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Beta-Lactam Antibiotics

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  1. Beta-Lactam Antibiotics Clinically Important β-Lactam Antibiotics Medicinal Chemistry Presentation David McLeod Southern Methodist University

  2. Introduction β-Lactam antibiotics are the most widely produced and used antibacterial drugs in the world, and have been ever since their initial clinical trials in 1941. β-Lactams are divided into several classes based on their structure and function; and are often named by their origin, but all classes have a common β-Lactam ring structure.

  3. History 1928- Alexander Fleming discovers a mold which inhibits the growth of staphylococcus bacteria 1940- penicillin is isolated and tested on mice by researchers at Oxford 1941- penicillin mass produced by fermentation for use by US soldiers in WWII 1950’s- 6-APA is discovered and semi-synthetic penicillins are developed. 1960’s to today- novel β-lactams/β-lactamase inhibitors are discovered and modified from the natural products of bacteria

  4. Target- Cell Wall Synthesis The bacterial cell wall is a cross linked polymer called peptidoglycan which allows a bacteria to maintain its shape despite the internal turgor pressure caused by osmotic pressure differences. If the peptidoglycan fails to crosslink the cell wall will lose its strength which results in cell lysis. All β-lactams disrupt the synthesis of the bacterial cell wall by interfering with the transpeptidase which catalyzes the cross linking process.

  5. Peptidoglycan Peptidoglycan is a carbohydrate composed of alternating units of NAMA and NAGA. The NAMA units have a peptide side chain which can be cross linked from the L-Lys residue to the terminal D-Ala-D-Ala link on a neighboring NAMA unit. This is done directly in Gram (-) bacteria and via a pentaglycine bridge on the L-lysine residue in Gram (+) bacteria.

  6. Mechanism

  7. Transpeptidase- PBP The cross linking reaction is catalyzed by a class of transpeptidases known as penicillin binding proteins A critical part of the process is the recognition of the D-Ala-D-Ala sequence of the NAMA peptide side chain by the PBP. Interfering with this recognition disrupts the cell wall synthesis. β-lactams mimic the structure of the D-Ala-D-Ala link and bind to the active site of PBPs, disrupting the cross-linking process.

  8. Mechanism of β-Lactam Drugs The amide of the β-lactam ring is unusually reactive due to ring strain and a conformational arrangement which does not allow the lone pair of the nitrogen to interact with the double bond of the carbonyl. β-Lactams acylate the hydroxyl group on the serine residue of PBP active site in an irreversible manner. This reaction is further aided by the oxyanion hole, which stabilizes the tetrahedral intermediate and thereby reduces the transition state energy.

  9. Mechanism of β-Lactam Drugs The hydroxyl attacks the amide and forms a tetrahedral intermediate.

  10. Mechanism of β-Lactam Drugs The tetrahedral intermediate collapses, the amide bond is broken, and the nitrogen is reduced.

  11. Mechanism of β-Lactam Drugs The PBP is now covalently bound by the drug and cannot perform the cross linking action.

  12. Bacterial Resistance Bacteria have many methods with which to combat the effects of β-lactam type drugs. Intrinsic defenses such as efflux pumps can remove the β-lactams from the cell. β-Lactamases are enzymes which hydrolyze the amide bond of the β-lactam ring, rendering the drug useless. Bacteria may acquire resistance through mutation at the genes which control production of PBPs, altering the active site and binding affinity for the β-lactam .

  13. Range of Activity β-Lactams can easily penetrate Gram (+) bacteria, but the outer cell membrane of Gram (-) bacteria prevents diffusion of the drug. β-Lactams can be modified to make use of import porins in the cell membrane. β-Lactams also have difficulty penetrating human cell membranes, making them ineffective against atypical bacteria which inhabit human cells. Any bacteria which lack peptidoglycan in their cell wall will not be affected by β-lactams.

  14. Toxicity β-Lactams target PBPs exclusively, and because human cell membranes do not have this type of protein β-lactams are relatively non toxic compared to other drugs which target common structures such as ribosomes. About 10% of the population is allergic (sometimes severely) to some penicillin type β-lactams.

