Date post: | 09-Jan-2017 |
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SWON AllianceCross council AMR collaborative
SWON: Multi-institutionalMulti-disciplinary team
• Sheffield, Southampton, Warwick, Oxford, Newcastle
• Complementary aspects of biochemistry, genetics, physiology
and molecular modeling in the area of PG metabolism, structure
and architecture, plus innovative lead molecules from Oxford
• Working with industry
Target organisms
• E. coli; resistance to penicillin by the acquisition of β-lactamases
and changes to membrane permeability/ efflux.
• S. aureus; resistance to methicillin by the acquisition of the low
affinity PBP2A.
• S. pneumoniae; penicillin resistance through horizontal gene
transfer to develop low affinity forms of PBPs 1A, 2X and 2B.
Objectives
1. Determine the fundamental mechanism of peptidoglycan
assembly
2. Determine how PBP activity is controlled at the molecular and
cellular level.
3. Determine how β-lactams impact upon the cell wall biosynthesis
machinery and discover non-lactam inhibitors to underpin new
chemotherapeutic regimes targeting PBPs.
Technologies
• Sheffield – super high resolution imaging• Southampton – molecular modeling• Warwick – reagent synthesis for assay
development (research and industry), fundamental biochemistry, structural biology
• Oxford – chemical synthesis, assay development• Newcastle – molecular microbiology, biochemistry
(protein protein interactions in vivo and in vitro), structural biology
1.1. How do PBPs interact with their substrates?
• This question has been unanswered for the past 70 years because of the lack of a quantitative assay for TP activity.
1.2. How are TG and TP activities co-ordinated?
2. Determine how PBP activity is controlled at the
molecular and cellular level.
• Ezra.yfp
Peptidoglycan Dynamics – Cellular heterogeneity
Consecutive 5 minutes labelling
Labelling patterns of sister cells: Labelling pattern of related cells:
Consecutive 30 minutes labelling
Cells progress through the cell cycle at different ratesSince sister cells behave differently this is unlikely to be genetically inherited
Total=231
Different Pattern16.45%
Same Pattern83.55%
Total=720
Different pattern5.69%
Same Pattern94.31%
SWoN
21 targets from S. aureus
Generate 95 constructs
Genes synthesised
Test expressions
OPPF
Large scale expression/purification & co-expression. Different construct/affinity tags for targets that fail to express
Standard crystallisation trials & LCP crystallography. Structure solution/phasing
Crystallisation with ligands (protein:protein complexes, antibiotics, pseudo/substrates, product etc). Characterise ligand interaction
OPPF
Lemo
Rosetta
• Different expression cells (Lemo/Rosetta) and induction (IPTG/AI) methods were tested• Membrane proteins solubilised in 1 % DDM• A sample of some of the results generated is given below
3. Investigating new ways of inhibiting PBPs and b-lactamases
-HTS for MBLs and SBLs, PBPs-Crystallography (VIM-2, IMP-1, BcII, SPM-1, NDM-1,-4-5; PBP-3...)-NMR, SPR new binding assays-MS, Tm shift, CD, SF, etc-Counter screen ...
PBPs: PBP-2a, -3, -4, -5, -6SBLs: TEM-1, AmpC, CTX-10, CTX-15, etc.MBLs: (B1) VIM-1, VIM-2, SPM-1, IMP-1, BcII, NDM-1 to -8; (B2) CphA, Imi-S; (B3) Fez-1, L-1, AIM-1, etc... -Several human metallo-enzymes, including human-metallo-β-lactamases (e.g. SNM1A and B, ETHE1, etc.)
NO
monobactams
NO
S
penicillins
NO
cephalosporins
S
NO
carbapenems
NH
CO2HR'
HN
CO2H CO2H
O
R
O
RR'
RHNR
OSO3H
N-acyl-D-Ala-D-AlaCO2H
NHHO
H
HN
O
R
NO
O
CO2H
OH
clavulanic acid
NO
O
HO2C
MeCOHN
O O
Lactivicn
VIM-2 (B1 MBL) BcII (B1 MBL) OXA-10 (Class D SBL)
Cyclic Boronic Acids are Potent SBL and MBL Inhibitors