e s and bo onic acids as well as e e sible
inhibi o s such as oxazoles, a new high- h oughpu sc eening
(HTS) campaign u ilized an assay moni o ing he ac i i y o
whole SaClpXP complex.11 The ac i i y was moni o ed by he
deg ada ion o g een luo escen p o ein (GFP) agged wi h a
Ss A pep ide sequence, equi ed o ClpX media ed
ecogni ion.12 In p inciple, his HTS could deli e 3 ypes o
inhibi o s: 1) inhibi o s o ClpP, 2) inhibi o s o ClpX and 3)
inhibi o s ha block he in e play be ween ClpX and ClpP. The
HTS e eled 6 alida ed hi s which we e all able o po en ly
inhibi he whole p o eoly ic complex. One inhibi o was
selec ed o ollow up s udies demons a ing ha i ac ed solely
on he ClpX chape one.11 Binding o his compound o ClpXP led
o a dis up ion o he oligome ic complex associa ed wi h a
disassembly o ClpX hexame s in o monome s.
Co espondingly, he compound inhibi ed ClpX ATPase ac i i y
bu had no e ec on ClpP.
He e, we ollow up on hese HTS esul s and in oduce, in
addi ion o al eady exis ing ClpP and ClpX inhibi o s, an
unp eceden ed compound which nei he in luences ClpP no
ClpX ac i i y bu po en ly blocks u no e o he whole complex.
Syn hesis o se e al de i a i es e eals insigh s in o he
s uc u e ac i i y ela ionship (SAR) and he p epa a ion o wo
enan iome s shows a clea p e e ence o one enan iome o
complex inhibi ion. Toge he wi h he mal shi assays, hese
a.
Technische Uni e si ä München, Cen e o In eg a ed P o ein Science Munich
(CIPSM), Lich enbe gs aße 4, 85748 Ga ching (Ge many)
b.
Cu en add ess: Helmhol z-Zen um ü In ek ions o schung, Inho ens aße 7,
38124 B aunschweig (Ge many)
† E-Mail: s ephan.siebe @ um.de
esul s sugges he exis ence o a de ined, s e eospeci ic binding
pocke loca ed on ClpP.
Compound
1
was iden i ied as one o he mos po en inhibi o s
o ClpXP ac i i y in a p e ious HTS (Figu e 1A).11 P io o u he
mechanis ic s udies o his compound, we pe o med a
seconda y assay o exclude a pu a i e ac i i y agains c ea ine
kinase, an enzyme equi ed in he HTS o egene a e ATP om
ADP in he GFP assay. In con as o a posi i e con ol wi h
iodoace amide, no signi ican educ ion o ac i i y could be
obse ed in he Kinase-Glo assay (Suppo ing Figu e 1).
In e es ingly,
1
did nei he inhibi ClpP pep idase ac i i y, as
measu ed by u no e o a luo escen subs a e (posi i e
con ol wi h a p e ious oxazole inhibi o ),9 no ClpX media ed
ATP hyd olysis (Figu e 1B, C). Howe e , when ClpXP ac i i y was
es ed ia he GFP deg ada ion assay, he compound e ealed
po en inhibi ion wi h an IC50 o 1.9 µM, a ema kable ac i i y
when conside ing ha 4 µM o enzyme (ClpP monome ) is
needed in his assay (Figu e 1D).
To u he localize i he compound binds o ClpP o ClpX we
pe o med he mal shi assays in he p esence o absence o
1
.
A s ong s abiliza ion as indica ed by a 9 K shi in mel ing
empe a u e was obse ed o ClpP bu no ClpX (Figu e 2A). As
expec ed by he s uc u e o
1
which lacks ob ious elec ophilic
moie ies, no co alen modi ica ion was obse ed on ClpP ia
mass spec ome y analysis (Figu e 2B). Analy ical size exclusion
ch oma og aphy showed ha
1
does nei he deoligome ize
ClpP no ClpX as well as he whole complex (Figu e 2C). This
indica es an unp eceden ed mode o inhibi ion o ClpXP ia
e e sible binding o solely ClpP while e aining he o e all
oligome ic assembly.
