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A s uc u e-based app oach owa ds he
iden i ica ion o no el an ichagasic compounds:
T ypanosoma c uzi ca bonic anhyd ase inhibi o s
Manuel A. Llanos, Ma ía L. Sba aglini, Ma ía L. Villalba, Ma ía D. Ruiz,
Ca olina Ca illo, Ca alina Alba So o, Alan Tale i, And ea Angeli, Seppo
Pa kkila, Claudiu T. Supu an & Luciana Ga e ne
To ci e his a icle: Manuel A. Llanos, Ma ía L. Sba aglini, Ma ía L. Villalba, Ma ía D. Ruiz,
Ca olina Ca illo, Ca alina Alba So o, Alan Tale i, And ea Angeli, Seppo Pa kkila, Claudiu T.
Supu an & Luciana Ga e ne (2020) A s uc u e-based app oach owa ds he iden i ica ion o no el
an ichagasic compounds: T ypanosoma�c uzi ca bonic anhyd ase inhibi o s, Jou nal o Enzyme
Inhibi ion and Medicinal Chemis y, 35:1, 21-30, DOI: 10.1080/14756366.2019.1677638
To link o his a icle: h ps://doi.o g/10.1080/14756366.2019.1677638
© 2019 The Au ho (s). Published by In o ma
UK Limi ed, ading as Taylo & F ancis
G oup.
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RESEARCH PAPER
A s uc u e-based app oach owa ds he iden i ica ion o no el an ichagasic
compounds: T ypanosoma c uzi ca bonic anhyd ase inhibi o s
Manuel A. Llanos
a
,Ma

ıa L. Sba aglini
a
,Ma

ıa L. Villalba
a
,Ma

ıa D. Ruiz
b
, Ca olina Ca illo
b
, Ca alina Alba So o
c
,
Alan Tale i
a
, And ea Angeli
d
, Seppo Pa kkila
e
, Claudiu T. Supu an
d
and Luciana Ga e ne
a
a
Labo a o y o Bioac i e Resea ch and De elopmen (LIDeB), Medicinal Chemis y, Depa men o Biological Sciences, Facul y o Exac Sciences,
Na ional Uni e si y o La Pla a, Buenos Ai es, A gen ina;
b
Ins i u o de Ciencias y Tecnolog

ıa D . Cesa Mils ein (ICT Mils ein), A gen inean
Na ional Council o Scien i ic and Technical Resea ch (CONICET), Buenos Ai es, A gen ina;
c
Depa amen o de Mic obiolog

ıa, Pa asi olog

ıae
Inmunolog
ıa, Facul ad de Medicina, Uni e sidad de Buenos Ai es, Ins i u o de In es igaciones en Mic obiolog

ıa y Pa asi olog

ıaM

edica (IMPaM),
UBA-CONICET, Buenos Ai es, A gen ina;
d
Neu o a ba Depa men , Sezione di Scienze Fa maceu iche e Nu aceu iche, Uni e si a degli S udi di
Fi enze, Ses o Fio en ino, Flo ence, I aly;
e
Facul y o Medicine and Heal h Technology, Uni e si y o Tampe e, Tampe e, Finland
ABSTRACT
T ypanosoma c uzi ca bonic anhyd ase (TcCA) has ecen ly eme ged as an in e es ing a ge o he design
o new compounds o ea Chagas disease. In his s udy we epo he esul s o a s uc u e-based i ual
sc eening campaign o iden i y no el and selec i e TcCA inhibi o s. The combina ion o p ope ly alida ed
compu a ional me hodologies such as compa a i e modelling, molecula dynamics and docking simula-
ions allowed us o ind high po ency hi s, wi h K
I
alues in he nanomola ange. The compounds also
showed ypanocidal e ec s agains T. c uzi epimas igo es and ypomas igo es. All he candida es a e
selec i e o inhibi ing TcCA o e he human iso o m CA II, which is encou aging in e ms o possible
he apeu ic sa e y and e icacy.