  15. Classes of β-Lactams The classes of β-lactams are distinguished by the variation in the ring adjoining the β-lactam ring and the side chain at the α position. Penicillin

  16. Modification of β-Lactams β-Lactam type antibiotics can be modified at various positions to improve their ability to: -be administered orally (survive acidic conditions) -be tolerated by the patient (allergies) -penetrate the outer membrane of Gram (-) bacteria -prevent hydrolysis by β-lactamases -acylate the PBPs of resistant species (there are many different PBPs)

  17. Penicillins- Natural Natural penicillins are those which can be obtained directly from the penicillium mold and do not require further modification. Many species of bacteria are now resistant to these penicillins. Penicillin G not orally active

  18. Penicillin G in Acidic Conditions Penicillin G could not be administered orally due to the acidic conditions of the stomach.

  19. Penicillin V Penicillin V is produced when phenoxyacetic acid rather than phenylacetic acid is introduced to the penicillium culture. Adding the oxygen decreases the nucleophilicity of the carbonyl group, making penicillin V acid stable and orally viable.

  20. Production All commercially available β-lactams are initially produced through the fermentation of bacteria. Bacteria assemble the penicillin molecule from L-AAA, L-valine, and L-cysteine in three steps using ACV synthase, IPN synthase, and acyltransferase. Modern recombinant genetic techniques have allowed the over expression of the genes which code for these three enzymes, allowing much greater yields of penicillin than in the past.

  21. Penicillin Biosynthetic Pathway

  22. o

  23. Semi-Synthetic Penicillins The acyl side chain of the penicillin molecule can be cleaved using enzyme or chemical methods to produce 6-APA, which can further be used to produce semi-synthetic penicillins or cephalosporins 75% of the penicillin produced is modified in this manner.

  24. Penicillins- Antistaphylococcal Penicillins which have bulky side groups can block the β-Lactamases which hydrolyze the lactam ring.

  25. Penicillins- Antistaphylococcal These lactamases are prevalent in S.aureus and S.epidermidis, and render them resistant to Penicillin G and V. This necessitated the development of semi-synthetic penicillins through rational drug design. Methicillin was the first penicillin developed with this type of modification, and since then all bacteria which are resistant to any type of penicillin are designated as methicillin resistant. (MRSA- methicillin-resistant S. aureus)

  26. Penicillins- Antistaphylococcal Methicillin is acid sensitive and has been improved upon by adding electron withdrawing groups, as was done in penicillin V, resulting in drugs such as oxacillin and nafcillin. Due to the bulky side group, all of the antistaphylococcal drugs have difficulty penetrating the cell membrane and are less effective than other penicillins.

  27. Penicillins- Aminopenicillins In order to increase the range of activity, the penicillin has been modified to have more hydrophilic groups, allowing the drug to penetrate into Gram (-) bacteria via the porins. Ampicillin R=Ph Amoxicillin R= Ph-OH

  28. Penicillins- Aminopenicillins These penicillins have a wider range of activity than natural or antistaphylococcal drugs, but without the bulky side groups are once again susceptible to attack by β-lactamases The additional hydrophilic groups make penetration of the gut wall difficult, and can lead to infections of the intestinal tract by H. pylori

  29. Penicillins- Aminopenicillins Due to the effectiveness of the aminopenicillins, a second modification is made to the drug at the carboxyl group. Changing the carboxyl group to an ester allows the drug to penetrate the gut wall where it is later hydrolyzed into the more polar active form by esterase enzymes. This has greatly expanded the oral availability of the aminopenicillin class.

  30. Penicillins- Extended Spectrum Extended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amine

  31. Penicillins- Extended Spectrum Like the aminopenicillins the extended spectrum drugs have an increased activity against Gram (-) bacteria by way of the import porins. These drugs also have difficulty penetrating the gut wall and must be administered intravenously if not available as a prodrug. These are more effective than the aminopenicillins and not as susceptible to β-lactamases

  32. Cephalosporins Cephalosporins were discovered shortly after penicillin entered into widespread product, but not developed till the 1960’s. Cephalosporins are similar to penicillins but have a 6 member dihydrothiazine ring instead of a 5 member thiazolidine ring. 7-aminocephalosporanic acid (7-ACA) can be obtained from bacteria, but it is easier to expand the ring system of 7-APA because it is so widely produced.

  33. Cephalosporins Unlike penicillin, cephalosporins have two side chains which can be easily modified. Cephalosporins are also more difficult for β-lactamases to hydrolyze.

