methicillin-resistant Staphylococcus aureus (MRSA) Kyra Gillard - - PowerPoint PPT Presentation

Identification of a pharmacophore capable of potentiating ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA)

Kyra Gillard

• Dr. Heather B. Miller and Dr. Meghan S. Blackledge

High Point University Department of Chemistry

Antibiotic Resistance

Clatworthy et. al. (2007) Nat. Chem. Biol. 3: 541-548

Methicillin-Resistant Staphylococcus aureus (MRSA)

• Kills over 20,000 people a year
• Employs several virulence factors,

including antibiotic resistance

• Giving it increased infectivity
• Making it extremely hard to treat

https://www.staph-infection-resources.com/recurring/mrsa-reinfection/

Ways to Combat Resistance

• 1. Build bigger stronger antibiotics
• Development of new antibiotics
• 2. Sneak antibiotics into cells
• Targeting resistance mechanisms

Antibiotic Adjuvants

• A Trojan horse approach to combating resistance
• Small molecules combined with antibiotics
• Do not have any antibiotic activity
• Non-toxic

Repotentiation is β-Lactam Specific

+ Amoxapine (150µM) Antibiotic MIC (µg/mL) MIC (µg/mL) Fold Reduction Oxacillin 32 2 16 Ampicillin 32 4 8 Penicillin 16 1 16 Cefazolin 16 0.5 32 V ancomycin 2 2 1

MIC 600 µM

• S. aureus

A TCC 43300

Fold Reduction =

MIC Antibiotic MIC Antibiotic+Compound

Wilson, T Blackledge, M Viguiera, P Heliyon 2018

FDA-Approved Antidepressants

Compound + 150 µM MIC (µg/mL) Fold Reduction + 75 µM MIC (µg/mL) Fold Reduction Amoxapine 2 16 8 4 Loxapine 8 4 32 1 Clothiapine

• 4

4 Clozapine 4 8 32 1 Olanzapine 32 1 32 1

• S. aureus

ATCC 43300

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

MRSA Resistance Mechanisms

• Three methods for resistance

1. Efflux pumps 2. Generate β-lactamase by blaZ

• Breaks down β-lactam antibiotics

3. Turn on penicillin-binding protein 2A (PBP2A) via mecA

• Has reduced affinity for β-lactam antibiotics

Regulation of the bla and mec operons in MRSA

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Nitrocefin Hydrolysis Assay

Whole Cell Nitrocefin Hydrolysis in MRSA 43300

20 40 60 80 100 120

• amoxapine

+ 75 uM amoxapine + 150 uM amoxapine A486 (% of Control)

• oxacillin

+ 4ug/ml oxacillin

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Whole Cell Nitrocefin Hydrolysis in MSSA 29213

20 40 60 80 100 120

• amoxapine

+ 75 uM amoxapine + 150 uM amoxapine A486 (% of Control)

• oxacillin

+ 4ug/ml oxacillin

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Amoxapine Induces Changes in Gene Expression

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Amoxapine Induces Changes in Gene Expression

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Amoxapine Induces Changes in Gene Expression

Gillard, K Miller, H Blackledge, M Chemical Biology and Drug Design submitted

Conclusion

• Structure activity relationship
• Methylation of piperazine ring decreases adjuvant activity
• Changing the bridge heteroatom from a ether to a thioether increases activity
• Aromatic halogenation may be required
• Mechanism of Action
• Not inhibiting β-lactamase nor preventing export
• Dampening transcriptional response to β-lactam antibiotic treatment
• Selectively affects the bla and mec operons

Future directions

• Identify the molecular target of amoxapine
• Upstream regulator?
• BlaR1 and MecR1?
• mRNA destabilization?
• Expand the structure activity relationship
• Synthesize more potent analogs

Acknowledgements

• Blackledge lab
• Dr. Heather Miller
• High Point University Chemistry Department
• High Point University Undergraduate Research and Creative Works