ARTICLE HISTORY
Recei ed 9 Sep embe 2019
Re ised 30 Sep embe 2019
Accep ed 1 Oc obe 2019
KEYWORDS
Chagas; ca bonic anhyd ase;
i ual sc eening;
sulphamides; sodium
cyclama e; acesulphame
In oduc ion
Ca bon dioxide (CO
2
) is a p oduc o many me abolic ae obic
p ocesses in di e en o ganisms. I has a ela i ely low solubili y
in wa e , bu i eac s wi h his sol en (a he neu al pH) o p o-
duce bica bona e anion and p o ons. These wo ions play a c ucial
ole in main aining pH homeos asis in li ing sys ems and hei
equilib a ion wi h CO
2
is con olled by he ac i i y o he enzymes
Ca bonic Anhyd ase (CA, EC 4.2.1.1)
1–5
. CAs a e ex emely e icien
ca alys s, and hey a e widesp ead in o ganisms all o e he phylo-
gene ic ee. So a , he e a e eigh gene ically dis inc amilies
ca ego ised as a-, b-, c-, d-, -, g-, H- and d-CAs
6–12
. In mammals,
he e a e 16 iso o ms known o da e, ha belong o he a-CA
amily and some o hem ha e been s udied as molecula a ge s
o ea human diseases
8–12
. T adi ionally, CA inhibi o s a e
employed as diu e ic and an iglaucoma d ugs, bu o he clinical
applica ions ha e been p oposed, including he ea men o can-
ce , obesi y and epilepsy
13–19
. Rega ding neglec ed p o ozoan dis-
eases, such as he Ame ican ypanosomiasis, CA is eme ging as a
new he apeu ic a ge , wi h i s inhibi o s being p obably able o
p o ide an inc eased e icacy and sa e y in compa ison wi h he
cu en ea men s
20
. Ame ican ypanosomiasis, also known as
Chagas disease, is caused by he pa asi e T ypanosoma c uzi
21
.I
was i s desc ibed by Ca los Chagas mo e han a cen u y ago and
i a ec s abou 8 million people wo ldwide
22
. The disease is cha -
ac e ised by wo phases: an ini ial acu e s age which usually goes
unde ec ed because i lacks speci ic symp oms and; a ch onic
s age ha may emain asymp oma ic bu e ol es o clinical mani-
es a ions in speci ic o gans (hea , diges i e sys em and/o ne -
ous sys em) in abou 30% o he pa ien s
23
. The he apeu ic
a senal o ea Chagas is composed o only wo d ugs, benznida-
zole and ni u imox
23
. They we e disco e ed mo e han 40 yea s
ago, and hey a e mos ly ac i e in he acu e phase o he disease.
Thei limi ed e icacy in he ch onic phase as well as hei consid-
e able oxici y os e ed he e o s o explo e he molecula biol-
ogy o T. c uzi aiming o disco e new he apeu ic a ge s. In his
scena io, he inhibi ion o p o ozoan CAs showed up ecen ly as
an in e es ing op ion o he u u e ea men o Chagas disease,
as well as o he neglec ed pa hologies like Leishmaniasis
20
.
A p esen , only one T. c uzi ca bonic anhyd ase (TcCA) has
been desc ibed in he genome o he pa asi e. TcCA has ecen ly
been cloned and cha ac e ised by Pan e al.
24
I belongs o he
a-CA amily and sha es he ac i e si e opology wi h he ca aly ic
a-iso o ms ound in mammals: he h ee His esidues coo dina ing
wi h one Zinc ion, and a ou h coo dina ion posi ion occupied by
a wa e molecule a acidic pH (<7) o by a hyd oxide ion a
highe pH alues
20,24
. The ole o he enzyme in he pa hogen is
s ill poo ly unde s ood, bu i is known ha TcCA shows a e y
high ca aly ic ac i i y
24,25–29
. I has been hypo hesised ha TcCA is
in ol ed in he p oli e a ion o he pa asi e when i is in he epi-
mas igo e s age
20
. This assump ion was suppo ed by some in
CONTACT Claudiu T. Supu an [email p o ec ed] Neu o a ba Depa men , Sezione di Scienze Fa maceu iche e Nu aceu iche, Uni e si a degli S udi di
Fi enze, Via U. Schi 6, Ses o Fio en ino, Flo ence, 50019, I aly; Luciana Ga e ne [email p o ec ed] Labo a o y o Bioac i e Resea ch and
De elopmen (LIDeB), Medicinal Chemis y, Depa men o Biological Sciences, Facul y o Exac Sciences, Na ional Uni e si y o La Pla a, 47 and 115, La Pla a, Buenos
Ai es, B1900BJW, A gen ina
Supplemen al da a o his a icle can be accessed he e.
ß2019 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 21–30
h ps://doi.o g/10.1080/14756366.2019.1677638
i o and ex i o s udies ha demons a ed he abili y o TcCA
inhibi o s o inhibi epimas igo e p oli e a ion.
Rega ding he chemical s uc u e o CA inhibi o s, sulphona-
mides a e he main class o compounds ha in e ac s wi h CAs in
mammals, especially wi h CA II (which is, by a , he mos s udied
iso o m)
13–19
. The classical mechanism o inhibi ion in ol es he
in e ac ion o he sulphonamide ni ogen a om wi h he Zinc ion,
which places he inhibi o s in he posi ion o iginally occupied by
wa e (o he hyd oxide ion)
13–19,30
.TcCA is also inhibi ed by o he
compound amilies, such as hiols and hyd oxama es
20
. Mo eo e ,
molecules om hese wo classes showed be e po ency and
selec i i y in i o compa ed o sulphonamides, as well as p omis-
ing in i o p o iles, by inhibi ing he h ee phases o he pa ho-
gen’s li e cycle ( ypomas igo e, amas igo e and epimas igo e)
24
.
Following hese p e ious in es iga ions, we in es iga e he e he
capaci y o a new se o compounds o inhibi TcCA. One o he
main challenges in he de elopmen o new CA inhibi o s is o
ind selec i i y agains he speci ic iso o ms in ol ed in he dis-
ease, o a oid ole ance p oblems and o imp o e he he apeu ic
sa e y. The e o e, he selec ion o he candida es o biological
assays was guided by he esul s o a i ual sc eening p o ocol
applied o a se o compounds ha sha e a zinc binding unc ion
(sulphamides, and hei bioisos e s sulphama es and sulphona-
mides) and ha e low inhibi o y ac i i y agains he ubiqui ous
human iso o m CA II, which could be conside ed as an an i- a ge .
