Download Towards the Discovery of New Antimicrobials: the Bifunctional

TowardstheDiscoveryofNewAnQmicrobials:
theBifuncQonalPenicillinBindingProteins
CarminaMicelli,AdrianJ.Lloyd,ChristopherG.DowsonandDavidI.Roper
SchoolofLifeSciences,GibbetHillCampus,TheUniversityofWarwick,Coventry,CV47AL
StructureandfuncQonofPBPs
Results
The major component of the bacterial cell
wall is a cross-linked glycopep@de polymer
calledpep@doglycan(PG).ThePGlayerisan
important an@microbial target, due to its
essen@alityanduniquenesstobacteria[1].
TPacQve
site(S459)
UB2H
H2O
D-Ala
D-Ala
D-Ala
D-Ala
m-Dap D-Ala m-Dap
m-Dap
D-Glu
D-Glu
D-Glu
L-Ala
L-Ala
L-Ala
20
40
60
GTase
80
100
0
120
0
D-Glu
GlcNAc-MurNAc
The bifunc@onal penicillin-binding proteins
(PBPs) func@on in the last stage of the PG
biosynthe@c pathway. Here, the precursor
Lipid II (undecaprenyl-pyrophosphorylM u r N A c ( p e n t a p e p @ d e ) - G l c N A c ) i s
polymerizedandcross-linkedintoPGbythe
glycosyltransferase(GT)andtranspep@dase
(TP) domain, respec@vely. The TP domain
can also catalyze DD-carboxypep@dase
reac@ons, resul@ng in release of the
terminalD-Ala[2].
100
120
30000
PBP1a+10μMMoenomycin
PBP1a+10μMMoenomycin
25000
20000
15000
PBP1a
10000
Fluorescence(521nm)
PBP1b
80
35000
β-lactaman@bio@csbindtotheTPdomain,
causing weakening of the cell wall and
eventually bacteria lysis. To escape the
ac@on of β-lactams bacteria have evolved
resistance mechanisms, for instance the
the produc@on of β-lactamases in Gramnega@vebacteria[3].
Escherichiacoli
(PDBid:3VMA)
60
L-Ala
Fluorescence(521nm)
5000
20000
15000
PBP1a
10000
5000
0
0
0
10
20
30
40
50
0
60
10
20
Time
30
40
50
60
Time
Con@nuous fluorescent GTase assay of Pa PBP1a (leb) and Ab PBP1a (right) at 2μM concentra@on.
MoenomycinisanMaffinityinhibitoroftheGTdomain,hereusedasacontrol.
0.5
0.5
PBP1a+20μMLipidIIm-DAP
0.4
Absorbance(555nm)
0.4
Aimoftheproject
0.3
0.2
PBP1a+0.1%TritonX-100
0.1
Absorbance(555nm)
PBP1a
Acinetobacterbaumannii
(PDBid:3UDX)
40
Elu@onvolume
25000
GTacQve
site(E233)
20
Purifica@onofPaPBP1a(leb,PDBid:4OON)andAbPBP1a(right,PDBid:3UDX).Crystalliza@onofthese
proteinsrequiresapoorlycontrolledproteolysisstep[6-7].
m-Dap
GlcNAc-MurNAcGlcNAc-MurNAcGlcNAc-MurNAc
GTacQve
site(E92)
100
Elu@onvolume
0
Post-proteolysis
150
50
100
D-Alam-Dap D-Ala
200
0
300
LipidIIprecursor
D-AlaD-Glu D-Ala
Absorbance(280)
Absorbance(280)
L-Ala
L-Ala TPase
CPase
200
400
Post-proteolysis
---GlcNAc-MurNAcGlcNAc-MurNAc---
Pre-proteolysis
250
500
TPacQve
site(S510)
ODD
Pre-proteolysis
600
PBP1a+20μMLipidIIm-DAP
0.3
0.2
0.1
PBP1a+0.1%TritonX-100
0
0
-0.1
-0.1
0
Structural and kine@c characteriza@on of bifunc@onal PBPs in Gram-nega@ve
bacteria, with a primary focus on Pseudomonas aeruginosa (Pa) and
Acinetobacter baumannii (Ab), two pathogens responsible for the majority of
nosocomialinfec@onsandwithreportedincreasingmul@-drugresistance[4].
This research will provide in vitro data that will help beier elucidate the
mechanis@c basis of in vivo catalyzed reac@ons and regula@on of PBPs and,
ul@mately, guide the op@miza@on of validated screening assays aimed at
discoveringnewPBPinhibitors.
5
10
15
20
25
30
35
40
0
Time
5
10
15
20
25
30
35
40
Time
Con@nuousspectrophotometricTPaseassayofPaPBP1a(leb)andAbPBP1a(right)at3μMconcentra@on.
