Bacterial Diseases Bubonic PlagueTuberculosisCholera SepsisLyme Disease Antibiotics 1929 – Penicillin discovered 1933 – Sulfa drugs synthesized 1969 – US surgeon Gen “end infectious diseases??? Today – bacteria with multi-drug resistance. Concern over resistance to ‘last resort’ antibiotics – Pasteur - Decay due to living organisms 1867 – Lister – phenol is disinfectant

Enterococcus faecalis A leading cause of hospital infections vanomycin = antibiotic of last resort E faecalis resistant strains for years can transfer resistance genes to Staphylococcus aureus in lab - MRSA virulent cause of pneumonia, endocarditis, sepsis etc.

Examples of Antibiotic Targets Cell Wall Formation - penicillin, cephalosporins, vancomycin Replication – novobiocin & DNA Gyrase Transcription – rifampicin & RNA Pol Translation – puromycin & ribosome ‘A’ Folate biosynthesis – sulfa drugs & DHPS Fatty Acid synthesis – triclosan & enoyl reductase

Killing Bacteria without Resistance Drastically alter Bacterial environment so that multiple systems become inoperative. Therefore, many genes would have to mutate to cause resistance. Bleach (NaOCl) – Oxidize multiple targets in bacteria Detergents/soap/alcohol – disrupt membrane Heat/pH extremes - denature proteins UV irradiation – grossly damage DNA Antimicrobial Peptides (AMPs) – lyse membranes

Bacteria chromosomeplasmids Plasmids in bacteria often contain genes critical for ….. antibiotic resistance, toxins, natural product metabolism F factor plasmid (for sexual transmission of plasmids) Bacteria can transfer antibiotic resistance plasmids between species

Practices that Foster Resistance 1. taking antibiotics for non-bacterial illness 2. not taking all of antibiotic 3. non-human use of antibiotics antibiotics as growth promoters in animals Resistant Bacteria ― strategies 1. mutated target enzyme – evasion strategy 2. enzyme to destroy antibiotic – attack strategy 3. efflux channel – bailout strategy

Fighting Back at Resistant Bacteria 3. Find new targets for Drugs 4. Find new classes of drugs 2. Develop ‘co’-drugs 1. Develop new drugs for same targets

Sulfthiazole resistance ― case study 1985 – 5 isolates of resistant Streptoccoccus Pyogenes saved from patients in Sweden Hospital 1990’s – Genomes from normal and resistant isolates compared – highly mutated genes cloned & expressed in E. coli. DHPS gene found to be mutated. (evasion strategy) Pathway genes: folC - folE - folP - folQ - folK folE = GTP cyclohydrolase folQ = dihydroneopterin aldolase folK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit folP = DHPS (dihydropteroate synthetase) adds PABA unit folC = dihydrofolate synthetase adds glutamate unit

H 2 N- -COOH H 2 N- N NN HN O O HN- -C- NH-CH-COO- CH 2 COO- Dihydrofolate Biosynthesis Pathway genes: folC - folE - folP - folQ - folK folE = GTP cyclohydrolase folQ = dihydroneopterin aldolase folK = hydroxymethydihydropterin pyrophosphatase converts GTP into dihydropteridin unit DHPS DHFS PABA → → dihydrofolate folP = DHPS (dihydropteroate synthetase) adds PABA unit ― 16% divergence folC = dihydrofolate synthetase adds glutamate unit

NH 2 O=S=O | NH 2 sulfanilamide sulfathiazole NH 2 N-H N S O=S=O

E. Coli - DHPS sulfonamide

E. Coli - DHPS

K M (inhib) = K M (1 + [I]/K i ) K M K i G1 (suscep) 0.7  M 0.2  M G56 (res) 2.5  M 27.4  M Difference 3.6x  137x  DHPS Kinetics

