MSOP1016 Antibacterial Agents - PDF

MSOP1016 Antibacterial Agents -lactam antibiotics (part 2) and beyond Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text An introduction to medicinal chemistry ISBN: 9780198749691 615.19 PAT (Drill Hall Lib...

MSOP1016 Antibacterial Agents -lactam antibiotics (part 2) and beyond Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text An introduction to medicinal chemistry ISBN: 9780198749691 615.19 PAT (Drill Hall Library) CHAPTER 19 Antimicrobials Classes to be covered today Cephalosporins Carbapenems Monobactams -lactamase inhibitors Learning outcomes At the end of this lecture, you should appreciate/understand:- Cephalosporins mechanism of action structure activity relationship resistance synthesis & development Carbapenems Monobactams -lactamase inhibitors clavulanic acid newer developments Cephalosporins Discovery Structure Activity Relationships Synthesis Generations Resistance Cephalosporins Discovery 1940s Cephalosporins Similarity to penicillins -lactam ring fused to 6-membered dihydrothiazine ring How does it compare to penicillins? potency? spectrum of activity? advantages? Cephalosporins Mechanism of action Cephalosporins inhibit the transpeptidase enzyme (cf. penicillins) Cephalosporins Structure activity relationship As with penicillins, expanded but still limited opportunities for modification Crucial things: -lactam ring free carboxylic acid at position 4 fused ring system stereochemistry of rings and substituents Cephalosporins Why make modifications? The aims are to: Improve acid stability Improve pharmacokinetics Broaden the spectrum of activity Increase the activity Resistance to bacterial enzymatic degradation Improved outer membrane penetration Higher affinity to the receptors (PBPs) Reduce allergy Increase tolerance after parenteral administration Cephalosporins Synthesis issues Fermentation route proved impossible! For semi-synthetic protocols 7-aminocephalosporonic acid (7-ACA) is the key intermediate (cf. 6-APA for penicillin semi-synthesis) Not available from fermentation! Cannot be obtained by hydrolysis of cephalosporin C! Cephalosporins Synthesis solution Chemical hydrolysis of cephalosporin C! Cephalosporins Classification There are around 50 cephalosporins in clinical use Classification into generations is based on Spectrum of anti-bacterial activity Chemical sophistication Chronology Cephalosporins First generation Cephalothin Cephaloridine Cefalexin Cefazolin Cephalosporins First generation issues Cephalothin was one of the most commonly used first generation cephalosporins The alcohol is less active, but acetoxy group is important to the mechanism of action! Also note that the acid and alcohol can react to give a lactone Cephalosporins First generation development 1 Esterase stability was obtained by changing the acetoxy group to a pyridinium group ➔ cephaloridine Note: exists as a zwitterion – still poorly absorbed Cephalosporins First generation development 2 Improve stability: add EWGs at carbon a to side chain amide Improve oral absorption: add methyl group at position-3 Cephalosporins Second generation 1 Cephamycins: methoxy group at position-7 Cefoxitin has a broader spectrum of activity (cf. 1st generation) Cephalosporins Second generation 2 Oximinocephalosporins: modification a to side chain amide Increases stability against -lactamase enzymes Cephalosporins Third generation 1 Aminothiazole ring enhances outer membrane penetration (Gram-negative bacteria) Cephalosporins Third generation 2 Substituent at position 3 ➔ variation in pharmacokinetics Major role due to activity against Gram-negative bacteria Cephalosporins Fourth generation Still oximinocephalosporins, but these are zwitterionic Dramatically improves outer membrane penetration Good affinity for transpeptidase enzyme Low affinity for a variety of -lactamase enzymes Cephalosporins Fifth generation Again, oximinocephalosporins Ceftolozane MRSA/MDRSP P. aeruginosa (app. 2014) Cephalosporins Resistance Activity of each type of cephalosporin depends on Ability to reach the bacterial transpeptidase Stability to -lactamases present Affinity for the target enzyme Example: Cephalosporins and MRSA Cephalosporins Summary 1 Strained -lactam ring fused to dihydrothiazine ring 1st generation versus penicillins: better stability against acid & -lactamases poor oral availability and lower activity Variation at 7-acylamino side chain alters activity Variation at position 3 alters pharmacokinetics/metabolism Methoxy groups can be added at C-7 Synthesis: semi-synthetic procedures from 7-ACA 7-ACA obtained by chemical hydrolysis of cephalosporins requires prior activation of the side chain Cephalosporins Summary 2 Metabolic deacetylation ➔ lowering of activity block with metabolically stable groups 3-methyl groups increase oral availability decrease in activity mediated by placing EWGs at the −position of the acyl side chain 3-methylcephalosporins can be synthesised from penicillins Cephamycins have a methoxy group (OMe) at C-7 Placing an oximino group at C-7 ➔ newer generations with increased potency and a broader spectrum of activity Other -lactam antimicrobials Carbapenems Example: Thienamycin (Merck, 1976) Potent, very broad range of activity Low toxicity and highly resistant to -lactamases Poor stability Not absorbed in GI tract Other -lactam antimicrobials Monobactams 1 Isolated from natural sources Is there anything unusual about their structures? Other -lactam antimicrobials Monobactams 2 Monocyclic -lactam ring Moderate activity in vitro against a narrow group of Gram- negative bacteria Active against P. aeruginosa Inactive against Gram-positive bacteria Different spectrum of activity from penicillins Thought to operate via a different mechanism to penicillins Low toxicity -lactamase inhibitors -lactamase enzymes There are two major classes of -lactamase enzymes Classes A, C & D Class B Chem. Commun. 