Ou in es iga ion ini ia ed wi h he cons uc ion o h ee-
dimensional models o TcCA, since he expe imen al s uc u e o
he a ge is no ye a ailable. A es se o 87 known TcCA inhibi-
o s was hen compiled om li e a u e and used o selec he
bes docking model o he i ual sc eening, based on he co el-
a ion be ween he docking sco e and he expe imen ally de e -
mined pK
I
. Finally, an in-house da ase o 255 compounds was
sc eened and 10 compounds we e es ed agains TcCA. An i-
ypanosomal e ec s we e also in es iga ed o he selec ed candi-
da es. In o ma ion abou he iden i y o he s uc u es o he es
se (Table S1) and he da ase o sc eening is gi en as suppo ing
in o ma ion (Table S2) (see Supplemen a y in o ma ion o de ails)
Ma e ials and me hods
P o ein s uc u e modelling
We applied he Meie e al.
31
algo i hm as implemen ed in he
HHP ed Se e
32
o cons uc ou ini ial model o he enzyme. The
sequence o TcCA was e ie ed om Unip o (Q4CVY4)
33
. A mul i-
empla e model was gene a ed combining he op 10 empla es
anked by i s alignmen sco e. The quali y o he model was e al-
ua ed wi h he me ics p o ided by he se e , Molp obi y and he
QMEAN sco ing unc ions
32
. Then, he mac os uc u e was submi -
ed o an i e a i e e inemen p ocess, combining Rose a and
Molecula Dynamics (MD) simula ions. Fo ha pu pose, we used
Rose a’s Fas Relax algo i hm wi h he me allop o eins modi ica-
ion, which imposes cons ain s on he Zinc ion and i s coo dina -
ing esidues o p ese e he coo dina ion geome y du ing he
simula ion
34–37
. B ie ly, he p o ocol consis s o al e na ing cycles
o o ame epacking and g adien -based ene gy minimisa ion,
inc easing he epulsi e con ibu ions wi hin each cycle. A e gen-
e a ing 100 new e ined models, he op sco ing decoy was
selec ed as he s a ing poin o MD simula ions.
MD simula ions we e ca ied ou wi h he Ambe 16 package
38
,
using he FF14SB o ce ield and he Zinc AMBER o ce ield
(ZAFF) modi ica ion designed o 4-coo dina ed Zinc me al
cen es
39
. Because he h ee His esidues ha coo dina e wi h he
zinc ion in human CA a e also conse ed in TcCA, we assumed a
e ahed al coo dina ion o he Zinc a om and a wa e molecule
was manually placed in he ou h e ex o ul il he coo dina ion
geome y. Thus, he cen e ID No. 6 in he ZAFF o ce ield
was selec ed o se he pa ame e s, which we e de i ed by Me z
e al. om human CA-II c ys al s uc u e 1CA2 using he
MCPB p og am
39
.
The s uc u e was sol a ed in an oc ahed al box o TIP3P wa e
molecules, and Cl

ions we e added o neu alise he sys em.
Minimisa ion o he sys em was pe o med in ou sequen ial
s eps. Fi s , only hyd ogen a oms we e minimised. Second, only
he sol en a ound he p o ein was allowed o mo e. Thi d, only
p o ein side chains and sol en we e minimised, ixing he pos-
i ion o he p o ein backbone a oms. Finally, he whole sys em
was minimised wi h no cons ain s. E e y minimisa ion s age
in ol ed 2500 s eepes descen s eps, ollowed by 7500 s eps o
conjuga e g adien . Then, he sys em was ca e ully hea ed o
298.15 K and equilib a ed in six consecu i e s eps, s a ing in NVT
condi ions and hen swi ching o an NPT ensemble o equilib a e
he sys em densi y, p og essi ely elie ing cons ain s o achie e a
p ope equilib a ion. A o al o 50ns was simula ed, moni o ing
he p o ein backbone RMSD as a simula ion con e gence c i e ion,
and 10 ep esen a i e snapsho s we e ex ac ed by clus e ing ana-
lysis. Fo ha pu pose, he whole ajec o y was clus e ed using
he K-means algo i hm implemen ed in cpp aj, ixing he numbe
o clus e s (K¼10) and choosing he clus e cen oid as he ep e-
sen a i e s uc u e in each case. Finally, all he selec ed snapsho s
we e subjec ed o a la e e inemen s ep using Rose a’s Fas
Relax p o ocol desc ibed be o e
34–37
, o sample high-quali y
decoys a ound he s a ing con o ma ion. These 10 inal TcCA
s uc u al models we e used o docking simula ion.
