Penicillin G and Penicillin Binding Protein 4: A Patient Case Related to Medicinal Chemistry and Drug Design
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A 26-year-old male was admitted to St. Vincent Hospital complaining of a cough, fever, and general fatigue. An echocardiogram and lab test showed the patient has infective endocarditis caused by Staphylococcus aureus. The man had been admitted to the hospital on multiple occasions for poor heath associated with IV drug use, which was believed to be the source of the infection as well. The physician ordered Penicillin G potassium, 4 million units IV every 4 hours. The pharmacist noticed the patient is also taking warfarin (an oral anticoagulant) for a previous condition and knew that Penicillin G can enhance the effect of warfarin. However, the antibiotic was needed to save the man's life, so the medication order was approved, along with a note to closely monitor the patient's International Normalized Ratio due to the drug-drug interaction. International Normalized Ratio, or INR, is a standardized measure of how long it takes a patient's plasma to clot. (1)
Penicillin is often described as one of the most important discoveries that the medicinal world has seen. It is quite ironic that this grand discovery was made by chance after Alexander Fleming returned to his lab after a vacation in 1928 to discover a mold inhibiting some microbial growth in a Petri dish he would left there. It took Fleming years of work for anyone to even see any potential in penicillin - clinical trials were not even started until 1941.(2)
Penicillin is a beta-lactam antibiotic, and thus inhibits bacterial wall synthesis in gram positive organisms. The characteristic structure of all beta-lactam antibiotics is the beta-lactam ring. Over the years, many gram positive bacteria have become resistant to penicillins. These resistant bacteria secrete beta-lactamases and use them to cleave the beta-lactam ring of penicillin through hydrolysis. Chemists have created new families of beta-lactams that have a less risk of being cleaved by beta-lactamases. Therefore, penicillin itself is not often used anymore.(3)
The patient from our case presented with infective endocarditis caused by Staphylococcus aureus. This infection was related to drug use. This makes sense, because S. aureus can be found on the skin as normal flora.(4) When a person fails to use sterile technique when using a needle to inject themselves, the bacteria on the skin goes straight into the bloodstream and to the right side of the heart.(5) This strain of S. aureus apparently did not secrete beta-lactamases, and therefore penicillin was used to treat it. Penicillin G tablets are poorly orally bioavailable, and the capsules are not much better. Therefore, it makes sense that our patient received Penicillin G by the intravenous route. Penicillin G does not readily cross the blood brain barrier, so there was no need to worry about neurological side effects. Penicillin G is partially metabolized in the liver, and mainly excreted by the kidney. Our patient had sufficient liver and kidney function, so no dosage adjustments were required. The half-life of penicillin is a little less than an hour, so every 4 hour dosing makes sense in our patient.(6)
Although our patient's infection was caused by S. aureus, there has been more research conducted on E. coli with regard to penicillin binding proteins, specifically penicillin binding protein 3 (PBP3). S. aureus and E. coli both contain PBP3 and penicillin binding protein 4 (PBP4), so the interactions with the drug are very similar. Therefore, E. coli and PBP3 will be further discussed in this section. E. coli has seven proteins that form a covalent bond with penicillin. The higher molecular weight binding proteins are essential for the cell, so these are the sites for beta-lactam antibiotics to inhibit. In particular, PBP3 is critical for the separation of dividing bacteria, so it inhibits the synthesis of bacterial walls.
The active site of the PBP3 in E. coli was found to be the amino acids numbers 298-310 in the sequence. This was determined since a major radioactive peak was seen at these points in the protein. The sequence is Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys. Serine-307 is the specific residue that forms a covalent bond with peptide substrates and beta-lactam antibiotics.(7) This covalent bond forms an acyl enzyme intermediate, but the beta-lactam ring inhibits the carboxy terminal end of the beta-lactam from being removed. Therefore, the incoming amino terminal end of the adjacent peptide cannot attack the acyl enzyme intermediate, and the transpeptidase cannot continue. This leads to autolysis. Lysine-310 is hypothesized to interact with the carboxyl group of the antibiotics. This particular section of the protein: serine-X-X-lysine is vital to the function of penicillin. As the transpeptidase active site of PBP3, this structure dictates the ability of the protein to react with enzymes in the cell membrane which are needed for cell wall biosynthesis and crosslinking of the peptidoglycan layer.(3)
As stated in the patient case, the only medication that the patient was taking that had drug-drug interaction potential was warfarin. Beta-lactam antibiotics, such as penicillin G, and multiple other classes of antibiotics can potentiate the effects of warfarin and lead to bleeding risks. Although the use of penicillin G and warfarin together are not absolutely contraindicated, the drug-drug interaction is considered to be major. The mechanism of action of warfarin is to inhibit synthesis of clotting factors that are dependent on vitamin K. Vitamin K is mainly synthesized by E. coli and B. fragilis in the intestines.(8) Penicillin G has the ability to bind to both E. coli and B. fragilis through PBP3 or PBP4 and the amino acid interactions discussed above, inhibiting their growth.(9) When penicillin inhibits intestinal bacteria from producing vitamin K in the intestines, even less clotting factors are synthesized. This leads to potentiated effects of warfarin, an increased INR in the patient, and an increased risk of bleeding. This interaction was studied and confirmed in a controlled case study, which concluded that 'patients on warfarin who received any antibiotic are twice as likely to be hospitalized for bleeding compared with matched controls on warfarin who were not exposed to antibiotics.' (10) Following is a further discussion of the interaction between PBP and penicillin G.
