ORGANIC CHEMISTRY
FRONTIERS
RESEARCH ARTICLE
Ci e his: O g. Chem. F on ., 2019, 6,
3127
Recei ed 30 h Ma ch 2019,
Accep ed 7 h July 2019
DOI: 10.1039/c9qo00453j
sc.li/ on ie s-o ganic
Hyd oxylammonium de i a i es o selec i e
ac i e-si e lysine modifica ion in he an i- i ulence
bac e ial a ge DHQ1 enzyme†
Ma ía Manei o,
a
Emilio Lence,
a
Ma a Sanz-Gai e o,
b
José M. O e o,
a
Ma k J. an Raaij,
b
Paul Thompson,
c
Alas ai R. Hawkins
c
and
Concepción González-Bello *
a
Ta ge ed i e e sible inhibi o s bea ing elec ophiles ha become ac i a ed owa ds co alen bond o -
ma ion upon binding o a specific p o ein/enzyme is an eme ging a ea in d ug disco e y. Ta ge ing lysine
esidues is challenging due o he in insically low eac i i y o he amino g oup a physiological pH.
He ein we epo he fi s example o a hyd oxylammonium de i a i e ha causes a specific co alen
modifica ion o an ac i e-si e and a s e ically inaccessible lysine esidue o an enzyme. The desc ibed
ligands, compounds 1–3, we e a ionally designed o be ac i a ed owa ds co alen bond o ma ion upon
binding o he ype I dehyd oquinase (DHQ1) enzyme o he de elopmen o new an i- i ulence agen s
o comba he widesp ead esis ance o an ibio ics. E idence in a omic de ail o he co alen modifi-
ca ions caused by he ligands o he ca aly ic Lys170 by he o ma ion o a s able seconda y amine is p o-
ided by he esolu ion a 1.08–1.25 Å o he c ys al s uc u es o DHQ1 om Salmonella yphi enzyme
adduc s. In addi ion, he fi s c ys al s uc u e o he addi ion in e media e adduc a 1.4 Å o a Schiffbase
o ma ion eac ion by using an analog o he na u al subs a e, compound 4, is also epo ed. Molecula
dynamics simula ion s udies on non-co alen enzyme/ligand complexes and a wo-dimensional QM/MM
umb ella sampling simula ion s udy sugges ed ha a di ec displacemen by Lys170 wi h he elease o
NH
2
OH would be easible. These s udies migh open up new oppo uni ies o he de elopmen o no el
lysine- a ge ed i e e sible inhibi o s bea ing a me hylhyd oxylammonium moie y as a la en elec ophile.
In oduc ion
Ta ge ing in ec ious diseases and cance wi h small molecules
ha co alen ly modi y he a ge is an eme ging a ea in d ug
disco e y.
1–9
The mo e de ailed knowledge a ailable oday on
he eal isks associa ed wi h his ype o compound, as well as
i s eno mous ad an ages (efficacy, selec i i y, and lowe sus-
cep ibili y o esis ance mechanisms), has led o a d ama ic
inc ease in hei p esence in an i-in ec i e and oncology dis-
co e y p og ams in ecen yea s.
10
Besides hese ad an ages,
he de elopmen o his ype o ligand ep esen s a huge chal-
lenge due o he need o combine eac i i y (co alen modi i-
ca ion) and selec i i y (sa e y) in a single chemical en i y. This
indica es he use o ligands bea ing weak elec ophilic g oups
which a e able o achie e po ency wi hou sac i icing selec i-
i y. In ecen yea s, effo s ha e been de o ed o he design o
i e e sible compounds capable o modula ing hei eac i i y
when complemen a i y wi h he speci ic a ge akes place.
8
These a e a ge ed i e e sible inhibi o s bea ing elec ophiles
which become ac i a ed owa ds co alen bond o ma ion
upon binding o a speci ic p o ein o enzyme. The in insically
low chemical eac i i y o hese ligands makes hem pa icu-
la ly desi able o d ug de elopmen since hey would be
hidden o non-speci ic a ge s and a e only ac i a ed o he
chemical modi ica ion eac ion upon binding. The majo i y o
hese ligands ha e been designed o modi y cys eine esidues.
Rele an examples include luo ome hylke ones, e minal
alkynes, ac ylamides, and disul ides (Fig. 1A).
11–18
Ta ge ing
†Elec onic supplemen a y in o ma ion (ESI) a ailable: Fig. S1–S7 and one able,
ull de ails o he Expe imen al sec ion (syn hesis, p o ein c ys alliza ion and
s uc u al de e mina ion, enzyme assays and compu a ional s udies) and NMR
spec a. Coo dina es and s uc u e ac o s a e a ailable om he P o ein Da a
Bank wi h accession codes 6H5C, 6H5D, 6H5G, and 6H5J. See DOI: 10.1039/
c9qo00453j
a
Cen o Singula de In es igación en Química Biolóxica e Ma e iais Molecula es
(CIQUS), Depa amen o de Química O gánica, Uni e sidade de San iago de
Compos ela, Jena o de la Fuen e s/n, 15782 San iago de Compos ela, Spain.
E-mail: concepcion.gonzalez.bel[email p o ec ed]
b
Depa amen o de Es uc u a de Mac omoléculas, Cen o Nacional de Bio ecnología
(CSIC), Campus Can oblanco, 28049 Mad id, Spain
c
Ins i u e o Cell and Molecula Biosciences, Medical School, Uni e si y o Newcas le
upon Tyne, Newcas le upon Tyne NE2 4HH, UK
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lysine esidues appea s mo e challenging because hei
ε-amino g oup is usually non-nucleophilic as i is p o ona ed
a physiological pH.