2011, 47, 4055-4061 -lactamase inhibitors Metallo--lactamase enzymes Major source of resistance to -lactam antibiotics Considerable diversity in amino acid sequence ➔ subclasses Subclasses B1 & B3 positive cooperativity in Zn binding catalytic species is a di-zinc system Subclass B2 catalytic species is a mono-zinc system inhibited by binding of a second zinc cation -lactamase inhibitors Clavulanic acid Isolated from Streptomyces clavuligerus Weak & unimportant antibacterial activity Powerful irreversible inhibitor of -lactamases Used as a “sentry drug” for amoxicillin Augmentin (co-amoxiclav) Timentin (ticarcillin + clavulanic acid) -lactamase inhibitors Clavulanic acid SAR Essentials strained -lactam ring enol ether Z-configuration at the C=C bond no substituent at C-6 (R)-stereochemistry at C-2/C-5 the carboxylic acid group -lactamase inhibitors Clavulanic acid MOA The mechanism of action (MOA) is as shown below -lactamase inhibitors Penicillanic acid sulphones Key examples are sulbactam & tazobactam Also suicide inhibitors of -lactamases Sulbactam: broader spectrum, but lower potency Unasyn (IV preparation with ampicillin) Tazobactam: broad spectrum, decent potency Tazosin/Zosyn (IV preparation with piperacillin)  -lactamase inhibitors Avibactam Approved in 2015 – combi therapy with ceftazidime (UTIs) First new -lactamase inhibitor to hit the market for 20 years! Chemical structures of ceftazidime and avibactam, which are approved for use in combination therapy for the treatment of complex urinary tract infections. -lactamase inhibitors Avibactam MOA Avibactam is not a “suicide” inhibitor Reaction with the -lactamase enzyme is reversible  -lactamase inhibitors Relebactam This drug was approved in July 2019 Combination therapy with imipenem/cilastatin Image result for cilastatin Image result for relebactam -lactamase inhibitors Vaborbactam This was a “first-in-class” -lactamase inhibitor Boronate ester within the structure Approvals FDA (8/17): cUTIs (including pyelonephritis) EMA (11/18) Cycloserine A miscellaneous cell-wall synthesis inhibitor Learning outcomes You should now appreciate & understand:- Cephalosporins mechanism of action structure activity relationship resistance synthesis & development Carbapenems Monobactams -lactamase inhibitors clavulanic acid newer developments MSOP1016 Antibacterial Agents -lactam antibiotics (part 2) and beyond Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text An introduction to medicinal chemistry ISBN: 9780198749691 615.19 PAT (Drill Hall Library) CHAPTER 19 Antimicrobials Classes to be covered today Cephalosporins Carbapenems Monobactams -lactamase inhibitors Learning outcomes At the end of this lecture, you should appreciate/understand:- Cephalosporins mechanism of action structure activity relationship resistance synthesis & development Carbapenems Monobactams -lactamase inhibitors clavulanic acid newer developments Cephalosporins Discovery Structure Activity Relationships Synthesis Generations Resistance Cephalosporins Discovery 1940s Cephalosporins Similarity to penicillins -lactam ring fused to 6-membered dihydrothiazine ring How does it compare to penicillins? potency? spectrum of activity? advantages? Cephalosporins Mechanism of action Cephalosporins inhibit the transpeptidase enzyme (cf. penicillins) Cephalosporins Structure activity relationship As with penicillins, expanded but still limited opportunities for modification Crucial things: -lactam ring free carboxylic acid at position 4 fused ring system stereochemistry of rings and substituents Cephalosporins Why make modifications? The aims are to: Improve acid stability Improve pharmacokinetics Broaden the spectrum of activity Increase the activity Resistance to bacterial enzymatic degradation Improved outer membrane penetration Higher affinity to the receptors (PBPs) Reduce allergy Increase tolerance after parenteral administration Cephalosporins Synthesis issues Fermentation route proved impossible! For semi-synthetic protocols 7-aminocephalosporonic acid (7-ACA) is the key intermediate (cf. 6-APA for penicillin semi-synthesis) Not available from fermentation! Cannot be obtained by hydrolysis of cephalosporin C! Cephalosporins Synthesis solution Chemical hydrolysis of cephalosporin C! Cephalosporins Classification There are around 50 cephalosporins in clinical use Classification into generations is based on Spectrum of anti-bacterial activity Chemical sophistication Chronology Cephalosporins First generation Cephalothin Cephaloridine Cefalexin Cefazolin Cephalosporins First generation issues Cephalothin was one of the most commonly used first generation cephalosporins The alcohol is less active, but acetoxy group is important to the mechanism of action! Also note that the acid and alcohol can react to give a lactone Cephalosporins First generation development 1 Esterase stability was obtained by changing the acetoxy group to a pyridinium group ➔ cephaloridine Note: exists as a zwitterion – still poorly absorbed Cephalosporins First generation development 2 Improve stability: add EWGs at carbon a to side chain amide Improve oral absorption: add methyl group at position-3 Cephalosporins Second generation 1 Cephamycins: methoxy group at position-7 Cefoxitin has a broader spectrum of activity (cf. 1st generation) Cephalosporins Second generation 2 Oximinocephalosporins: modification a to side chain amide Increases stability against -lactamase enzymes Cephalosporins Third generation 1 Aminothiazole ring enhances outer membrane penetration (Gram-negative bacteria) Cephalosporins Third generation 2 Substituent at position 3 ➔ variation in pharmacokinetics Major role due to activity against Gram-negative bacteria Cephalosporins Fourth generation Still