Subsequen SAR s udies aimed o deciphe he mos impo an
s uc u al elemen s o
1
esponsible o inhibi ion. Fo his he
compound was dissec ed in o h ee pa s, namely he
hydan oin, linke and hiophene moie ies (Figu e 3A). All
analogs we e ei he syn hesized o ob ained om comme cial
sou ces and hei co esponding s uc u es a e displayed in
Figu e 3B. The syn hesis s a ed wi h educ i e amina ion o
subs i u ed hienca baldehyde wi h alkylamines (Scheme 1).
The ob ained seconda y amines we e acyla ed wi h 2-
b omoace yl b omide. 5,5-Dia ylhydan oins we e syn hesized
om he co esponding 1,2-dia yl-1,2-diones and u ea.
Regioselec i e N-alkyla ion o 5,5-dia ylhydan oins wi h 2-
4.55.0 5.56.0 6.57.0 7.58.0
0
20
40
60
80
100
-log c [c/M]
ClpP Pep idaseAc i i y[%]
286
1
DMSO
nega i e
1-1
00
µM
0
20
40
60
80
100
120
ClpX ATPase Ac i i y [%]
4.04.5 5.05.5 6.06.5 7.0
0
20
40
60
80
100
-log c 1 [c/M]
ClpXPP o easeAc i i y[%]
A)
D)
B)
C)
1
O
N
HO
N
N
H
N
H
OS
286
Figu e 1 In luence o compound 1 on c ea ine kinase, ClpP, ClpX and ClpXP ac i i y. A)
Chemical s uc u e o ClpXP inhibi o 1. B) 1 does no al e ClpP pep idase ac i i y in a
luo escen assay (1 µM ClpP; mean ± s anda d e o ). The non-co alen ClpP inhibi o
286 was used as a posi i e con ol. C) ClpX ATPase ac i i y assay is no inhibi ed by 1
(100 µM inal compound concen a ion; mean ± s anda d de ia ion). D) 1 shows a dose-
dependen inhibi ion o he ClpXP p o ease (mean ± s anda d e o ).
22600 22800 23000 23200 23400
0
50
100
ClpP + 1
MW [Da]
Rela i eIn ensi y[%]
22600 22800 23000 23200 23400
0
50
100 ClpP + DMSO
MW [Da]
Rela i eIn ensi y[%]
6810 12 14 16
0
20
40
60
80
100
V [mL]
Rela i eUV Abso bance[%]
1
DMSO
ClpX6ClpX2-3
6810 12 14 16
0
20
40
60
80
100
V [mL]
Rela i eUV Abso bance[%]
DMSO
1
ClpXP
ClpP14
6810 12 14 16
0
20
40
60
80
100
V [mL]
Rela i eUV Abso bance[%]
DMSO
1
ClpP14
30 35 40 45 50 55 60 65 70
-1500
-1000
-500
0
500
1000
1500
Tempe a u e[°C]
-d(RFU)/dT
ClpP +DMSO: 46.9 ± 0.2 °C
ClpP +1: 55.9 ± 0.2 °C
ClpX +DMSO: 40.4 ± 0.3 °C
ClpX +1: 41.0 ± 0.5 °C
A) C)
B)
ClpP
ClpX
ClpXP
unmodi ied ClpP
unmodi ied ClpP
Figu e 2 In e ac ion o 1 wi h ClpP and ClpX. A) The mal-shi assay depic s s ong
s abiliza ion o ClpP while he mel ing poin o ClpX emains unal e ed. Expe imen s we e
conduc ed wi h 10 µM enzyme concen a ion in PBS bu e and DMSO o 60 µM o 1. B)
No co alen modi ica ions o ClpP a e de ec able by in ac -p o ein mass spec ome y
upon ea men wi h 1 a 100 µM (100- old excess). C) Size-exclusion ch oma og aphy
expe imen s show no subs an ial change o ClpP- e adecame , ClpX-hexame and
ClpXP-complex upon ea men wi h 100 µM 1.