19
Rema kable examples include a oma ic
sul onyl luo ides, inyl sul ones, a yl luo osul a es and
ni iles (Fig. 1B).
20–26
The selec i e modi ica ion o ac i e-si e
lysine esidues is mo e affo dable, because hey ei he ha e a
lowe pK
a
o hei nucleophilici y/basici y is modula ed by
o he amino acid esidues (Asp and His) in he icini y, which
migh also pa icipa e in he ca alysis.
We became in e es ed in he design o ligands able o cause
he co alen modi ica ion o he ype I dehyd oquinase
(DHQ1) enzyme. DHQ1 is an aldolase (dehyd a ase) enzyme
ha does no ha e any coun e pa in human cells, and has
been pinpoin ed as a p omising a ge in he sea ch o new
an i- i ulence agen s.
27
I is belie ed ha DHQ1 may ac as a
i ulence ac o in i o since he dele ion o he a oD gene,
which encodes DHQ1 om Salmonella yphi and Shigella
lexne i, has been p o en o affo d sa is ac o y li e o al
accines.
28–30
Ta ge ing he bac e ial capaci y o p oduce in ec-
ion ( i ulence), a he han he mos widely used dis up ion
o bac e ial su i al, is an inno a i e s a egy which is inc eas-
ingly being explo ed o comba he wo ldwide inc easing
appea ance o “supe bugs”(mul i-d ug esis an bac e ia),
which a e esis an o mos an ibio ics in clinical use.
Compounds ha a ge bac e ial i ulence signalling pa hways
would c ea e an in i o scena io simila o accina ion wi h a
li e a enua ed s ain in which he bac e ia a e e en ually
clea ed by he hos immune sys em. DHQ1 is p esen
in se e al pa hogenic bac e ia such as Esche ichia coli,
S aphylococcus au eus and Salmonella yphi. This enzyme,
which is a dime , has 8 lysine esidues in each chain and ca a-
lyzes he e e sible syn dehyd a ion o wa e in 3-dehyd oqui-
nic acid o o m 3-dehyd oshikimic acid by a mul i-s ep
mechanism which in ol es he o ma ion o Schiffbase
species.
31
We epo he e he use o hyd oxylammonium de i a i es,
compounds 1–3, o he speci ic co alen modi ica ion o he
ca aly ic lysine esidue o DHQ1 om S. yphi (S -DHQ1)
(Fig. 1C). E idence in a omic de ail o he co alen modi i-
ca ions caused by compounds 1–3 o he essen ial Lys170 by
he o ma ion o a s able amine is p o ided by he esolu ion
o high quali y c ys al s uc u es o S -DHQ1 ( esolu ion ange:
1.08–1.25 Å) chemically modi ied by 1–3. The epo ed com-
pounds we e also designed o p o ide non-labile enzyme
adduc s by being able o a oid he in insic dehyd a ion eac-
ion pe o med by he enzyme. The chemical basis o he la e
was inspi ed by he he ein epo ed Molecula Dynamics (MD)
simula ion s udies on he Michaelis and subs a e Schiffbase
complexes, as well as by he esolu ion o he i s ime o he
c ys al s uc u e o he addi ion in e media e adduc a 1.4 Å
o he subs a e Schiffbase o ma ion eac ion o S -DHQ1 by
using an analog o he na u al subs a e, compound 4.
Resul s and discussion
Ligand design
I has been p oposed ha he eac ion ca alyzed by DHQ1 is
ini ia ed by he ac i a ion o he C3 ca bonyl g oup o he sub-
s a e om he Re ace by he essen ial His143, and subsequen
nucleophilic a ack o he ε-amino g oup o he essen ial
Lys170 om he opposi e ace o affo d ini ially he addi ion
in e media e adduc (Fig. 2A).
32
I has been shown p e iously
ha epoxide 6causes he chemical modi ica ion o S -DHQ1
by he o ma ion, a e dehyd a ion, o a Schiffbase wi h he
essen ial Lys170, adduc II (Fig. 2B).
32
This p ocess was e i-
denced by he esolu ion o he c ys al s uc u e o he S -
DHQ1/6adduc ob ained by co-c ys alliza ion (PDB en y
4CLM). On he o he hand, de i a i e 5p o ed o cause he
co alen modi ica ion o Lys170 h ough he ini ial o ma ion
o an amine, adduc I, as e ealed by he c ys al s uc u e o
he enzyme adduc ob ained by soaking apo-S -DHQ1 c ys als
o 30 s (PDB en y 4UIO).
33
In solu ion, his ini ial adduc I
Fig. 1 Selec ed examples o la en elec ophiles and a ge ed ligands. (A and B) La en elec ophiles o modifica ion o cys eine (A) and lysine esi-
dues (B). The posi ion ha is co alen ly modified is indica ed wi h an as e isk. (C) Designed quinic acid-based hyd oxylammonium compounds 1–3
and subs a e analog 4. The chemical modifica ions o he ca aly ic Lys170 o DHQ1 om S. yphi iden ified by X- ay c ys allog aphy a e also shown.
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unde goes a dehyd a ion eac ion o affo d also he imine
adduc II (S. yphi).
