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