oximinocephalosporins, but these are zwitterionic Dramatically improves outer membrane penetration Good affinity for transpeptidase enzyme Low affinity for a variety of -lactamase enzymes Cephalosporins Fifth generation Again, oximinocephalosporins Ceftolozane MRSA/MDRSP P. aeruginosa (app. 2014) Cephalosporins Resistance Activity of each type of cephalosporin depends on Ability to reach the bacterial transpeptidase Stability to -lactamases present Affinity for the target enzyme Example: Cephalosporins and MRSA Cephalosporins Summary 1 Strained -lactam ring fused to dihydrothiazine ring 1st generation versus penicillins: better stability against acid & -lactamases poor oral availability and lower activity Variation at 7-acylamino side chain alters activity Variation at position 3 alters pharmacokinetics/metabolism Methoxy groups can be added at C-7 Synthesis: semi-synthetic procedures from 7-ACA 7-ACA obtained by chemical hydrolysis of cephalosporins requires prior activation of the side chain Cephalosporins Summary 2 Metabolic deacetylation ➔ lowering of activity block with metabolically stable groups 3-methyl groups increase oral availability decrease in activity mediated by placing EWGs at the −position of the acyl side chain 3-methylcephalosporins can be synthesised from penicillins Cephamycins have a methoxy group (OMe) at C-7 Placing an oximino group at C-7 ➔ newer generations with increased potency and a broader spectrum of activity Other -lactam antimicrobials Carbapenems Example: Thienamycin (Merck, 1976) Potent, very broad range of activity Low toxicity and highly resistant to -lactamases Poor stability Not absorbed in GI tract Other -lactam antimicrobials Monobactams 1 Isolated from natural sources Is there anything unusual about their structures? Other -lactam antimicrobials Monobactams 2 Monocyclic -lactam ring Moderate activity in vitro against a narrow group of Gram- negative bacteria Active against P. aeruginosa Inactive against Gram-positive bacteria Different spectrum of activity from penicillins Thought to operate via a different mechanism to penicillins Low toxicity -lactamase inhibitors -lactamase enzymes There are two major classes of -lactamase enzymes Classes A, C & D Class B Chem. Commun. 2011, 47, 4055-4061 -lactamase inhibitors Metallo--lactamase enzymes Major source of resistance to -lactam antibiotics Considerable diversity in amino acid sequence ➔ subclasses Subclasses B1 & B3 positive cooperativity in Zn binding catalytic species is a di-zinc system Subclass B2 catalytic species is a mono-zinc system inhibited by binding of a second zinc cation -lactamase inhibitors Clavulanic acid Isolated from Streptomyces clavuligerus Weak & unimportant antibacterial activity Powerful irreversible inhibitor of -lactamases Used as a “sentry drug” for amoxicillin Augmentin (co-amoxiclav) Timentin (ticarcillin + clavulanic acid) -lactamase inhibitors Clavulanic acid SAR Essentials strained -lactam ring enol ether Z-configuration at the C=C bond no substituent at C-6 (R)-stereochemistry at C-2/C-5 the carboxylic acid group -lactamase inhibitors Clavulanic acid MOA The mechanism of action (MOA) is as shown below -lactamase inhibitors Penicillanic acid sulphones Key examples are sulbactam & tazobactam Also suicide inhibitors of -lactamases Sulbactam: broader spectrum, but lower potency Unasyn (IV preparation with ampicillin) Tazobactam: broad spectrum, decent potency Tazosin/Zosyn (IV preparation with piperacillin)  -lactamase inhibitors Avibactam Approved in 2015 – combi therapy with ceftazidime (UTIs) First new -lactamase inhibitor to hit the market for 20 years! Chemical structures of ceftazidime and avibactam, which are approved for use in combination therapy for the treatment of complex urinary tract infections. -lactamase inhibitors Avibactam MOA Avibactam is not a “suicide” inhibitor Reaction with the -lactamase enzyme is reversible  -lactamase inhibitors Relebactam This drug was approved in July 2019 Combination therapy with imipenem/cilastatin Image result for cilastatin Image result for relebactam -lactamase inhibitors Vaborbactam This was a “first-in-class” -lactamase inhibitor Boronate ester within the structure Approvals FDA (8/17): cUTIs (including pyelonephritis) EMA (11/18) Cycloserine A miscellaneous cell-wall synthesis inhibitor Learning outcomes You should now appreciate & understand:- Cephalosporins mechanism of action structure activity relationship resistance synthesis & development Carbapenems Monobactams -lactamase inhibitors clavulanic acid newer developments MSOP1016 Antibacterial Agents 1. Glycopeptides and cell wall synthesis 2. Agents that impair bacterial translation (part 1) Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text An introduction to medicinal chemistry ISBN: 9780198749691 615.19 PAT (Drill Hall Library) CHAPTER 19 Learning outcomes At the end of this lecture, you should appreciate & understand:- the use of carbohydrates in medicine glycopeptides origin, mechanism(s) of action, development difficulties impairment of bacterial translation processes chloramphenicol tetracyclines origin, mechanism of action, synthesis & development aminoglycosides Origin, mechanisms of action, development difficulties, resistance Carbohydrates in medicine Antibacterials Biologically active natural products Antibacterials Glycopeptides Aminoglycosides Macrolides & Ketolides Carbohydrates in medicines General background There is a large number of carbohydrate-containing natural products which are biologically active Many families of structurally-related compounds have been identified and have a wide range of pharmaceutical application: Antibiotics anti-tumour/anti-cancer anti-parasitic anti-fungal Antibiotic and anti-tumour compounds are by far the most common form of biological activity currently identified in carbohydrate-containing