Conside ing ha he o ma ion o an imine g oup does no
ep esen he bes scena io o disabling a a ge , e en aking
in o accoun ha i p o ed o be a e y s able Schiffbase, ou
effo s we e de o ed o no only explo ing he po en ial o a
me hylhyd oxylammonium g oup as a no el la en elec o-
phile, bu also a oiding possible dehyd a ion p ocesses by
using ano he ype o co alen linkage wi h he p o ein. The
hyd oxylammonium g oup was chosen because: (i) in MD
simula ion s udies on he S -DHQ1/5complex, i was obse ed
ha he ammonium g oup in 5es ablishes, du ing a signi i-
can pa o he simula ion, a weak elec os a ic in e ac ion
(∼3.4 Å) wi h he conse ed Asp114 esidue (Fig. 2C); and (ii)
he inco po a ion o an OH g oup in he lea ing g oup would
enhance he la e in e ac ion and he e o e would ix he
a angemen o he side chain o nucleophilic a ack by
Lys170 om he opposi e si e.
Mo eo e , MD simula ion s udies ca ied ou wi h he
addi ion in e media e adduc e ealed ha he C3 hyd oxyl
g oup in he la e adduc emained ixed du ing he whole
simula ion (50 ns) wi h he p o on engaged in an in a-
molecula hyd ogen bond wi h one o he oxygen lone pai s o
he C1 hyd oxyl g oup and wi h one o he oxygen lone pai s
accep ing a hyd ogen bond om he guanidinium g oup o
A g48 (Fig. 2D and S1†). The C1 hyd oxyl g oup seems o be
he e o e employed by DHQ1 o achie e he app op ia e
a angemen o he C3 hyd oxyl g oup o an effec i e sub-
sequen dehyd a ion eac ion o affo d he subs a e Schiff
base. Reasoning ha he dehyd a ion eac ion would be dis a-
o ed by using compounds lacking he e ia y C1 hyd oxyl
g oup, compound 4was syn hesized o s uc u al s udies ha
could con i m his hypo hesis.
Syn hesis o ligands 1–4
Ligand 4was p epa ed as ou lined in Fig. 3A. Fi s ly, hyd oge-
nolysis o he p o ec ed 3-epi-shikimic acid 7
34
affo ded com-
pound 8dias e eoselec i ely. I is impo an o highligh ha
ha ing he Scon igu a ion in he seconda y alcohol is c ucial
o achie e good s e eocon ol o he educ ion om he Re ace
o he double bond. The con igu a ion o he new chi al cen e
in 8was con i med by he coupling cons an s in he co es-
ponding
1
H NMR spec a (Fig. S2†). Basic hyd olysis o 8 ol-
lowed by alkyla ion wi h benzyl b omide ga e es e 10.
Oxida ion o 10 using Dess–Ma in pe iodinane ollowed by
acid hyd olysis o he ace al g oup in 11 and inal hyd ogenoly-
sis o he esul ing benzyl es e 12 ga e acid 4.
Compound 1was syn hesized om aldehyde 18, which was
p epa ed in ou s eps om alcohol 8(Fig. 3B). Fi s ly, PDC-
oxida ion o 8 ollowed by nucleophilic addi ion o inyl mag-
nesium b omide o he esul ing ke one 13 ga e dias e eoselec-
i ely he inyl de i a i e 16. The egioselec i i y o he eac ion
was con i med by NOE expe imen s. In e sion o he signal o
H6 led o an enhancemen o he signal o he H1′ inyl g oup
(3.1%). Ha ing obse ed ha he ozonolysis o alkene 16
affo ded complex eac ion mix u es and a e y low yield o he
desi ed compound, p o ec ion o he e ia y alcohol in 16 as a
ime hylsilyl e he was ca ied ou . In his way, ozonolysis o
he esul ing p o ec ed alkene 17 and subsequen educ i e
wo kup using dime hyl sul ide ga e he desi ed aldehyde 18.
Finally, educ i e amina ion o aldehyde 18 wi h hyd oxyl-
amine ollowed by hyd olysis o me hyl es e 19 unde acidic
condi ions wi h concomi an emo al o he p o ec ing g oups
led o he a ge compound 1.
Compounds 2and 3we e p epa ed om p e iously
epo ed alkene 15
32
and compound 14 using aldehydes 22
and 24 as key in e media es, espec i ely (Fig. 3B). Alkene 14
was syn hesized by me hylena ion o he ke one 15.
Subsequen con e sion o he alkenes 15 and 14 o he
equi ed aldehydes 22 and 24, espec i ely, was achie ed in
wo s eps. Fi s ly, hyd obo a ion–oxida ion ook place om he
less hinde ed Si ace o he ex e nal double bond o affo d
compounds 20 and 21 dias e eoselec i ely, bo h o which ha e
he CH
2
OH g oup in he axial disposi ion. Secondly, oxida ion
o he p ima y alcohols in 20 and 21 using Dess–Ma in pe i-
odinane o PDC wi h subsequen ull epime iza ion o he
he modynamic equa o ial aldehydes by hea ing in he p es-
ence o py idine ga e he desi ed aldehydes 22 and 24, espec -
i ely. The s e eochemis y o he new chi al cen e c ea ed in
compounds 20–22 and 24 was con i med by he coupling con-
Fig. 2 Mechanis ic and s uc u al basis o ligand design. (A) Reac ion
ca alyzed by DHQ1. The ole o he essen ial esidues Lys170 and His143
in he o ma ion o he subs a e-Schiffbase in he enzyma ic mecha-
nism is shown. (B) Chemical modifica ion o S -DHQ1 by 5and 6iden -
ified by X- ay c ys allog aphy. (C) A angemen o 5 ela i e o Asp114.
(D) Subs a e addi ion in e media e adduc a e 50 ns o dynamic simu-
la ion (yellow) ob ained by compu a ional s udies.
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s an s in he co esponding
1
H NMR spec a and by NOE
expe imen s (Fig. S2†). Finally, aldehydes 22 and 24 we e con-
e ed o he a ge de i a i es 2and 3in he same way as ha
o compound 1 om aldehyde 18.