Cons uc ion o he da ase and alida ion o he
docking models
A alida ion se o 87 molecules es ed agains he TcCA was com-
piled om li e a u e
20
. I includes compounds wi h di e en
chemical s uc u es bu mos ly p esen ing sulphonamides, hiols
and hyd oxama es as Zinc binding unc ions. The ac i i y alues
(K
I
) o he compounds ange be ween 0.51 and 84,000 nM. These
epo ed ac i i ies we e ans o med o pK
I
alues (Table S1).
Benzoxabo oles desc ibed by Nocen ini e al.
40
we e excluded
om he da ase because he docking o ce ield does no include
pa ame e s o bo on a oms. Fo each molecule, he mos abun-
dan species a pH 7.4 was calcula ed wi h ChemAxon
41
, conside -
ing au ome iza ion and esonance e ec s. Finally, 5000 s eps o
geome y op imisa ion we e ca ied ou wi h he MMFF94S o ce
ield, o ge easonable s a ing geome ies. I is wo h no icing
ha hiol compounds we e p edic ed o be dep o ona ed a he
gi en pH, wi h he nega i e cha ge s abilised by a ni ogen a om
om an adjacen he e oa oma ic ing ha is p esen in hese mol-
ecules. The docking calcula ions we e un wi h Au oDockZN
42
,a
so wa e ha includes modi ica ions o he classic Au oDock4
o ce ield wi h speci ic pa ame e s o he zinc ion. This o ce ield
has p o en o o e come he pe o mance o he o iginal
Au oDock4
43
o docking small molecules in o Zinc me allop o-
eins, in bo h binding ene gy es ima ion and pose p edic ion. The
g id maps we e cons uc ed cen ed on he Zinc a om o e e y
TcCA model wi h Au og id 4.2.6
43
. They co e ed 60 poin s in each
dimension om he Zinc cen e, wi h a spacing o 0.375Å. The
docking simula ions we e un by conside ing a popula ion size o
300 and an eli ism o 1. We pe o med 100 uns o he
Lama ckian Gene ic Algo i hm wi h Au oDock4.2
43
. The sco ing
22 M. A. LLANOS ET AL.
powe o e e y docking model (combina ion o docking p o-
g amme and TcCA s uc u al model) was e alua ed by calcula ing
he Pea son co ela ion coe icien (Pea son’s ) be ween he dock-
ing sco es and he pK
I
alues o all he compounds in he alid-
a ion se . Addi ionally, we e alua ed he anking powe o he
models by calcula ing Kendall’s ank co ela ion coe icien (Tau B)
and Spea man’s ank co ela ion coe icien (Rho).
In o de o e alua e he model’s pe o mance as a classi ie ,
compounds we e de ined ei he as ac i e o inac i e based on
he epo ed K
I
alue. To his end, compounds wi h a K
I
alue
lowe han 1 lM we e conside ed as ac i es. Then, he A ea
Unde he Recei e Ope a ing Cha ac e is ic Cu e (ROC-AUC)
was calcula ed as a me ic o e alua e he disc imina ing powe
o he model. Figu e 1 shows a schema ic ep esen a ion o he
me hods used in his in es iga ion o c ea e and alida e he
s uc u e-based p edic i e model o TcCA o he i ual sc een-
ing campaign.
Vi ual sc eening
The p e iously desc ibed docking p o ocol was selec ed o sc een
an inhouse lib a y o 255 compounds (Table S2). Some o hese
compounds we e syn hesised in ou lab
44–46
, whils o he s we e
comme cial compounds like he a i icial swee ene s cyclama e
and acesulphame. Among op sco ing hi s, 10 s uc u es we e
selec ed o biological e alua ion. All he candida es we e es ed
be o e as human CA II inhibi o s and hey showed poo inhibi o y
po ency in his mammalian iso o m
46–48
. As p e iously men ioned,
CA II is ubiqui ous in humans and i was conside ed as an an i-
a ge in his in es iga ion, since we a e looking o selec i e
inhibi o s o he pa asi e iso o m.
Biological assays
TcCA inhibi ion s udies
An Sx.18M -R Applied Pho ophysics (Ox o d, UK) s opped- low
ins umen has been used o assay he ca aly ic ac i i y o a ious
CA isozymes o CO
2
hyd a ion eac ion
49
. Phenol ed (a a con-
cen a ion o 0.2 mM) was used as indica o , wo king a he
abso bance maximum o 557nm, wi h 10 mM Hepes (pH 7.5) as
bu e , 0.1 M Na
2
SO
4
( o main aining cons an ionic s eng h), ol-
lowing he CA-ca alyzed CO
2
hyd a ion eac ion o a pe iod o
10 s a 25 C. The CO
2
concen a ions anged om 1.7 o 17 mM
o he de e mina ion o he kine ic pa ame e s and inhibi ion
cons an s. Fo each inhibi o a leas six aces o he ini ial 5–10%
o he eac ion ha e been used o de e mining he ini ial eloci y.
The unca alyzed a es we e de e mined in he same manne and
sub ac ed om he o al obse ed a es. S ock solu ions o inhibi-
o s (10 mM) we e p epa ed in dis illed-deionised wa e and dilu-
ions up o 1nM we e done he ea e wi h he assay bu e .