natural products This is perhaps due to the nature of glycan-lectin interactions Carbohydrates in medicines Antibiotics in clinical use Glycopeptides complex polypeptides attached to a variety of mono-, di- and tetra- saccharides some members also have anti-tumour activity for treatment of Gram-positive bacteria (too big to get into cell of Gram- negative) Aminoglycosides characterized by the presence of an aminocyclitol ring linked to amino-sugars in their structure broad antimicrobial spectrum Macrolides classified according to their aglycone ring size (12-, 14- or 16-membered rings) and contain two monosaccharides excellent antibiotic activity against Gram-positive bacteria. inhibition of protein synthesis Glycopeptides Vancomycin Introduced in 1956 “Drug of last resort” Has been used to treat bacterial infections that do not succumb to other antibiotics (e.g. MRSA) Glycopeptides Vancomycin biosynthesis Involves many reaction steps Cyclisation reactions ➔ very rigid structure Glycopeptides Vancomycin mechanism of action 1 Glycopeptides Vancomycin mechanism of action 2 Glycopeptides Vancomycin mechanism of action 3 Glycopeptides Problems with vancomycin Size Unable to cross outer membrane of G-negative bacteria Unable to cross inner cell membrane of G-positive bacteria Resistance Slow to develop, but exists (1989 VRE, 1996 VRSA) Glycopeptides Teicoplanin This is actually a mixture of 5 similar structures Glycopeptides Teicoplanin mechanism of action Does not dimerise Less toxic than vancomycin Teicoplanin A2-5 Glycopeptides Dalbavancin This is a second-generation teicoplanin Teicoplanin A2-5 Dalbavancin Glycopeptides Dalbavancin development story 1 Dalbavancin is a semisynthetic lipoglycopeptide designed to improve upon the natural glycopeptide analogue teicoplanin It was shown to have in vitro activity against a variety of Gram-positive pathogens, including MRSA and MRSE 2005: A once-weekly, two-dose antibiotic acquired by Pfizer (ex-Vicuron Pharmaceuticals). Entered Phase 3 clinical trial for adults with complicated skin infections Dec 2007: the FDA required more data before approval could be granted Sept 2008: Pfizer announced that it will withdraw all marketing applications in order to conduct another Phase 3 clinical trial Dec 2009: Durata Therapeutics acquired the rights to Dalbavancin Glycopeptides Dalbavancin development story 2 Sept 2011: Durata announces second global, pivotal Phase 3 study for the treatment of acute bacterial skin & skin structure infections (abSSSI) “Due to its unique features and PK profile, dalbavancin offers the significant convenience of once-a-week dosing and short, 30-minute infusion time.” “……has the potential to set the bar for activity against important Gram-positive bacterial infections, including those due to MRSA.” Sept 2012: Ongoing phase 3 clinical trials and initial top line data available in 2013… By Feb 2015: Dalbavancin approved for use in USA as Dalvance® for injection Durata Therapeutics bought by Actavis (Nov 2014) March 2015: Dalbavancin approved for use in Europe as XYDALBA® Glycopeptides Eremomycin, telavancin & oritavancin LY 33328 = Oritavancin Glycopeptides Oritavancin development story 1 Originally discovered and developed by Eli Lilly 2001: Acquired by InterMune 2005: acquired by Targanta Therapeutics in 2005 Dec 2008: the FDA decline to approve it, EU application withdrawn 2009: development rights were acquired by The Medicine Company 2012: “Oritavancin is an innovative, investigational hospital-based antibiotic with potent bactericidal (killing) activity against a broad spectrum of Gram-positive bacteria, including staphylococcal strains with resistance to methicillin (MRSA) and vancomycin. The safety and efficacy has not been established by the FDA for any use; however, further clinical studies are expected to commence in 2009 to evaluate oritavancin” Glycopeptides Oritavancin development story 2 2013: Clinical trials for a possible new FDA application Glycopeptides Oritavancin development story 3 August 2014: approved by the FDA for skin infections (USA) ➔ ORBACTIVTM (IV administration) March 2015: approved in Europe http://www.themedicinescompany.com/ Glycopeptides Oritavancin development story 4 December 2019: Melinta Therapeutics files for bankruptcy protection……… April 2020: Melinta emerges from bankruptcy and continues to operate Glycopeptides Pros, Cons & Future(?) Glycopeptides offer very good targeting and selectivity But it is difficult to synthesise new analogues One possible solution is to reduce structural complexity Glycopeptides Oritavancin mechanism of action 1 Thought to have three different mechanisms of action Glycopeptides Oritavancin – 3 MOAs Bacterial cell-wall synthesis Three mechanisms of action: 1. Inhibiting tranglycosylation 2. Inhibiting transpeptidation 3. Disruption of bacterial membrane integrity Glycopeptides Further resources Design and discovery of new antibacterial agents: Advances, perspectives, challenges Jampelik J, Current Medicinal Chemistry 2018, 25, 4972-5006 Glycopeptide antibiotics: Back to the future Butler MS et al., Journal of Antiobiotics 2014, 67,631-644. Recent advances in the synthesis of new glycopeptide antibiotics Ashford PA et al., Chemical Society Reviews 2012, 41, 957-978 New Lipoglycopeptides A Comparative Review of Dalbavancin, Oritavancin and Telavancin Zhanel G et al., Drugs 2010, 70 ,859-886. The half-life of dalbavancin ranges from 147 to 258 hours, which allows for once-weekly dosing, the half-life of oritavancin of 393 hours may allow for one dose per treatment course, while telavancin requires daily administration Impairing translation Stopping protein synthesis Bacteria need to synthesise many different types of protein to survive As in humans this is done via the process of translation mRNA is produced through