X- ay c ys al s uc u es o S -DHQ1/1–4 adduc s
S -DHQ1/4 adduc . The X- ay c ys al s uc u e o he S -
DHQ1/4adduc was ob ained by soaking apo-S -DHQ1 c ys als
and he s uc u e was sol ed a 1.4 Å (Fig. 4). C ys als we e
moun ed in o c yoloops and we e di ec ly lash ozen by apid
imme sion in liquid ni ogen. X- ay diff ac ion da a we e col-
lec ed om c ys als c yo-cooled in a s eam o cold ni ogen
gas (100 K) a ambien p essu e using synch o on adia ion
and he da a we e subsequen ly p ocessed. The s uc u e was
de e mined by molecula eplacemen , using he p e iously
desc ibed s uc u e o he educed o m o he S -DHQ1
p oduc –Schiffbase in e media e (PDB en y 1QFE
28
)asa
sea ch model, and he s uc u e was e ined. A summa y o he
s a is ical da a ollowing da a educ ion and p ocessing is p o-
ided in Table S1.†
Unbiased, calcula ed elec on densi y maps showed clea
and high quali y elec on densi y o he enzyme-modi ied
ligand molecule 4. The s uc u e e ealed ha he addi ion
adduc o he ε-amino g oup o he Lys170 esidue and he
ke one g oup in 4was ob ained and, mo e impo an ly, de-
hyd a ion o he C3 hyd oxyl g oup did no ake place since
clea elec on densi y was obse ed o his hyd oxyl g oup.
The NZ a om o he modi ied Lys170 esidue es ablishes a
s ong in e ac ion wi h he conse ed esidue Asp114 ia a
wa e molecule (s uc u al, W23) –an in e ac ion ha is ound
in all ela ed c ys al s uc u es epo ed p e iously. The essen-
ial His143 esidue in e ac s by hyd ogen bonding wi h he
C3 hyd oxyl g oup o he modi ied ligand. In addi ion, he
C3 hyd oxyl and he C1 ca boxyla e g oups o he modi ied
ligand a e linked ia b idges in ol ing wa e molecules (W81
and W104). The modi ied ligand is also ancho ed o he ac i e
si e by he same ype o elec os a ic and hyd ogen bonding
in e ac ions as he na u al subs a e, speci ically, esidues
Gln236 and Se 232 (subs a e-co e ing loop), and A g213,
Se 21, A g48, A g82 and Glu46.
Mo eo e , MD simula ion s udies ca ied ou wi h he
addi ion in e media e adduc S -DHQ1/4clea ly showed ha
he oxygen lone pai s o he C3 hyd oxyl g oup would no be
engaged by he guanidinium g oup o A g82 (Fig. S3†). Thus,
his g oup would o a e eely, and he guanidinium g oup o
A g82 would mainly in e ac wi h he C4 hyd oxyl g oup o he
modi ied ligand 4ins ead o in e ac ing wi h i s C3 hyd oxyl
g oup. Du ing he simula ion, a la ge a iabili y o he di-
Fig. 3 Syn hesis o compounds 4(A) and 1–3(B). Reagen s and condi ions: (a) H
2
(g), Pd/C (10%), MeOH, RT. (b) 1. LiOH, THF, RT; 2. Ambe li e
IR-120 (H
+
). (c) 1. E
3
N, DMAP, Bu
4
NI, DMF, RT; 2. BnB , RT. (d) Dess–Ma in pe iodinane, DCM, RT. (e) TFA/H
2
O (20 : 1), RT. ( ) H
2
, Pd/C (10%), E OAc,
RT. (g) PDC, 4 Å MS, DCM, RT. (h) Zn dus , CH
2
I
2
, TiCl
4
, THF, 0 °C. (i) CH
2
CHMgB , THF, −78 °C. ( j) TMSCl, (TMS)
2
NH, Py, RT. (k) 1. O
3
, DCM,
−78 °C. 2. SMe
2
,−78 °C. (l) 1. NH
2
OH.HCl, NaOAc, 4 Å MS, MeOH, RT; 2. NaBH
3
CN, MeOH, RT. (m) HCl (0.3 M), 100 °C. (n) 1. BH
3
-THF, THF, 0 °C;
2. NaBO
3
. (o) o 20 : 1. Dess–Ma in pe iodinane, DCM, RT; 2. Py, MeOH, 60 °C. (p) o 21 : 1. PDC, 4 Å MS, DCM, RT; 2. Py, MeOH, 60 °C.
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hed al angle among he C2, C3, O3 and HO3 a oms o he
modi ied ligand 4is obse ed (Fig. S3C†). When he same ana-
lysis was ca ied ou wi h he addi ion in e media e adduc
ob ained wi h he na u al subs a e, which has a hyd oxyl
g oup in C1 (Fig. S1C†), no o a ion o he C3 hyd oxyl g oup
was obse ed as i s oxygen lone pai s we e held by A g82
(Fig. 2D). Fu he mo e, he ε-ni ogen a om o he modi ied
Lys170 by he modi ied na u al subs a e would be in an
app op ia e an i-pe iplana a angemen o subsequen de-
hyd a ion o affo d he subs a e-Schiffbase. Thus, du ing he
simula ion he a e age alue o he dihed al angle among he
O3, C3, NZ and CE a oms o he modi ied subs a e would be
−61.8° (Fig. S4†). A less efficien an i-pe iplana con o ma ion
was obse ed in he modi ied ligand 4, since he la e di-
hed al angle has a alue o −68.1° in he c ys al s uc u e and
an a e age alue o −72.6° du ing he simula ion (Fig. S4†).