Enzyme and inhibi o solu ions we e p e-incuba ed oge he o
15 min (s anda d assay a oom empe a u e) p io o assay, o
allow o he o ma ion o he enzyme–inhibi o complex. The
inhibi ion cons an s we e ob ained by non-linea leas -squa es
me hods using PRISM 3 and he Cheng–P uso equa ion, as
epo ed ea lie
40,50–72
. All CAs we e ecombinan p o eins p o-
duced as epo ed ea lie by ou g oup
24
.
Biological ac i i y agains T. c uzi epimas igo es
Fo all biological assays, s ock and wo king solu ions o he candi-
da e d ugs we e p epa ed using DMSO as sol en and all condi-
ions we e es ed in iplica e.
Epimas igo es o he Y s ain o T. c uzi we e cul u ed a 28 C
in BHT medium supplemen ed wi h 20 lg/ml haemin, 10% hea -
inac i a ed oe al bo ine se um (FBS), 100 mg/ml s ep omycin and
Figu e 1. Schema ic illus a ion o he me hods in ol ed in he cons uc ion o he model o he i ual sc eening. The p og ess o he in es iga ion is desc ibed as a
cyan ci cle in each s ep o he molecula modelling.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 23
100 U/ml penicillin. The an ip oli e a i e ac i i y o he candida es
was es ed a 50 mM concen a ion in cul u es ini ia ed a 10
7
cells/ml. Un ea ed con ols we e pe o med unde he same cul-
u e condi ions wi h equal concen a ions o DMSO as o candi-
da e d ugs. A e 3 and 7 d, he numbe o iable pa asi es was
coun ed using a haemocy ome e chambe unde ligh mic o-
scope and esul s we e exp essed as pe cen age espec o he
un ea ed con ols
73
.
Biological ac i i y agains T. c uzi ypomas igo es
T ypomas igo es o T. c uzi we e pu i ied a he pa asi emia peak
om pe iphe al blood o mice in ec ed wi h he RA s ain.
T ypomas igo es (1 10
5
pe well) we e cul u ed in a 96 well-pla e
( inal olume 200ll) in RPMI medium supplemen ed wi h 10%
FBS a 37 Cin5%CO
2
a mosphe e. A e 24 h mo ile pa asi es
we e coun ed in haemocy ome e chambe unde he ligh mic o-
scope
74
. Resul s we e exp essed as % iabili y o ypomas igo es
( espec o con ol) a 20 mM o he hi s. Con ols we e pe o med
unde he same cul u e condi ions wi h equal concen a ions o
DMSO as o candida e d ugs. The nega i e con ol was cul u ed
wi h PBS and he posi i e con ol was cul u ed wi h Benznidazole
(20mM). The animal ca e o he expe imen al p o ocols was con-
duc ed in acco dance wi h he guidelines o he ca e and use o
labo a o y animals app o ed by he E hical Commi ee o he
Facul y o Medicine o he Uni e si y o Buenos Ai es.
Resul s and discussion
Cons uc ion o TcCA models
TcCA has been ecen ly cloned and cha ac e ised by Pan e al.
24
As men ioned be o e, he h ee His esidues coo dina ing he Zinc
ion a e conse ed in he ac i e si e o TcCA, wi h he ou h
coo dina ion posi ion occupied by a wa e molecule (o by a
hyd oxide ion, depending on he pH). Like human CA II, TcCA
exe s a high ca aly ic ac i i y, and he ga ekeeping esidues
Glu106 and Th 199 a e well conse ed
8–12
. Howe e , he p o on
shu le His64 is absen in TcCA iso o m. Despi e se e al CA s uc-
u es o many o ganisms ha e been c ys allised so a , he e is no
a good empla e o pe o m a compa a i e modelling o TcCA.
The e a e 924 s uc u es o CAs deposi ed in he P o ein Da a
Bank (PDB)
75
, bu all o hem ha e sequence iden i ies lowe han
30% wi h TcCA. In his si ua ion, he combina ion o mul iple em-
pla es gene ally inc eases he quali y o he esul an model, bu
only when he co ec empla es a e combined since he e is a
ade-o be ween he numbe o sequences included and he
noise in oduced in he es ain s. One possible app oach o
add ess his challenge was ecen ly p oposed by Meie e al.
31
and used in his in es iga ion. They in oduced a modi ica ion in
he MODELLER algo i hm, using p obabili y heo y o combine he
densi y unc ions o indi idual empla e es ain s
76
.
The mul iple sequence alignmen is p o ided as Suppo ing
In o ma ion (Figu e S1), including he PDB ID and UNIPROT ID o
each empla e selec ed. The inal model is 261 amino acids long,
spanning om esidue 56 o 316 o he a ge sequence. The
s uc u al quali y me ics a e summa ised in Table 1 o e e y
in e media e s uc u e o he modelling pipeline, om he aw
model gene a ed by he HHP ed se e o he e ined MD snap-
sho selec ed as he bes TcCA model o docking ( he c i e ia o
his selec ion was included in he nex sec ion). The quali y o he
models was es ima ed wi h he QMEAN sco ing unc ion, and i s
s e eochemical co ec ness and all-a oms con ac s we e e alua ed
wi h he Molp obi y so wa e.