the process of transcription The mRNA is then “translated” at the ribosome to create the desired protein Impairing translation Ribosomes: Us versus “them” Impairing translation Different drugs act at different stages Impairing translation Tetracyclines These are broad-spectrum, bacteriostatic agents Impairing translation Tetracyclines SAR Structure shows two distinct regions Impairing translation Tetracyclines – ribosomal binding Extensive hydrogen bonding plus metal ion coordination Cytosine-1054 also involved in − stacking with D ring Impairing translation Tetracyclines – development 1 Minocycline and tigecycline Image result for minocycline Impairing translation Tetracyclines – development 2 Aminomethyl derivatives Omadacycline (aka amadacycline) Activity against drug resistant pathogens: MRSA, PRSP and MDRSP, VRE & extended spectrum β-lactamase producing enterobacteriaceae (ESBL) NUZYRATM FDA approved Oct 2018 – CABP & ABSSSI https://paratekpharma.com/science/  Impairing translation Tetracyclines – most recent breakthrough A cost-effective and feasible synthesis!!! Liu & Myers, Current Opinion in Chemical Biology 2016, 32, 48-57. https://doi.org/10.1016/j.cbpa.2016.03.011 Eravacycline (Xerava) first fluoro-tetracycline Granted fast-track approval by FDA https://www.tphase.com/ Impairing translation Tetracyclines & pharmacokinetics Perhaps more important in explaining selectivity, as opposed to selective binding to bacterial ribosomes In Gram-negative bacteria, tetracyclines cross the outer membrane via passive diffusion Their passage across inner membrane depends on a pH gradient, suggesting the involvement of a proton-driven carrier All this leads to a higher concentration more quickly in bacterial cells (compared with mammalian cells) Impairing translation Chloramphenicol Originally isolated from Streptomyces venezuelae Note: two stereo-centres – only one diastereomer is active Impairing translation Chloramphenicol – mechanism of action Binds to the A-site of the 50S ribosomal subunit Image result for oxazolidinones mechanism of action Inhibits movement of ribosome along mRNA (inhibition of peptidyl- transferase) Note: cannot use with macrolides & lincosamides Carbohydrates in medicine Antibacterials – more Biologically active natural products Antibacterials Glycopeptides Aminoglycosides Macrolides & Ketolides Impairing translation Aminoglycosides 1 These are bactericidal agents Streptomycin (1944) Gentamicin C1a (from Streptomyces griseus) (also a natural product) Impairing translation Aminoglycosides 2 Other natural product examples include Neomycin B topical formulations for treatment of skin, ear, eye and nose infections Tobramycin similar activity to gentamicin inhalation formulation for treatment of chronic pulmonary infections (P. aeruginosa) in cystic fibrosis Paromomycin 1950s Orphan drug approval 2005 Leishmaniasis treatment 2006 India Semi-synthetic analogues (1970s onwards) include Amikacin: better stability ( to enzymes) than gentamicin 2nd line defence against MDRTB Impairing translation Aminoglycosides - 3 Aminoglycosides work best at slightly alkaline pH, where they are positively charged This aids absorption through Gram-negative bacterial outer membrane Transport across the cell membrane is driven by an energy-dependent process Drug becomes trapped inside cell and accumulates Impairing translation Aminoglycosides - MOA Three potential mechanisms of action Impairing translation Aminoglycosides – a recent addition Plazomicin (Achaogen) August 2018: FDA approval for treatment of cUTIs But only in patients without other alternatives! April 2019: Achaogen files for bankruptcy Impairing translation Aminoglycosides – a pipeline? Apramycin (Juvabis) Licensed for oral use in animals in 1980 First resistance warning in 1986… Impairing translation Aminoglycosides – resistance 1 This occurs via a number of mechanisms: Aminoglycoside-modifying enzymes (AMEs) Mutations to nucleotides in the target binding site Structural changes to the outer membrane in G- bacteria Gaining of more effective efflux pumps Action of membrane proteases “Mechanisms of resistance to aminoglycoside antibiotics: overview & perspectives” S. Garneau-Tsodikova & K.J. Labby Med. Chem. Commun. 2016, 7, 11-27 Impairing translation Aminoglycosides – resistance 2 Reactions catalysed by “aminoglycoside modifying enzymes” (AMEs) acetylation of amino groups phosphorylation of hydroxyl groups addition of ADP to hydroxyl groups Learning outcomes Now, you should appreciate & understand:- the use of carbohydrates in medicine glycopeptides origin, mechanism(s) of action, development difficulties impairment of bacterial translation processes chloramphenicol tetracyclines origin, mechanism of action, synthesis & development aminoglycosides Origin, mechanisms of action, development difficulties, resistance MSOP1016 Antibacterial Agents 1. Agents that impair bacterial translation (part 2) 2. Agents that impair bacterial transcription & replication 3. Antimicrobial resistance Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text An introduction to medicinal chemistry ISBN: 9780198749691 615.19 PAT (Drill Hall Library) CHAPTER 19 Learning outcomes At the end of this lecture, you should appreciate & understand the chemistry of:- Macrolides & Ketolides Oxazolidinones Fluoroquinolones Aminoacridines Miscellaneous antimicrobials Antimicrobial resistance – a cause for concern Carbohydrates in medicine Antibacterials Biologically active natural products Antibacterials Glycopeptides Aminoglycosides Macrolides & Ketolides Macrolides General background Macrolide classification is based on the aglycone ring size (12-, 14- or 16- membered rings) They contain two monosaccharides which are crucial for activity Erythromycin (1952) (Streptomyces erythreus) One of the safest antibiotics Macrolides