  34. Mechanism of Cephalosporins The acetoxy group (or other R group) will leave when the drug acylates the PBP.

  35. Cephalosporins- Classification Cephalosporins are classified into four generations based on their activity. Later generations generally become more effective against Gram (-) bacteria due to an increasing number of polar groups (also become zwitterions.) Ceftazidime (3rd gen) in particular can cross blood brain barrier and is used to treat meningitis. Later generations are often the broadest spectrum and are reserved against penicillin resistant infections to prevent the spread of cephalosporin resistant bacteria.

  36. Carbapenems Carbapenems are a potent class of β-lactams which attack a wide range of PBPs, have low toxicity, and are much more resistant to β-lactamases than the penicillins or cephalosporins.

  37. Carbapenems Thienamycin, discovered by Merck in the late 1970’s, is one of the most broad spectrum antibiotics ever discovered. It uses import porins unavailable to other β-lactams to enter Gram (-) bacteria. Due to its highly unstable nature this drug and its derivatives are created through synthesis, not bacterial fermentation.

  38. Carbapenems Thienamycin was slightly modified and marked as Imipenem. Due to its rapid degradation by renal peptidase it is administered with an inhibitor called cilastatin under the name Primaxin. Imipenem may cause seizures or sever allergic reactions. Other modifications of Thienamycin have produced superior carbapenems called Meropenem and Ertapenem, which are not as easily degraded by renal peptidase and do not have the side effects of Imipenem.

  39. Monobactams The only clinically useful monobactam is aztreonam. While it resembles the other β-lactam antibiotics and targets the PBP of bacteria, its mechanism of action is significantly different. It is highly effective in treating Gram (-) bacteria and is resistant to many β-lactamases

  40. β-Lactamases β-Lactamases were first discovered in 1940 and originally named penicillinases. These enzymes hydrolyze the β-lactam ring, deactivating the drug, but are not covalently bound to the drug as PBPs are. Especially prevalent in Gram (-) bacteria. Three classes (A,C,D) catalyze the reaction using a serine residue, the B class of metallo-β-lactamases catalyze the reaction using zinc.

  41. β-Lactamase Inhibitors There are currently three clinically available β-lactamase inhibitors which are combined with β-lactams; all are produced through fermentation. These molecules bind irreversibly to β-lactamases but do not have good activity against PBPs. The rings are modified to break open after acylating the enzyme.

  42. β-Lactam/Inhibitor combinations Aminopenicillins: ampicillin-sulbactam = Unasyn amoxicillin-clavulante = Augmentin Extended-Spectrum Penicillins piperacillin-tazobactam = Zosyn ticarcillin-clavulanate = Timentin

  43. Summary β-Lactam antibiotics have dominated the clinical market since their introduction in the 1940’s and today consist of nearly ¾ of the market. Development of natural products such as penicillin G into more potent forms through rational modification has increased the range of activity of these drugs, although this has led to some toxicity problems. Widespread use of β-lactams has led to the development of resistant strains, new modifications are necessary in order for β-lactams to remain viable.

  44. Assigned reading: Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition. New York: Oxford University Press, 2009. 388-414. Print.

  45. Optional References/ Reading Brunton, Laurence L. et al. Goodman and Gillman’s Pharmaceutical Basis of Therapeutics 11th Edition. McGraw-Hill, 2006 1134- 52. Print. Bush, Karen. β-Lactamase Inhibitors from Laboratory to Clinic. Clinical Microbiology Reviews, Jan. 1988, p. 109-123. Web. Elander, R.P. Industrial production of β-Lactam antibiotics. Journal of Applied Microbiology and Biotechnology (2003) 61:385–392. Web. Hauser, Alan R. Antibiotic Basics for Clinicians: Choosing the Right Antibacterial Agent. Philadelphia: Lippincott, 2007. 18-46. Print. Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition. New York: Oxford University Press, 2009. 388-420. Print. Rolinson, George N. Forty years of β-lactam research. Journal of Antimicrobial Chemotherapy (1998) 41, 589–603. Web.

  46. Questions 1. What are two ways by which a bacteria could become resistant to carbapenems? 2. How were the natural penicillins modified to be orally available? 3. How are extended spectrum penicillins modified to be orally available? 4. What are two ways that the β-lactam can be protected from β-lactamases?