The aw model p oduced by Modelle shows he poo es qual-
i y, whe eas Rose 
as e ined model o he bes snapsho
(17423_ e ined in Table 1) is he mos co ec in bo h geome ic
and ene ge ic e alua ions. The no malised QMEAN4 alue o he
inal model is simila o he sco es achie ed in high esolu ion X-
ay s uc u es o compa able size, and he pe - esidue ene gy ana-
lysis shows ha he p oblema ic po ions o he model a e
beyond he de ined docking si e (Figu e S2). Figu e 2 shows he
o e all a chi ec u e o he inal TcCA model, which was con-
s uc ed based on he mul i- empla e s a egy and subsequen ly
e ined by i e a i e Rose a-MD simula ions. Coo dina es o he
model ha e been included as Suppo ing In o ma ion.
Valida ion o he docking p o ocol o TcCA i ual sc eening
Once we ob ained an ensemble o TcCA models, he nex s ep
was he selec ion o he bes docking condi ions o he i ual
sc eening. Fo ha pu pose, we analysed he abili y o he so -
wa e o co ela e he docking sco es wi h he pK
I
alues o he
compounds o he alida ion se . We docked he alida ion se
Table 1. Values o he pa ame e s achie ed in he s uc u e quali y e alua ion o he di e en models o TcCA.
Me ic/model HHP ed aw HHP ed e ined Snap 17423 Snap 17423_ e ined
QMEAN4 6.04 1.60 4.06 0.96
Molp obi y 3.57 1.83 2.04 1.81
Ramachand an allowed 95.4 98.1 95.8 98.1
Clash sco e 136.37 5.43 2.71 4.93
Figu e 2. Final model o TcCA achie ed om mul i- empla e compa a i e model-
ling and subsequen i e a i e Rose a-MD e inemen . His idine esidues o he
ac i e si e a e highligh ed in g een o ca bon a oms and in blue o ni ogen
a oms. The zinc ion is ep esen ed as a non-bonded sphe e in g ey.
24 M. A. LLANOS ET AL.

in o he inal Rose a’s e ined models o he a ge . Boo s ap
sampling was applied o he da a o calcula e median alues and
i s associa ed con idence in e als o compa e be ween di e en
models. The lowes ene gy pose om all he uns was chosen as
he binding mode o each s uc u e. O he c i e ia would be o
conside he lowes ene gy pose om he mos popula ed clus e ,
bu we ound ha hese con o ma ions o ien ed he zinc-binding
unc ion ou side he binding si e o some compounds o
he da ase .
We did no expec a pe ec co ela ion be ween he Ki alues
and he sco es due o he known limi a ions o docking sco ing
unc ions o p edic absolu e binding ene gies, especially when
using p o ein models ins ead o c ys al s uc u es. Mo eo e , he e
a e expe imen al e o s and biological a iabili y inhe en o all
bioac i i y da a ha impac s on he inal co ela ions.
Ne e heless, hese me ics can be applied o compa e be ween
di e en p o ein/docking models o selec he bes one unde di -
e en condi ions.
Figu e 3 shows he s a is ical pa ame e s in a condi ional iolin
plo o e e y p o ein model, conside ing Pea son’sRbe ween he
docking sco es and he pK
I
alues. Acco ding o he esul s, we
decided o use as a ge model he snapsho 17423_ , since i
achie ed he bes co ela ion me ics o he s uc u es o he al-
ida ion se . All he me ics calcula ed o e e y p o ein model a e
summa ised in Table S3.
Addi ionally, ROC cu es we e cons uc ed o e alua e he cap-
aci y o he models o disc imina e known inhibi o s om non-
inhibi o s. The ROC cu es plo he sensi i i y o he model ( ue
posi i e a e) as a unc ion o he alse posi i e a e
(1 speci ici y) a a ious h eshold se ings. I could be in e -
p e ed as he p obabili y ha a classi ie (like he docking sco e)
anks a andomly chosen ac i e compound highe han a an-
domly chosen inac i e one
77,78
. Acco dingly, a pe ec classi ica-
ion o compounds would be ep esen ed in he g aph by a line
ha s a s om he o igin, eaches e ically he uppe le co ne ,
and hen goes o he uppe igh co ne
78
. The a ea unde he
cu e (AUC) will be equal o one o his ideal pe o mance while
an AUC o 0.5 will ep esen a andom selec ion o ac i e com-
pounds. Figu e 4 shows he ROC cu e ob ained o he bes
docking model selec ed o he i ual sc eening. The black line
ep esen s a andom classi ie whose AUC-ROC is 0.5, whe eas he
AUC-ROC o ou model is 0.915, which is e y close o he op i-
mal alue.
The i ual sc eening o ou da abase wi h he e ined model
17423_ iden i ied 42 s uc u es as possible TcCA inhibi o s.