Taken oge he , he esul s o ou s uc u al and compu-
a ional s udies con i m ou hypo hesis abou he key ole o
he C1 hyd oxyl g oup in con olling he co ec a angemen
o he gene a ed C3 hyd oxyl g oup in he addi ion in e medi-
a e adduc o dehyd a ion o affo d he subs a e-Schiffbase.
S -DHQ1/1–3 adduc s. The c ys al s uc u es o S -DHQ1/1–3
adduc s we e ob ained by co-c ys alliza ion (4 weeks) and he
s uc u es we e sol ed a high esolu ions o 1.25, 1.04 and
1.14 Å, espec i ely (Fig. 5). The s uc u es we e de e mined by
molecula eplacemen , using he p e iously desc ibed s uc-
u es o S -DHQ1 (PDB en ies 4UIO and 4CNO) as sea ch
models, and he esul ing s uc u es we e e ined. See he
Expe imen al sec ion and Table S1† o u he de ails. Fo all
cases, unbiased, calcula ed elec on densi y maps e ealed
clea and well-de ined elec on densi y o he enzyme-modi-
ied ligand molecules 1–3, wi h he co alen modi ica ion o
Lys170 h ough he o ma ion o an amine. I is no ewo hy
ha o ligand 1, which is he only one ha has a hyd oxyl
g oup in he C3 posi ion, dehyd a ion eac ions did no ake
place as clea elec on densi y o he C3 hyd oxyl g oup was
obse ed. In con as o he S -DHQ1/4adduc , o S -DHQ1/
1–3adduc s, a s ong in e ac ion be ween he His143 side
chain and he NZ a om o he modi ied Lys170 esidue (dis-
ance: 2.8–2.9 Å measu ed be ween hea y a oms) is obse ed
ega dless o he p esence o absence o a hyd oxyl g oup a C3
o he modi ied ligand. In addi ion, an in e ac ion be ween he
modi ied Lys170 and he ca boxyla e side chain o Asp114 ia
he s uc u al wa e molecule was no obse ed. In gene al, he
es o he in e ac ions in ol ing he C4 and C5 hyd oxyl
g oups and he C1 ca boxyla e g oup a e simila o hose
obse ed in he S -DHQ1/4adduc . Mo eo e , Klean hous
e al.
35
epo ed ha he co alen a achmen o he eac ion
p oduc molecule (as educed om a seconda y amine) o DHQ1
om Esche ichia coli, which was e ealed o ha e a mel ing
empe a u e 40 °C highe han ha o he unmodi ied p o ein,
causes a d ama ic inc ease in he s abili y o he p o ein
agains p o eolysis. A simila beha io would be expec ed o
he epo ed chemically modi ied S -DHQ1 enzyme.
Inhibi ion s udies
The capaci y o he epo ed hyd oxylammonium de i a i es o
cause he i e e sible inhibi ion o he DHQ1 enzyme om
S. yphi (S -DHQ1) and S. au eus (Sa-DHQ1) was analyzed by
incuba ion o bo h enzymes wi h ligands 1–3 o a 24 h pe iod
(PPB, pH 7.0, 25 °C). The ac i i y was p og essi ely de e mined
by UV spec oscopy unde he s anda d assay condi ions using
aliquo s om he incuba ion samples and he con ol. The
ligands 1–3we e ound o be slow ime-dependen i e e sible
inhibi o s o he wo enzymes (Fig. S5†). The inac i a ion
p o ed o be mo e efficien o Sa-DHQ1 han o S -DHQ1. Fo
he S. yphi enzyme, compound 2p o ed o be he mos
efficien o he h ee ligands (4 h, ∼50% inac i a ion,
∼@1 : 120 enzyme/ligand a io), whe eas compound 1was o
Fig. 4 C ys al s uc u e o S -DHQ1 co alen ly modified by 4. (A) O e all iew o he s uc u e o S -DHQ1 co alen ly modified by 4. (B) Unbiased
elec on densi y o he modified ligand 4(yellow) and i s co alen a achmen o Lys170 (g een) o S -DHQ1 (g ay). A maximum-likelihood weigh ed
2F
o
−F
c
map con ou ed a 1σis shown up o 1.6 Å a ound he ligand molecule. The final model, including he ligand molecule, is supe imposed
on o he map. (C) In e ac ions o he modified ligand 4wi h S -DHQ1. Hyd ogen bonding and elec os a ic in e ac ions be ween he ligand and
S -DHQ1 a e shown as dashed lines. Rele an esidues and wa e molecules and dis ances a e shown and labeled.
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Sa-DHQ1 (2.5 h, ∼50% inac i a ion, ∼@1 : 120 enzyme/ligand
a io).