Penicillin forms an irreversible covalent bond with PBP4. This acyl bond formation permanently inactivates the protein, and thus prevents it from its functions related to cell wall formation.(7) This deactivation is the function that inevitably kills the bacterial cell. PBP4 consists of three domains, with the active serine SER62 residue lying within domain one. Along with the active serine residue, there are several other residues that contribute to the binding pocket of PBP4 via hydrogen bonding. The residues aiding in the binding pocket include PHE160, SER306, ASN308, THR418, and SER420.
The various hydrogen bonds at a distance of six angstroms from the center of penicllin are responsible for the structure of the binding site as well as many of the interactions between pencillin and PBP4 as follows. The carboxylic acid of the pencillin hydrogen bonds with SER420 of PBP4. The benzene ring of penicillin bonds with PHE160. The nitrogen as part of the ring bonds SER306. One of the many carbonyls bonds with ASN308 while another bonds THR418. (11)
As previously discussed, penicillin covalently binds to PBP4 receptors within various microbes, including S. aureus and E. coli, which leads to weakened cell walls and, ultimately, autolysis. Domain I of S. aureus PBP4 contains both the N-terminal and the C-terminal ends. The N-terminus participates in transpeptidase activity, which includes a five-stranded antiparallel β-sheet positioned between two helical clusters.(12) One helical cluster has seven helices and the other helical cluster has two helices. The transpeptidase activity is based on the active serine residue (serine-X-X-lysine) located in the larger α-helix of the N-terminal. The C-terminal is primarily made up of two antiparallel β-sheets, which are responsible for carboxypeptidase activity and binding penicillin based on serine-X-asparagine and lysine-threonine(serine)-glycine residues.(12) These three main residues make up the active site of PBP4. When penicillin reacts with these residues it can lead to particularly precarious situations with medications such as warfarin, as illustrated in the aforementioned patient case. Warfarin action relies heavily on stable levels of vitamin K within the body as its mechanism antagonizes vitamin K epoxide reductase, which inhibits the synthesis of clotting factors II, VII, IX and X. Normal flora within the intestines, including E.coli, synthesizes vitamin K which is absorbed through the intestinal lumen.(13) As penicillin binds to PBP4 receptors present in E. coli, the cell wall is weakened to the point of autolysis, yielding less microbes and less vitamin K synthesis and absorption. This reduction of vitamin K in the patient's body results in toxic levels of warfarin, leading to spikes in INR values with respect to a typical goal range of 2 to 3. While this drug-drug interaction is not direct, the consequences may be severe. Increased levels of free warfarin in the body, without Vitamin K to counter its anticoagulant effects, further inhibits the clotting cascade, leading to an increased risk of hemorrhagic events. The patient presented earlier received orders from the pharmacist for strict monitoring due to this indirect interaction and its effects on bleeding risks.
1. Black, JG. Microbiology: Principles and Explorations. 6th ed. Hoboken, NJ: John Wiley & Sons, Inc; 2005. 686.
2. Ligon BL. Penicillin: its discovery and early development. Pediatr. Infect. Dis. J. 2004; 15(1): 52-57.
3. Golan DE, Tashjiaan AH, Armstrong EH, Armstrong AW, eds. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. 3rd ed. Baltimore, MD: Lippincott Williams & Wilkins; 2012. 609-610.
4. Baron S, eds. Medical Microbiology. 4th ed. Galveston, TX: University of Texas Medical Branch at Galveston; 1996. 78-80.
5. Ashley EA, Niebauer J. Cardiology Explained. London: Remedica; 2004. 167-170.
6. Micromedex Healthcare Series [Internet database]. Greenwood Village, Colo: Thomson Reuters (Healthcare) Inc. Updated periodically. Accessed January 25, 2014.
7. Hirota Y, Nicholas RA, Strominger JL, Suzuki H. Identification of the active site in penicillin-binding protein 3 of Escherichia coli. J. Bacteriol. 1985; 164(1): 456.
8. Bentley R, Meganathan R. Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol. Rev. 1982; 46(3): 273-274.
9. Ayala J, Criado J, Quesada A, Piriz S, Vadillo S. Penicillin-binding proteins of Bacteroides fragilis and their role in the resistance to imipenem of clinical isolates. J. Med. Microbiol. 2005; 54(11): 1055-1064.
10. Baillargeon J, Holmes HM, Lin YL et al: Concurrent use of warfarin and antibiotics and the risk of bleeding in older adults. Am J Med. 2012; 125(2):183-189.
11. PDB ID: 2EX8. Kishida H, Unzai S, Roper DI, Lloyd A, Park SY, Tame JRH. Crystal structure of penicillin binding protein 4 (dacB) from Escherichia coli, both in the native form and covalently linked to various antibiotics. J. Biochem. 2006; 45: 783-792.
12. Navratna V, Nadig S, Sood V, Prasad K, Arakere G, Gopal B. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance. J. Bacteriol. 2010; 192(1): 134-144.
13. Ramakrishna B. Nutrition and the digestive system: role of the gut microbiota in human nutrition and metabolism. J. Gastroenterol. Hepatol. 2013; 28(4): 9-17.