Among hem, 10 compounds we e selec ed o biological e alu-
a ion (Figu e 5). The c i e ia o choice we e he docking sco es
and he chemical s uc u e o he candida es. I is impo an o
no e ha human examina ion o he hi s is highly ecommended
o he selec ion o he candida es, since i helps o educe alse
posi i es due o docking sco e a e ac s and can lead o he dis-
co e y o mo e po en hi s han sco e p io i isa ion alone.
Compounds 1–8 a e aminoes e -de i ed sulphamides wi h di e -
en subs i uen s in he second ni ogen a om o he sulphamide
unc ion. These compounds we e es ed be o e as human CA II
inhibi o s, wi h poo esul s
50–54
. The biological esul s agains
TcCA o his amily would gi e us he oppo uni y o ge in o ma-
ion abou he s uc u e-ac i i y ela ionships. The se was com-
ple ed wi h wo a i icial swee ene s, acesulphame and sodium
cyclama e, which also ha e low po ency as human CA II inhibi o s
and di e en sca olds han he o he selec ed candida es
47,48
.
Addi ionally, hey a e comme cially a ailable compounds
app o ed o human consump ion.
Biological assays
Table 2 shows he esul s o he biological assays agains he
enzyme and he pa asi e. Inhibi ion alues agains TcCA we e
epo ed as inhibi ion cons an s (K
I
) and he co esponding alues
agains human CA II we e also included o compa ison. The
e ec s o he candida es wi h espec o he un ea ed con ols
agains T. c uzi epimas igo es (Y-s ain) and ypomas igo es (RA-
s ain) we e also measu ed and included in he able.
Figu e 3. Pea son’sRob ained om docking he alida ion se on e e y e ined MD snapsho . The whi e do ep esen s he median alue, and he black line heigh
ep esen s he dis ance be ween he lowe qua ile (Q1) o he uppe qua ile (Q3), which is called he in e qua ile ange (IQR). Violin plo s show he p obabili y dens-
i y o he da a a di e en alues as a ke nel densi y es ima ion.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 25
Rega ding he in e ac ion wi h TcCA, mos o he s uc u es
showed in e es ing inhibi o y e ec agains he enzyme, wi h K
I
alues in he nanomola ange. These esul s e idence he
p edic i e capaci y o he docking model o iden i y TcCA inhibi-
o s. Pa icula ly, sulphamide 5is he mos po en inhibi o o he
se , wi h a K
I
alue o 0.26 lM. Sodium cyclama e, a widely used
Figu e 4. ROC cu e ob ained o he inal model applied in he i ual sc eening.
Figu e 5. Candida es selec ed om he i ual sc eening.
26 M. A. LLANOS ET AL.
swee ene , showed o be a po en TcCA inhibi o oo. Conce ning
selec i i y, bo h compounds a e abou 30 imes mo e ac i e
agains he pa asi e a ge han he human CA II, which ep esen
an impo an aspec o be conside ed in u u e in es iga ions.
Figu e 6 shows he ep esen a ion o he binding in e ac ion o
compound 5wi h he ac i e si e. The docking p og amme o ien s
one ni ogen a om o he sulphamide g oup owa ds he zinc ion,
whe eas oxygen a oms ac s as hyd ogen bond accep o s o
THR201 and THR202 sidechain hyd oxyl g oups. Simila in e ac-
ions we e achie ed be ween he second ni ogen a om o he
sulphamide and he Ty 4 side chain. This esidue is also in ol ed
in posi i e a oma ic in e ac ions wi h he inhibi o .
Simila ly, Figu e 7 shows he ep esen a ion o he in e ac ion
p oposed by docking be ween cyclama e and he ac i e si e o
TcCA. The ni ogen a om o he sulphama e unc ion in e ac s
wi h he Zinc ion and hei oxygen a oms beha e as hyd ogen
bonding accep o s om he Th esidues o he ac i e si e. The
cyclohexyl subs i uen a ached o he N a om con ibu es o he
binding h ough hyd ophobic in e ac ions.
The e ec o he candida es agains T. c uzi epimas igo es,
he non-in ec i e and p oli e a i e s age, was es ed a 50 mM
(Table 2). Among he bes wo TcCA inhibi o s o he se , sodium
cyclama e showed 20% o inhibi ion on he p oli e a ion o epi-
mas igo es a he es ed concen a ion (7 h day). When sodium
cyclama e was es ed agains ypomas igo e, one o he clinically
ele an s ages o T. c uzi, his candida e inhibi ed pa asi e iabil-
i y by 50% a 20 mM.
O he compounds also showed good inhibi ion p o iles agains
bo h o ms o he pa asi e, bu hey we e poo e TcCA inhibi o s.
The lack o co ela ion be ween he inhibi o y e ec s on he
enzyme and he ypanocidal e ec s on he pa asi e may sugges
ha o he mechanisms o ac ion besides CA inhibi ion a e p esen .
O he possible explana ions o he disc epancy may ela e wi h
he d ugs up ake kine ics by he pa asi e and/o bio ans o m-
a ion/inac i a ion o he candida es.