Compu a ional s udies
The binding modes o ligands 1–3in he ac i e si e o
S -DHQ1 we e i s s udied by molecula docking using he
p og am GOLD 5.2.2
36
and u he analyzed by MD simula ion
s udies (100 ns). The monome o he bina y S -DHQ1/1–3
p o ein complexes in a unca ed oc ahed on o wa e mole-
cules ob ained wi h he molecula mechanics o ce ield
AMBER was employed. These simula ion s udies we e ca ied
ou conside ing he wo possible p o ona ion s a es o His143
(δand dual). Howe e , he la e possibili y (dual) was uled
ou because i caused an opening o he ac i e si e and a dis-
placemen o he ligands. The esul s e ealed ha he con-
se ed Asp114 esidue would be he main agmen espon-
sible o he exquisi e con ol o he a angemen o he
lea ing g oup and consequen ly o he me hylene g oup a
which co alen modi ica ion occu s (Fig. 6A–C). In pa icula ,
he hyd oxyl g oup o he hyd oxylammonium moie y would
acili a e he es ablishmen o a s ong and s able hyd ogen
bonding in e ac ion wi h he ca boxyla e g oup o Asp114. The
a e age dis ance be ween Asp114 (OD1 a om) and he NH
2
OH
g oup (H04 a om) in 1–3du ing he whole simula ion was
∼1.6 Å in all cases (Fig. S6†). In addi ion, he hyd ogen
bonding in e ac ion o he ligand side chain wi h he essen ial
His143 would also g ea ly con ibu e owa ds eezing he con-
o ma ion o he C3 side chain (Fig. S6†). Mo eo e , he
a e age dis ances be ween His143 (NE2 a om) and he NH
2
OH
g oup (HZ a om) in 1–3we e calcula ed o be ∼2.5, 2.1 and
2.3 Å, espec i ely. Fo ligands 2–3, a s ong and s able hyd o-
gen bonding in e ac ion wi h he s uc u al wa e (a e age dis-
ance o ∼2.1 Å) is he hi d con ac which ixes he con o -
ma ion o he lea ing g oup. This con ac was no obse ed o
ligand 1which seems o be caused by in amolecula hyd ogen
bonding be ween he C3 hyd oxyl g oup and he NH
2
OH g oup
o p o ide a diffe en a angemen o he NZ a om.
In an effo o ge an insigh in o how he epo ed ligands
cause he co alen modi ica ion o he ca aly ic lysine esidue,
a combined quan um mechanics/molecula mechanics (QM/
MM) umb ella sampling simula ion s udy was ca ied ou .
Modelling o eac ions in enzymes wi h QM/MM me hods can
p o ide knowledge in a omic de ail abou he mechanism, he
key in e ac ions o he ligands, eac ion in e media e(s) and
ansi ion s a e(s).
37,38
By using hese me hods, he elec onic
ea angemen s in ol ed in he b eaking/making o chemical
Fig. 5 C ys al s uc u es o S -DHQ1 co alen ly modified by 1–3. (A, B, and C) Unbiased elec on densi y o he modified ligands 1(A, iole ),
2(B, cyan), and 3(C, o ange), and hei co alen a achmen o Lys170 o S -DHQ1 (g een). F om he model ob ained by molecula eplacemen and
be o e he inclusion o he inhibi o molecule, efinemen was pe o med o ob ain unbiased densi y o he inhibi o molecule and o he model
changes. A maximum-likelihood weigh ed 2F
o
−F
c
map con ou ed a 1σis shown up o 1.6 Å a ound he ligand molecule, he s uc u al wa e mole-
cule, and Lys170, His143 and Asp114 side chains (g ay). The final model, including he ligand molecule, is supe imposed on o he map. (D, E, and F)
In e ac ions o he modified ligands 1( iole ), 2(cyan), and 3(o ange) wi h S -DHQ1 (g ay). Hyd ogen bonding and elec os a ic in e ac ions
be ween he ligands and S -DHQ1 a e shown as dashed lines. Rele an esidues a e shown and labeled.
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bonds (QM) a e s udied in a dynamic en i onmen (MM)
which mimics he con o ma ional changes o he enzyme
occu ing du ing he eac ion, which a e also impo an o
he eac ion ene ge ics. In addi ion, by using umb ella
sampling echniques, he explo a ion o egions along he eac-
ion coo dina e which would o he wise ha e insufficien
sampling is achie ed. These QM/MM umb ella sampling simu-
la ion s udies we e pe o med wi h compound 2and di e se
geome ies on he non-co alen bina y S -DHQ1/2complex
we e ob ained by MD simula ion s udies. Th ee possible eac-
ion coo dina es we e explo ed: (i) b eakdown o he C–N bond
in 2; (ii) o ma ion o he C(2)–N(K170) bond; and (iii) a linea
combina ion o (i) minus (ii). Two possible p o ona ion s a es
o His143 (δand dual) we e employed. Howe e , he la e
possibili y (dual) was uled ou because i caused an opening
o he ac i e si e. The bes esul s we e ob ained wi h he wo-
dimensional US (iii) (see he ESI† o ull de ails). The esul -
ing ee ene gy p o ile a he SCC–DFTB/ff14SB le el indica es
ha he S
N
2 nucleophilic subs i u ion by Lys170 wi h he
elease o NH
2
OH would be easible, wi h an ene gy ba ie o
27.2 kcal mol
−1
o he TS (TS1), which would be he a e-de e -
mining s ep (Fig. 6D–G). The eac ion would in ol e he o -
ma ion o an in e media e INT wi h an ene gy ba ie o
11.8 kcal mol
−1
which would unde go a subsequen dep o o-
na ion by neu al His143 ha ing a e y low eac ion ba ie
(0.9 kcal mol
−1
ela i e o INT, TS2) (Fig. S7†).