Erythromycin mechanism of action Erythromycin binds to the 50S ribosomal subunit, inhibiting translocation extensive van der Waals interactions (exit tunnel) desosamine -OH forms a hydrogen bond to an adenine base Macrolides Stability 1 Erythromycin is unstable to stomach acids ➔ need for “special” oral formulations Sensitivity caused by presence of a ketone and two alcohol groups One method to prevent this is to protect one of the hydroxyl groups ➔ clarithromycin (OMe group) (1991) Macrolides Stability 2 Second method to increase stability is to remove the keto group Example 1: roxithromycin (1987) Introduction of an alkoxyimine functionality Macrolides Stability 3 Second method to increase stability is to remove the keto group Example 2: azithromycin (1986) Incorporation of an N-methyl group ➔ 15-membered ring Ketolides General background Semi-synthetic derivatives of 14-membered ring macrolides Cladinose sugar (position 3 in erythromycin) removed and replaced with a keto group Nilius, A. M.; Ma, Z. K., Current Opinion in Pharmacology 2002, 2, 493-500. Ketolides Telithromycin Note the masking of OH groups Position 6 as OMe Position 12 as a (cyclic) carbamate Ketek - once a day, oral European market 2001 FDA 2004 FDA restrictions 2007 Ketolides Cethromycin 1 Restanza – once a day, oral 2009: proven safe, but NOT YET approved by FDA for CAP Ketolides Cethromycin 2 Restanza – once a day, oral Shown to have higher in vitro potency and a broader range of activity than macrolides against Gram-positive bacteria associated with respiratory tract infections Appears to be effective against penicillin-, macrolide- and fluoroquinolone-resistant bacteria Also being investigated for the prophylactic treatment of inhalation of anthrax post-exposure (and other high priority biodefense pathogens, including plague and tularemia) Ketolides Cethromycin 3 Restanza FDA gave orphan drug status for treatment after exposure to anthrax Orphan drug a pharmaceutical agent developed specifically to treat a rare medical condition a matter of public policy in many countries allows for medical breakthroughs that may not have otherwise been achieved (economics!) by conventional drug discovery routes Easier to gain marketing approval for an orphan drug (USA/EU) There may also be other financial incentives, such as extended exclusivity periods Ketolides Solithromycin 1 Currently in Phase III clinical trials (but only in Japan, NDA April 2019) Key structural elements (vis-à-vis telithromycin) 1,2,3-triazole (not imidazole) ➔ improved metabolic stability/higher bioavailability Aminobenzene (not pyridine) ➔ reduced side effects (visual/muscular/liver) 2-F (not Me) ➔ third ribosomal binding site (expected to limit resistance development) Impairing translation Oxazolidinones Linzolid This is a relatively new class of antibacterial agents Linezolid Reached the market in 2000 Sales of £716 million/year by 2010 Oxazolidinones Mechanism of action 1 They bind to the 50S sub-unit and prevent it from combining with 30S sub-unit to give the full bacterial ribosome unit No formation of the 70S ribosome needed for translation They do not suffer same resistance problems Oxazolidinones Mechanism of action 2 Binding modes have been revealed through X-ray crystallography Mainly van der Waals interactions & p-p stacking Acetamide interacts with RNA backbone (H-bond) Oxazolidinones Newer derivatives Tedizolid (approved 2014) higher potency pyridine & tetrazole groups ➔ additional interactions ABSSSIs Radezolid (clinical trials) higher potency (10000 x more than linezolid) additional interactions Qualified infectious disease product – bacterial vaginosis Melinta Therapeutics… Inhibiting transcription & replication (Fluoro)quinolones 1 Nalidixic acid (1962) was the first therapeutically useful quinolone derivative Breakthrough in the 1980s with enoxacin, then norfloxacin and ciprofloxacin (most active of the class against Gram-negative bacteria) Inhibiting transcription & replication (Fluoro)quinolones 2 Ciprofloxacin synthesis Inhibiting transcription & replication (Fluoro)quinolones 3 Mechanism of action Stabilises DNA-topoisomerase complexes ➔ Inhibition of replication & transcription Gram-positive: topoisomerase IV Gram-negative: topoisomerase II (DNA-gyrase) Inhibiting transcription & replication (Fluoro)quinolones – generation 3/4 Began to be developed in 1990s to overcome issues Finafloxacin was approved in 2014 Inhibiting transcription & replication Aminoacridines 1 An example is proflavin Topical agents (used a lot in WWII) The best example in this class are completely ionised at pH 7 They interact through direct intercalation with bacterial DNA Not suitable for systemic delivery – toxic to the host cells! Inhibiting transcription & replication Aminoacridines 2 Mechanism of action Inhibiting transcription & replication Nitrofurantoin and nitroimidazoles 1 Nitrofurantoin (1953) UTIs Metronidazole (1959) Originally used as an anti-protozoal Anti-bacterial use from the 1970s Inhibiting transcription & replication Nitrofurantoin and nitroimidazoles 2 Mechanism of action Nitro group is reduced in bacterial cell ➔ concentration gradient established Free radicals that are toxic to the bacterial/protozoal cell are produced Antimicrobial resistance An apocalypse? https://www.weforum.org/agenda/2016/09/this-is-how-many-people-will-die-from-antimicrobial-resistance-every-year-by-2050-if-nothing-is-done/ Antimicrobial resistance Dark ages? Antimicrobial resistance Which bacteria? Inhibiting transcription & replication Priority pathogens 1 Inhibiting transcription & replication Priority pathogens 2 Antimicrobial resistance A new thing? “It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body.” “The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and, by exposing his microbes to non-lethal quantities of the