Scheme 1 Syn hesis o 1 analogs. Reagen s and Condi ions: a) u ea, 40% aq. NaOH, E OH,
e lux, 3 h, hen HCl, . ., 41 – 66%; b) R2NH2, HOAc, THF, MeOH, 0° C, 4 h, hen . ., 18
h, 92–94%; c) NaBH4, E OH, 2 h, . ., 86 – 91%; d) 2-b omoace yl b omide, CH2Cl2, 0° C
o . ., 1 h, 38 – 76%; e) Cs2CO3, DMF, . ., 3 h, 25 – 57%
b omoace amides using Cs2CO3 as a base led o he desi ed
p oduc s.13 Al e a ions in he hiophene sec ion o he molecule
by exchange o he halogen subs i u ed hiophene wi h a phenyl
g oup (
2
) esul ed in a en old lowe IC50 alue in he ClpXP
p o ease assay (Figu e 3C). P opyla ion (
3
) ins ead o
me hyla ion (
4
) o he ni ogen loca ed in he linke almos
comple ely p e en ed inhibi ion. Howe e , sho ening o he
linke and emo al o he ni ogen (
5
) s ill e ained some
ac i i y, al hough no ull inhibi ion could be ob ained.
Replacemen o one phenyl g oup by a me hyl subs i uen a he
hydan oin co e s uc u e (
6
) esul ed in a 40- old d op in
po ency. This sugges s ha s e ically demanding g oups a e
necessa y o po en inhibi ion. Hence, he ole o addi ional
me hyl subs i u ed phenyl g oups was u he elucida ed.
Disubs i u ed compound
1
exhibi ed he highes po ency while
compound
7
, bea ing wo equally me hyl subs i u ed phenyl
moie ies, showed comple e loss o ac i i y. This esul indica es
ha only one enan iome o
1
is binding o ClpP as he me hyl
subs i uen seems o clash wi h he binding si e in one o he
wo possible con o ma ions. Consequen ly, enan iome s we e
sepa a ed by chi al high-pe o mance liquid ch oma og aphy.
Due o he small amoun o isola ed enan iome s he absolu e
con igu a ion could no be assigned by X- ay analysis.
Indi idual es ing o bo h enan iome s in he ClpXP p o ease
assay e ealed ha only
1
-E1 showed inhibi ion, while he o he
enan iome (
1
-E2) was comple ely inac i e (Figu e 3C). This
sugges s he exis ence o a de ined, s e eospeci ic and
d uggable binding pocke enabling u he s udies o imp o e
he in si u ac i i y ia pa ame e s such as up ake and s abili y.
S udies in o i ulence we e pe o med ia Wes e n blo analysis
and LC-MS/MS. Only a sligh educ ion o alpha-hemolysin, a
majo i ulence ac o linked o ClpXP ac i i y, was obse ed o
he acema e and
1
-E2 bu no o
1
-E1 ea ed samples
(Suppo ing Figu e 2). Howe e , sec e ome analysis o
1
enan iome s by LC-MS/MS showed no change o alpha-
hemolysin compa ed o DMSO ea ed samples (Suppo ing
Table 1). We hus conclude ha physicochemical pa ame e s
need o be op imized o mo e e ec i e s udies on whole cells.
Conclusions
In conclusion,
1
cons i u es a new class o ClpXP inhibi o s ha
nei he ac on pep idase no chape one ac i i ies bu equi es
he whole p o eoly ic complex o impai ing i s subs a e
u no e . The compound binds s e eospeci ically in o a ye
uniden i ied binding pocke on ClpP, which acco ding o ou SAR
da a exhibi s a de ined shape allowing only es ic ed
modi ica ions. The elucida ion o his pocke in u u e s udies
may u he un a el he p ecise mechanism o ac ion and guide
he design o imp o ed inhibi o s also bea ing in si u ac i i ies.