Conclusions
In summa y, we ha e demons a ed ha quinic acid-based
hyd oxylammonium de i a i es can be used o he selec i e
co alen modi ica ion o he ca aly ic lysine esidue o he
DHQ1 enzyme, which is a p omising a ge o an i- i ulence
d ug disco e y. This was e idenced by he esolu ion o he
c ys al s uc u es o DHQ1 om S. yphi ( esolu ion ange:
1.08–1.25 Å) chemically modi ied by he epo ed ligands 1–3
showing ha he modi ied ligands a e linked o he lysine
esidue h ough an amine g oup. The esul s ob ained wi h
compounds 2and 3 e eal ha he p esence o a hyd oxyl
g oup in posi ion C3 o he epo ed scaffold is no equi ed o
achie e co alen modi ica ion allowing he use o qui e simple
ligands such as compound 3. The esul s om he MD and
wo-dimensional QM/MM umb ella sampling simula ion
s udies sugges ed ha he co alen modi ica ion mechanism
migh ake place by he di ec nucleophilic a ack o he
ε-amino g oup o he lysine esidue wi h he elease o NH
2
OH
ollowed by dep o ona ion o he lysine adduc by he essen ial
Fig. 6 Co alen modifica ion mechanism o Lys170 by ligands 1–3. (A, B, and C) P oposed binding mode o ligands 1(A, pink), 2(B, cyan) and
3(C, o ange) in he ac i e si e o S -DHQ1 ob ained by MD simula ion s udies. The pose o he ligands shown co esponds o ha ob ained a e
90 ns o simula ion. The side chains o he mos ele an esidues a e shown and labeled. The esidue Asp114 (g een), which is key in he geome ic
con ol o he side chain o he ligand, he essen ial Lys170 ( ed), he essen ial His143 (yellow) and he ca aly ic wa e , as well as he mos ele an
pola in e ac ions, a e highligh ed. (D, E, F, and G) QM/MM MD simula ion s udies o he co alen modifica ion mechanism o S -DHQ1 by com-
pound 2. Rep esen a i e geome ies o TS1 (D) and in e media e INT (E). Rele an esidues and wa e molecules a e shown and labeled. Key hyd o-
gen bonding in e ac ions and bonds b oken/ o med (dis ances included) a e indica ed as blue and ed dashed lines, espec i ely. The esidues
Lys170 (o ange) and His143 (yellow) a e highligh ed. Geome ies we e aken om he ee ene gy su ace (F). (G) F ee ene gy p ofile ob ained using
umb ella sampling simula ions a he SCC-DFTB/ff14SB le el o he whole eac ion.
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his idine. To ou knowledge, his is he i s example o hyd o-
xylammonium de i a i es ha cause he speci ic co alen
modi ica ion o a ca aly ic and s e ically inaccessible lysine
esidue o an enzyme. These s udies migh open up new
oppo uni ies o he de elopmen o no el lysine- a ge ed i -
e e sible inhibi o s bea ing a hyd oxylammonium moie y as a
la en elec ophile.
Conflic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
Financial suppo om he Spanish Minis y o Economy and
Compe i eness (SAF2016-75638-R), he Xun a de Galicia
[Cen o singula de in es igación de Galicia acc edi a ion
2016-2019 (ED431G/09) and ED431B 2018/04] and he
Eu opean Union (Eu opean Regional De elopmen Fund –
ERDF) is g a e ully acknowledged. MM and EL hank he
Spanish Minis y o Educa ion, Cul u e and Spo and he
Xun a de Galicia o hei espec i e FPU and pos doc o al el-
lowships. We a e g a e ul o he ALBA synch o on (Ba celona,
Spain) o he p o ision o beam ime and o he Cen o de
Supe compu ación de Galicia (CESGA) o use o he Finis
Te ae compu e .
No es and e e ences
1 R. A. Baue , D ug Disco e y Today, 2015, 20, 1061–1073.
2 D. S. Johnson, E. Wee apana and B. F. C a a , Fu u e Med.
Chem., 2010, 2, 949–964.
3 D. C. Swinney, Na . Re . D ug Disco e y, 2004, 3, 801–808.
4 J. Singh, R. C. Pe e , T. A. Baillie and A. Whi y, Na . Re .
D ug Disco e y, 2011, 10, 307–317.
5 F. M. Fe guson and N. S. G ay, Na . Re . D ug Disco e y,
2018, 17, 353–377.
6 K. Sande son, Na . Re . D ug Disco e y, 2013, 12, 649–651.
7 J. A. Blai , D. Rauh, C. Kung, C.-H. Yun, Q.-W. Fan,
H. Rode, C. Zhang, M. J. Eck, W. A. Weiss and
K. M. Shoka , Na . Chem. Biol., 2007, 3, 229–238.
8 J. Singh, R. C. Pe e and A. F. Kluge, Cu . Opin. Chem.
Biol., 2010, 14, 475–480.
9 Q. Liu, Y. Sabnis, Z. Zhao, T. Zhang, S. J. Buh lage,
L. H. Jones and N. S. G ay, Chem. Biol., 2013, 20, 146–159.
10 C. Jös , C. Ni sche, T. Scholz, L. Roux and C. D. Klein,
J. Med. Chem., 2014, 57, 7590–7599.
11 M. S. Cohen, C. Zhang, K. M. Shoka and J. Taun on,
Science, 2005, 308, 1318–1321.
12 R. Ekkebus, S. I. an Kasa e en, Y. Kula hu, A. Schol en,
I. Be lin, P. P. Geu ink, A. de Jong, S. Goe dayal, J. Nee jes,
A. J. R. Heck, D. Komande and H. O aa, J. Am. Chem. Soc.,
2013, 135, 2867–2870.
13 M. Hagel, D. Niu, T. S Ma in, M. P. Shee s, L. Qiao,
H. Be na d, R. M. Ka p, Z. Zhu, M. T. Labenski,
P. Cha u edi, M. Nach , W. F. Wes lin, R. C. Pe e and
J. Singh, Na . Chem. Biol., 2011, 7,22–24.
14 B. R. Lanning, L. R. Whi by, M. M. Dix, J. Douhan,
A. M. Gilbe , E. C. He , T. O. Johnson, C. Joslyn,
J. C. Ka h, S. Niessen, L. R. Robe s, M. E. Schnu e,
C. Wang, J. J. Hulce, B. Wei, L. O. Whi ely, M. M. Haywa d
and B. F. C a a , Na . Chem. Biol., 2014, 10, 760–767.