0.1%TritonX-100isthedetergentusedtosolubilizeLipidIIm-DAP,hereusedasacontrol.
Conclusionsandfuturework
The published crystalliza@on condi@ons for Pseudomonas aeruginosa and
AcinetobacterbaumanniiPBP1ahavebeentried,butnocrystalshavegrownsofar.
Next,crystalliza@onaiemptswillbefocusedontheGTdomainoftheseproteins.
The soluble constructs of PBP1a from Pseudomonas aeruginosa and Acinetobacter
baumanniihaveshowntobeac@veinboththeGTaseandTPaseassays.
Next,thefull-lengthversionsofPBP1awillbeinves@gated,andwhetherornotthe
presence of the transmembrane helix affects the ac@vity of PBP1a in vitro will be
evaluated.
Methods
Fragment-basedscreeningbyproteincrystallography
Absorbance(280)
References
Elu@onvolume
Screeningof
fragmentlibraries
Crystalliza@on
Expressionandpurifica@on
[1] Brown, L., Wolf, J., Prados-Rosales, R., and Casadevall, A. (2015). Through the wall: extracellular vesicles in Gram-posi@ve bacteria, mycobacteria and fungi. Nature Reviews
Microbiology,13(10),pp.620-630.
[2]Sauvage,E.,Kerff,F.,Terrak,M.,Ayala,J.andCharlier,P.(2008).Thepenicillin-bindingproteins:structureandroleinpep@doglycanbiosynthesis.FEMSMicrobiologyReviews,
32(2),pp.234-258.
[3]Zapun,A.,Contreras-Martel,C.,andVernet,T.(2008).Penicillin-bindingproteinsandbeta-lactamresistance.FEMSMicrobiologyReviews,32(2),pp.361-385.
[4]Zavascki,P.,Carvalhaes,G.,Picão,C.,andGales,C.(2010).Mul@drug-resistantPseudomonasaeruginosaandAcinetobacterbaumannii:resistancemechanismsandimplica@onsfor
therapy.ExpertReviewofAnD-InfecDveTherapy,8(1),pp.71-93.
[5]Schwartz,B.,Markwalder,J.,Seitz,S.,Wang,Y.,andStein,R.(2002).Akine@ccharacteriza@onoftheglycosyltransferaseac@vityofEschericiacoliPBP1banddevelopmentofa
con@nuousfluorescenceassay.Biochemistry,41(41),pp.12552-12561.
[6]Starr,J.,Brown,M.,Aschenbrenner,L.,Caspers,N.,Che,Y.,Gerstenberger,B.,Huband,M.,Knafels,J.,Lemmon,M.,Li,C.,McCurdy,S.,McElroy,E.,Rauckhorst,M.,Tomaras,A.,
Young,J.,Zaniewski,R.,Shanmugasundaram,V.,andHan,S.(2014).Siderophorereceptor-mediateduptakeoflac@[email protected]
Chemistry,57(9),pp.3845-3855.
[7] Han, S., Caspers, N., Zaniewski, R., Lacey, B., Tomaras, A., Feng, X., Geoghegan, K., and Shanmugasundaram, V. (2011). Dis@nc@ve aiributes of β-lactam target proteins in
Acinetobacterbaumanniirelevanttodevelopmentofnewan@[email protected],133(50),pp.20536-20545.
Hitiden@fica@on
Medicinalchemistry
InvitroPGsynthesisassays
L-Ala
m-Dap D
m-Dap
D
D-Ala
D-Ala
D-Ala
Highfluorescence
Lowfluorescence
D-Ala
---GlcNAc-MurNAc---
Noenzyme
GTase
C o n @ n u o u s G T a s e
assay with dansylated
LipidIIsubstrate[5].
Time
---GlcNAc-MurNAc---
L-Ala
L-Ala
TPase
m-Dap
D-Glu
L-Ala
---GlcNAc-MurNAc---
D-Glu
m-Dap D-Ala
D-Ala
m-Dap
D-Glu
Absorbance
D-Ala
m-Dap D-Ala
D-Glu
D-Ala
L-Ala
D-Ala
D-Ala
GlcNAc-MurNAc
D-Glu
m-Dap D
muramidase
PG
chains
D-Glu
D-Glu
GTase
GlcNAc-MurNAc
L-Ala
Fluorescence
---GlcNAc-MurNAc
CPase/TPase
Nolipid
L-Ala
---GlcNAc-MurNAc---
Time
Con@nuous CPase/TPase assay with Lipid II mDAPsubstratedevelopedbyDr.AdrianJ.Lloyd.
Acknowledgements
ThisresearchisfundedbyBBSRC.ThanktoalltheC10labmembersfortheirsupport