Con lic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
This wo k was suppo ed by he Deu sche Fo schungsgemein-
scha SI1096/8-1 (ClpP) and CIPSM.
No es and e e ences
1. R. T. Saue and T. A. Bake , Annu. Re . Biochem., 2011, 80,
587-612.
2. K. Liu, A. Ologbenla and W. A. Hou y, C i . Re . Biochem.
Mol. Biol., 2014, 49, 400–412.
3. T. A. Bake and R. T. Saue , Biochim. Biophys. Ac a, 2012,
1823, 15-28.
4. D. F ees, S. N. Qazi, P. J. Hill and H. Ingme , Mol. Mic obiol.,
2003, 48, 1565-1578.
5. D. F ees, K. So ensen and H. Ingme , In ec . Immun., 2005,
73, 8100-8108.
6. A. Cole, Z. Wang, E. Coyaud, V. Voisin, M. G onda, Y.
Ji ko a, R. Ma son, R. Hu en, S. Babo ic, N. Maclean, I.
Res all, X. Wang, D. V. Jeya aju, M. A. Sukhai, S. P abha, S.
Bashi , A. Ramak ishnan, E. Leung, Y. H. Qia, N. Zhang, K. R.
Combes, T. Ke ela, F. Lin, W. A. Hou y, A. Aman, R. Al-Awa ,
W. Zheng, E. Wienholds, C. J. Xu, J. Dick, J. C. Wang, J.
Mo a , M. D. Minden, C. J. Ea es, G. D. Bade , Z. Hao, S. M.
Ko nblau, B. Raugh and A. D. Schimme , Cance cell, 2015,
27, 864-876.
7. T. Bö che and S. A. Siebe , J. Am. Chem. Soc., 2008, 130,
14400-14401.
8. M. W. Hackl, M. Lakemeye , M. Dahmen, M. Glase , A.
Pahl, K. Lo enz-Baa h, T. Menzel, S. Sie e s, T. Bo che , I.
An es, H. Waldmann and S. A. Siebe , J. Am. Chem. Soc.,
2015, 137, 8475-8483.
9. A. Pahl, M. Lakemeye , M. T. Vielbe g, M. W. Hackl, J.
Vomacka, V. S. Ko o ko , M. L. S ein, C. Fe ze , K. Lo enz-
Baa h, K. Rich e , H. Waldmann, M. G oll and S. A. Siebe ,
Angew. Chem. In . Ed., 2015, 54, 15892-15896.
10. F. Ye, J. Li and C. G. Yang, Mol. Biosys ., 2016, 13, 23-31.
11. C. Fe ze , V. S. Ko o ko , R. Thane , K. M. Lee, M.
Neuenschwande , J. P. on K ies, E. Medina and S. A.
Siebe , Angew. Chem. In . Ed., 2017, 56, 15746-15750.
12. S. Go esman, E. Roche, Y. Zhou and R. T. Saue , Genes
De ., 1998, 12, 1338-1347.
13. N. T. Tz e ko , H. Eule and C. E. Mülle , Beils ein J. O g.
Chem., 2012, 8, 1584–1593.
Figu e 3 S uc u e-ac i i y ela ionship s udies o 1 analogs in ClpXP-p o ease assay. A)
Dissec ion o 1 in o 3 pa s. B) Chemical s uc u es o 1 analogs. C) Inhibi ion da a o all
compounds in he ClpXP p o ease assay.
A)
B)
O
NCl
S
N
HN
O
O
linke hiophenehydan oin
34567
0
25
50
75
100
-log c [c/M]
ClpXPP o easeAc i i y[%]
1
1 - E1
1 - E2
C)
4567
0
25
50
75
100
-log c [c/M]
ClpXPP o easeAc i i y[%]
2
3
4
4567
0
25
50
75
100
-log c [c/M]
ClpXPP o easeAc i i y[%]
6
5
7