drug, make them resistant” Antimicrobial resistance How does it happen? https://www.cdc.gov/antibiotic-use/community/images/how-AR-happens.jpg Antimicrobial resistance Spreading? https://www.cdc.gov/antibiotic- use/community/images/howAR- spreads.jpg Antimicrobial resistance Pathways? https://doi.org/10.1016/j.bmcl.2017.08.027 Antimicrobial resistance Mutation Antimicrobial resistance Genetic transfer: transduction Antimicrobial resistance Genetic transfer: conjugation Antimicrobial resistance Other factors Popularity Variance Learning outcomes Now, or on reflection, you should appreciate & understand the chemistry of:- Macrolides & Ketolides Oxazolidinones Fluoroquinolones Aminoacridines Miscellaneous antimicrobials Antimicrobial resistance – a cause for concern MSOP1016 Advanced Skills & Techniques (Analytical) Dr Andrew J Hall Senior Lecturer in Chemistry Room A120, Anson Building [email protected] Recommended text CHAPTERS 14 & 16 High-performance capillary electrophoresis Methods used in the quality control of biotechnologically produced drugs Learning outcomes At the end of this lecture, you should appreciate/understand the principle behind a selection of advanced instrumental techniques and how they are used in the analysis of biotechnologically-produced drugs Electrophoretic techniques Size Exclusion Chromatography Binding assays Protein sequencing using the Edman reaction Knowledge of protein structure is taken to be already understood Proteinaceous drugs “Old” peptide drugs Peptide Physical Source Therapeutic action(s) characteristics Glucagon 30 amino acids Bovine or Reversal of insulin-induced porcine hypoglycaemia pancreas or synthetic Insulin 51 amino acids Porcine thyroid, Diabetes 2 chains linked by 2 salmon or sulfide bridges synthesis Human growth 191 amino acids Human Treatment of short stature due hormone pituitary gland to hormone deficiency Vaccines High molecular Infective Stimulation of immune system weight proteins organisms to develop B-lymphocyte memory cells Immunoglobulins 150 kDa or greater Pooled human Antibody mixture offering short- blood term resistance to pathogens, e.g. hepatitis A Proteinaceous drugs Some best-selling protein drugs (2018) Brand name Production Company Sales Indications method ($ billions) Humira CHO AbbVie 20.2 Rheumatoid and (adalimumab) psoriatic arthritis, Crohn’s disease, ulcerative colitis Avastin CHO cells Roche 6.4 Metastatic colorectal, (bevacizumab) non-small-cell lung, glioblastoma & metastatic kidney cancers Herceptin CHO cells Roche 6.4 HER2-positive breast and (trastuzumab) metastatic gastric cancers Remicade Mouse myeloma Johnson & 6.3 Crohn’s disease, (infliximab) cells Johnson ulcerative colitis, rheumatoid arthritis Keytruda CHO cells Merck 6.1 Melanoma, non-small- (prembrolizamab) cell lung cancer Capillary electrophoresis Principles Analyte separation is achieved by applying a high potential (10-30 kV) to a fused-silica capillary tube filled with mobile phase The mobile phase generally contains an aqueous component and MUST contain an electrolyte Analytes migrate in the applied electric field at rates depending on their charge and their ionic radius Neutral analytes also migrate through the capillary column due to electro-osmotic flow (usually towards the cathode) Capillary electrophoresis Applications Accurate and precise technique for drug quantification in all types of formulation Particularly useful in quality control of peptide drugs Highly selective and very effective in separating enantiomers Very effective for impurity profiling due to high resolving power Very effective for analysing drugs/metabolites in biological fluids Capillary Electrophoresis Some theoretical background I Separation achieved by differences in analyte velocities in an electric field, with ion velocity (n) given by n = eE e is electrophoretic mobility E is the applied electric field e = Electric force (FE) / Frictional drag (FF) FE = q E where q is the charge on the molecule For spherical ions FF = -6   r where h is the viscosity of the medium used, r is the ion radius and n is the ion velocity Capillary Electrophoresis Some theoretical background II When the frictional drag and the electric field experienced by the ion are equal, i.e. FE = FF qE = -6  r If we substitute this into the first equation and re-arrange, we get e = q / 6   r If the applied electric field is increased beyond the point where FE = FF the ion will begin to migrate Capillary Electrophoresis Consequence of the theory e = q / 6   r The greater the charge on the ion, the higher its mobility The smaller the ion, the greater its mobility As the equation relates to a spherical ion, the more closely an ion approximates to a sphere (lower surface area), the greater is the mobility Capillary Electrophoresis Further theoretical considerations Ion mobility can be affected by the analyte’s pKa value – the more ionised the analyte, the greater its mobility molecular shape in solution Beware: degree of ionisation can have a bearing on the shape in solution ➔ potential for behaviour to be complex! Variation in pH is only one part of the story regarding CE separations… Capillary Electrophoresis Electro-osmotic flow (EOF) Consider the wall of the fused-silica capillary to have similar chemistry to surface of silica gel Laminar flow caused by drag (HPLC) Electro-osmotic flow Capillary Electrophoresis Ion migration in CE As there is EOF, everything will move towards the cathode (regardless of charge) Cations: rate of movement = ion mobility + EOF Neutral: rate of movement = EOF Anions: rate of movement = EOF - ion mobility Rate of EOF > rate at which anions move towards the anode by ca. 10 times Capillary Electrophoresis Ion migration in CE - schematic Capillary Electrophoresis Migration in CE Variable Effects on EOF Comments Buffer pH EOF increases