They are analogs of the peptide component of the bacterial cell wall penicillins and cephalosporins are antibiotic classes that possess lactam ring  -lactamases of varying specificities are often found in ‘R’ plasmids of resistant bacteria. penicllin and  -lactams inhibit the cell wall synthesis in bacteria Lactams contain a 4-membered ring with an amide nitrogen and a keto group.  -lactamases destroy  -lactams by cleaving (O=C ― N) in lactam structure. Attack strategy destroys antibiotic before it can kill bacteria. Penicillin inhibits last connection in making bacterial cell wall … Glycopeptide Transpeptidase

Glycopeptide transpeptidase  -lactam antibiotic

Glycopeptide transpeptidase  -lactam antibiotic

Polysaccharide X-X-X-A-A G-G-G-G-G X-X-X-A-A G-G-G-G-G Peptidoglycan

Bacterial Cell Wall Completion X-X-X-A G-G-G-G-G X-X-X-A-A

R C = O H - N S CH 3 C - C C - CH 3 C - N C O COO - CH 3 CH 3 - N - C - C - N - C O COO - penicillin -D-Ala-D-Ala mimics AA seq of peptide linker  -lactamase

R C = O H - N S CH 3 C - C C - CH 3 C - N C O COO - penicillin O CH 3 C - C C - CH 3 C - N C O COO - O clavulanate given along with penicillin it will inhibit penicillinase

OH OO Cl N N COCO OH N O HOOC O N O OH N O N-CH 3 O NH 2 HO Vancomycin binds to D-Ala – D-Ala peptide unit Resistance due to target mutation in peptidoglycan – D Ala to D – lactate giving 3x less drug affinity due to missing H-bond. replacing C=O with CH 2 produces 100x activity to mutant retains only 3% activity to sensitive bacteria. C&E News Feb 13, 2006

Vancomycin (blue) D-Ala – D-Ala

Efflux Pumps ― bailout strategy Many efflux pumps expel a broad range of compounds – may have normal anti-toxin function. efflux pump inhibitors, like  -lactamase inhibitors, could well be analogs of the original antibiotic and have mild antibiotic activity as well.

E. Coli ACRB Multi-drug efflux transporter

Efflux Pumps antibiotic bacteria cell membrane antibiotic target efflux pump EP inhibitor

Pdb – 2f2m EmrE tetraphenylphosphonium

Cl O O Triclosan inhibits enoyl reductase

Fatty Acid Synthesis acetylCoA + HCO ATP  malonyl CoA +ADP acetylCoA + ACP  acetyl-ACP + CoA malonylCoA + ACP  malonyl-ACP + CoA acetyl-ACP +malonyl-ACP  acetoacetyl-ACP + CO 2 + ACP acetoacetyl-ACP + NADPH  hydroxybutyryl-ACP + NADP + hydroxybutyryl-ACP  Crotonyl-ACP + H 2 O Crotonyl-ACP + NADPH  butyryl-ACP + NADP + Enoyl-ACP reductase

Enoyl reductase ( step in fatty acid synthesis) triclosan

Enoyl reductase ( step in fatty acid synthesis) triclosan

Parikh et. al. (2000) Biochemistry 39, Triclosan inhibits enoyl-ACP reductase from Mycobacterium Tuberculosis K i ~ 0.22  M for crotonyl-ACP & NADH Y 158  F K i ~ 47 & 36  M M 161  V triclosan resistant K i ~ 4.3  M also less sensitive to isoniazid triclosan could stimulate TB resistant strains of mycobacterium

New Antibiotics oxazolidimones (linezolid) – binds 30S subunit of ribosome and prevents mRNA & fMet-tRNA binding. gemifloxacin – DNA gyrase inhibitor used on respiratory tract infections daptomycin – blocks peptidoglycan and lipoteichoic acid synthesis (cell wall formation) works on vanomycin resistant enterococci BPI Protein - (bacterial permeability increasing) naturally found in bactria killing wbc’s – good in combo Antimicrobial Peptides – defensins & protegrins may function as voltage-gated pores specific for acidic phospholipids found only in bacteria

New Targets for Antibiotics sortase – cleaves loosely bound surface proteins in gram (+) bacteria to activate infectivity proteins. (doesn’t kill bacteria) deformylase – removes formyl group from amino end of bacterial polypeptides – includes actinonen (natural cpd) Efflux Pump Inhibitors