Conclusions
We epo he esul s o a a ge -based i ual sc eening o he
disco e y o new inhibi o s o TcCA wi h poo in e ac ion wi h
hCA II. As he expe imen al s uc u e o he a ge is no a ailable,
we u ned o he ca e ul cons uc ion o mul i- empla e h ee-
dimensional models, o selec he bes mac os uc u e o docking
simula ion. The i e a i e combina ion o Rose a and MD
Table 2. Biological da a and docking sco es achie ed o compounds o he se .
Compound K
I
TcCA (nM) K
I
hCA II (nM)
a
Selec i i y a io Docking sco e
% P oli e a ion. Epimas igo es
b
% Viabili y. T ypomas igo es 24 h
c
3 d day o g ow h 7 h day o g ow h
1594.4 >10,000 >16.8 8.38 86 92 73
2604.6 >10,000 >16.5 9.16 79 102 66
3752.5 4957 >6.6 9.30 107 94 91
4950.5 >10,000 >10.5 8.48 57 55 72
5261.4 8528 >32.6 9.13 73 96 105
6951.8 >10,000 >10.5 8.14 103 104 65
77250 8884 1.22 8.79 103 121 76
8448.8 >10,000 >22.3 7.97 72 114 98
Acesulphame 2242 >20,000 >8.9 7.05 92 89 101
Sodium cyclama e 348.1 >10,000 >28.7 7.46 89 78 52
a
Inhibi ion cons an s agains hCAII we e aken om li e a u e
28–30
.
b
Epimas igo es o he Y s ain o T. c uzi (candida es es ed a 50 mM).
c
T ypomas igo es o he RA s ain o T. c uzi (candida es es ed a 20 mM).
Figu e 6. (A) Bes docking solu ion o he in e ac ion o compound 5wi hin he ac i e si e o TcCA. His idine and impo an esidues o he ac i e si e a e highligh ed
in g ey o ca bon a oms, in ed o oxygen a oms and in blue o ni ogen a oms. The zinc ion is ep esen ed as a non-bonded sphe e in cyan. Colou code o com-
pound 5: Ca bon a oms in g een, Ni ogen a oms in blue, sulphu a oms in yellow and oxygen a oms in ed. (B) Schema ic ep esen a ion o he in e ac ions be ween
compound 5and he ac i e si e o he TcCA model.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 27
simula ions allowed us o signi ican ly imp o e he quali y o he
s a ing model, as e lec ed by he epo ed me ics. I is impo -
an o no e ha we ha e achie ed a ge models wi h high qual-
i y despi e he low sequence iden i y o he empla es ound o
hei cons uc ion.
The docking calcula ions we e ully alida ed h ough a es se
o compounds wi h epo ed ac i i y agains TcCA, which led us
o ind a s uc u al model wi h he bes sco ing powe . By he
applica ion o he model in a i ual sc eening campaign we iden-
i ied sulphamides wi h high po ency and selec i i y agains he
ubiqui ous human CAII iso o m. Addi ionally, he model selec ed
wo comme cial and widely used a i icial swee ene s wi h abun-
dan oxicological da a a ailable. They sha e he sulphama e unc-
ion, a bioisos e ic pa ne o sulphamide as zinc-binding unc ion.
The assays in pa asi es iden i ied sodium cyclama e as he mos
p omising s uc u e in e ms o ypanocidal ac i i y, educing y-
pomas igo e iabili y by 48% a 20 mM. Fu he molecula model-
ling and SAR s udies will be pe o med in u u e in es iga ions, o
achie e a deepe knowledge abou he molecula de e minan s o
he po ency and selec i i y agains TcCA.
Acknowledgemen s
C. Alba So o, C. Ca illo, L. Ga e ne and A. Tale i, a e membe s o
Consejo Nacional de In es igaciones Cien 
ı icas y T
ecnicas de la
Rep
ublica A gen ina (CONICET). M. Llanos and M. Ruiz a e ellow-
ship holde o CONICET. The au ho s g a e ully acknowledge he
suppo o NVIDIA Co po a ion wi h he dona ion o he Quad o
M5000 and he Ti an Xp GPUs used o his esea ch.
Disclosu e s a emen
No po en ial con lic o in e es was epo ed by he au ho s.
Funding
This esea ch was suppo ed in pa h ough g an s om Agencia
de P omoci
on Cien 
ı ica y Tecnol
ogica [PICT 2013-0520], CONICET,
and Uni e sidad Nacional de La Pla a, A gen ina.
ORCID
And ea Angeli h p://o cid.o g/0000-0002-1470-7192
Claudiu T. Supu an h p://o cid.o g/0000-0003-4262-0323
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Figu e 7. (A) Bes docking solu ion o he in e ac ion o cyclama e wi h he ac i e si e o TcCA. His idine and impo an esidues o he ac i e si e a e highligh ed in
g ey o ca bon a oms, in ed o oxygen a oms and in blue o ni ogen a oms. The zinc ion is ep esen ed as a non-bonded sphe e in cyan. Colou code o cycla-
ma e: Ca bon a oms in iole , Ni ogen a oms in blue, sulphu a oms in yellow and oxygen a oms in ed. (B) Schema ic ep esen a ion o he in e ac ions be ween cyc-
lama e and he ac i e si e o he TcCA model.
28 M. A. LLANOS ET AL.