with pH Convenient method for controlling EOF Buffer strength EOF decreases with Higher ionic strength ➔ increased current increasing buffer strength ➔ heating Low ionic strength ➔ greater analyte absorption to capillary wall Temperature Increased temperature Easy to control reduces viscosity ➔ increased flow Electric field Increased electric field Lowering applied field my reduce efficiency, increases EOF raising it may cause heating Surfactant Absorbs to capillary wall Depends on nature of surfactant and surface concentration, but cationic surfactants can reduce EOF or reverse flow towards the anode Anionic surfactants increase EOF Covalent wall Can raise or lower EOF Neutral coatings reduce EOF, while ionic coating depending on coating coatings will have marked effects on EOF Capillary Electrophoresis Instrumentation Capillary Electrophoresis Controlling separation Migration time Dispersion Longitudinal diffusion Injection plug length Joule heating Solute/wall interactions Electro-dispersion Capillary Electrophoresis Example 1: Separation of NSAIDs based on ionic radius 1 = sulindac 2 = indomethacin 3 = piroxicam 4 = tiaprofenic acid 5 = aceclofenac Buffer: 0.075 M glycine adjuste to pH 9.1 with triethanolamine Bechet et al. 1994 J Pharm Biomed Anal 13, 497-503 Capillary Electrophoresis Example 2: Separation of proteins Capillary electrophoresis of human cerebrospinal fluid from (A) a patient with multiple sclerosis approaching the malignant phase, (B) a patient with cerebral infarction in the recovery phase, and (C) a patient with neurosis with no organic damage in the central nervous system. Peak a, g-globulin; b, b2-globulin; c, b1-globulin; d, a2-globulin; e, a1-globulin; f, albumin; g, prealbumin. Huck & Bonn from Methods in Molecular Biology, 384: Capillary Electrophoresis Capillary Electrophoresis Example 3: Comparison of HPLC-MS and HPCE-MS Aviptadil: synthetic injectable formulation of human vasoactive intestinal peptide Treatment of acute respiratory distress syndrome, asthma & COPD Stefanik et al. 2023 https://doi.org/10.1002/elps.202300141 Capillary electrophoresis Strengths and limitations Potentially many times more efficient than HPLC in separating power Shorter analysis times than in HPLC Cheaper columns than in HPLC Negligible solvent consumption Robustness needs to be improved More parameters ➔ more optimisation required than in HPLC Electrophoresis Sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) The principal of migration is same as in capillary electrophoresis All proteins pick up the same proportion of SDS on a w/w basis ➔ identical negative charge regardless of their size But we’ve added a gel so the smaller the protein is, the easier it will move through the gel network under influence of applied electric field Electrophoresis SDS-PAGE apparatus Electrophoresis Example of an SDS-PAGE separation Different Recombinant Human Erythropoietin Copy Products Halim et al. 2014 Pharmaceutical Research 31, 1210-1218 Electrophoresis Isoelectric focusing SDS-PAGE is a rather low-resolution technique Isoelectric focusing is a variation of SDS-PAGE using the same gel system BUT the protein mixture is not pre-treated with SDS Gel is prepared so that there is a pH gradient across the gel Separation occurs based on the pI values of the proteins A protein migrates under the influence of the applied electric field to a particular point (pH) where it is charge neutral. The stains used in SDS-PAGE are used for visualisation Electrophoresis An example of separation using isoelectric focusing Analysis of glycoforms of generic EPO from various sources Federici et al. 2013 Biologicals 41, 131-137 Electrophoresis Real-world pharmacopoeial analyses Alteplase glycoform analysis Molgramostim identification test Monoclonal antibody testing Interferon alfa-2 analysis Characterisation of allergens used in allergy testing SEC Separation based on molecular size Larger molecules elute first! SEC Separation of serum proteins Performed using a (diol SEC column) Binding assays Enzyme-linked immunosorbent and radio- immunoassays (ELISA/RIA) Each technique uses an antibody to the protein drug Antibody binds specifically to the protein drug The amount of antibody bound is then quantified by measuring enzyme activity radioactivity Downside: can suffer from a lack of specificity Binding assays Different types of ELISA Binding assays Radio-immunoassay (RIA) Concept is simpler than for ELISA Radio-labelled version of the antigen is used (typically using radioactive iodine – undergoes ready reaction with tyrosine residues) Mix radiolabelled antigen with equivalent amount of specific antibody Add sample of antigen to be analysed (this is unlabelled) Competition between labelled and unlabelled antigen ➔ proportional displacement of radiolabelled version The amount of displaced radiolabelled antigen is measured in the supernatant solution after precipitation of the antibody-antigen complex ELISA/RIA examples Real-world pharmacopoeial analyses ELISA Checking for contaminated proteins in a number of products: Hepatitis B surface antigen in factor VIII Identification of antigenic components in adsorbed pertussis vaccine Identification of antigenic components in polio vaccine Determining effectiveness of foot and mouth disease vaccine RIA Determination of pro-insulin in human, porcine and bovine insulin Identification of hepatitis surface B surface antigen in urokinase and fibrinogen Protein sequencing The Edman reaction Protein sequencing Sequencing of halicylindramide C Hahn et al. 2022 Scientific Reports 12, 10285 Learning outcomes Take-home points You should now appreciate/understand the principles behind Electrophoretic techniques SEC Binding assays Protein sequencing using the Edman reaction and how they are used in the analysis of biotechnologically-produced drugs