15 D. W. F y, A. J. B idges, W. A. Denny, A. Dohe y,
K. D. G eis, J. L. Hicks, K. E. Hook, P. R. Kelle ,
W. R. Leopold, J. A. Loo, D. J. McNama a, J. M. Nelson,
V. She wood, J. B. Smaill, S. T umpp-Kallmeye and
E. M. Bob usin, P oc. Na l. Acad. Sci. U. S. A., 1998, 95,
12022–12027.
16 A. H. Chan, W.-G. Lee, K. A. Spaso , J. A. Cisne os,
S. N. Kudalka , Z. O. Pe o a, A. B. Buckingham,
K. S. Ande son and W. L. Jo gensen, P oc. Na l. Acad.
Sci. U. S. A., 2017, 114, 9725–9730.
17 W. Zhou, D. E can, L. Chen, C.-H. Yun, D. Li, M. Capelle i,
A. B. Co o , L. Chi ieac, R. E. Iacob, R. Pade a, J. R. Engen,
K.-K. Wong, M. J. Eck, N. S. G ay and P. A. Jänne, Na u e,
2009, 462, 1070–1074.
18 J. M. Os e m, U. Pe e s, M. L. Sos, J. A. Wells and
K. M. Shoka , Na u e, 2013, 503, 548–551.
19 J. Pe inge , K. Jones and M. D. Cheeseman, Angew. Chem.,
In . Ed., 2017, 56, 15200–15209.
20 Q. Zhao, X. H. Ouyang, X. B. Wan, K. S. Gajiwala, J. C. Ka h,
L. H. Jones, A. L. Bu lingame and J. Taun on, J. Am. Chem.
Soc., 2017, 139, 680–685.
21 N. P. G ims e , S. Connelly, A. Ba anczak, J. Dong,
L. B. K asno a, K. B. Sha pless, E. T. Powe s, I. A. Wilson
and J. W. Kelly, J. Am. Chem. Soc., 2013, 135, 5656–5668.
22 E. Anscombe, E. Meschini, R. Mo a-Vidal, M. P. Ma in,
D. S aun on, M. Gei mann, U. H. Danielson, W. A. S anley,
L. Z. Wang, T. Reuillon, B. T. Golding, C. Cano,
D. R. Newell, M. E. M. Noble, S. R. Wedge, J. A. Endico
and R. J. G iffin, Chem. Biol., 2015, 22, 1159–1164.
23 D. E. Mo enson, G. J. B igh y, L. Pla e, G. Ba e, W. Chen,
S. Li, H. Wang, B. F. C a a , S. Fo li, E. T. Powe s,
K. B. Sha pless, I. A. Wilson and J. W. Kelly, J. Am. Chem.
Soc., 2018, 140, 200–210.
24 P. A. Smi h, e al.,Na u e, 2018, 561, 189–194.
25 P. Ma ín-Gago and C. A. Olsen, Angew. Chem., In . Ed.,
2019, 58, 957–966.
26 L. H. Jones, ACS Med. Chem. Le ., 2018, 9, 584–586.
27 C. González-Bello, Fu u e Med. Chem., 2015, 7, 2371–2383.
28 D. G. Gou ley, A. K. Sh i e, I. Polika po , T. K ell,
J. R. Coggins, A. R. Hawkins, N. W. Isaacs and L. Sawye ,
Na . S uc . Biol., 1999, 6, 521–525.
29 A. Ka nell, P. D. Cam, N. Ve ma and A. A. Lindbe g,
Vaccine, 1993, 11, 830–836.
30 M. Malco a, D. Ka aso a and I. Rychlik, FEMS Mic obiol.
Le ., 2009, 291,44–49.
31D.A.Dil s,I.Riesen eld-O n,J.P.Fulgini i,E.Ekwall,
C. G ane , J. Nonenmache , R. N. B ey, S. J. C yz, K. Ka lsson,
Resea ch A icle O ganic Chemis y F on ie s
3134 |O g. Chem. F on .,2019,6,3127–3135 This jou nal is © he Pa ne O ganisa ions 2019
Open Access A icle. Published on 23 July 2019. Downloaded on 4/13/2020 9:33:56 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
K.Be gman,T.Thompson,B.Hua,A.H.B ückne and
A. A. Lindbe g, Vaccine, 2000, 18, 1473–1484.
32 L. Tizón, M. Manei o, A. Peón, J. M. O e o, E. Lence,
S. Poza, M. J. an Raaij, P. Thompson, A. R. Hawkins and
C. González-Bello, O g. Biomol. Chem., 2015, 13, 706–716.
33 C. González-Bello, L. Tizón, E. Lence, J. M. O e o, M. J. an
Raaij, M. Ma inez-Gui ian, A. Becei o, P. Thompson and
A. R. Hawkins, J. Am. Chem. Soc., 2015, 137, 9333–9343.
34 B. Blanco, V. P ado, E. Lence, J. M. O e o, C. Ga cia-Do al,
M. J. an Raaij, A. Llamas-Saiz, H. Lamb, A. R. Hawkins
and C. González-Bello, J. Am. Chem. Soc., 2013, 135, 12366–
12376.
35 C. Klean hous, M. Reilly, A. Coope , S. Kelly, N. C. P ice
and J. R. Coggins, J. Biol. Chem., 1981, 266, 10893–10898.
36 h ps://www.ccdc.cam.ac.uk/solu ions/csd-disco e y/com-
ponen s/gold/(las access Ma ch 15, 2019).
37 H. M. Senn and W. Thiel, Angew. Chem., In . Ed., 2009, 48,
1198–1229.
38 M. W. Van de Kamp and A. J. Mulholland, Biochemis y,
2003, 42, 2708–2728.
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