Ertapenem: A Patient Case Related to Medicinal Chemistry and Drug Design
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Escherichia Coli is an example of carbepenem-resistant enterobacteriaceae (CRE). This family of bacteria is difficult to treat due to their resistance to antibiotics. Although E. coli is part of the normal human flora, sometimes it may cause serious infections where antibiotics are needed. One class of antibiotics that is very potent and used for these infections are carbapenems; however, CRE- related therapy issues can arise. Although these cases are rare, CREs are very dangerous and incidents are increasing.(1)
A 35 year old male patient was admitted to Froedtert Hospital. He had been in and out of the hospital during the past months with little improvement due to bacterial infections. He had been taking multiple antibiotics to treat his previous infections. In the past few days, he had developed a high fever of 103 degrees Fahrenheit and severe diarrhea. Labs show he was positive for E. coli induced enteritis, which is swelling and inflammation of the small intestine. The doctor ordered that the patient receive ertapenem 1g over 30 minutes IV infusion once a day. The patient was on ertapenem for five days with no signs of improvement. Labs were taken daily and showed that he tested positive for E. coli throughout the duration of ertapenem therapy. After further analysis, the doctor concluded that the patient had CRE and is asking if ertapenem is an appropriate treatment choice.
Ertapenem, a carbapenem antibiotic approved by the FDA in 2001, has a longer half-life than the other carbapenems. Ertapenem is the drug of choice for fighting infections caused by gram-negative bacteria, like E. coli. The indications for this drug include: acute pelvic infections, community-acquired pneumonia, and complicated intra-abdominal, skin, or urinary tract infections.(2) Even though Ertapenem is the most effective carbapenem, it also has a higher affinity for gram-negative efflux systems and cannot pass easily through the porins of non-fermenting bacteria.(3) Although most beta-lactamase producing bacteria remain sensitive to ertapenem, we currently know there are resistant strains such as in the genera Escherichia, Salmonella, Enterobacter, and Pseudomonas.
E. coli is notorious for its contributions to hospital acquired infections that can be particularly detrimental in immunocompromised patients. An issue that often arises with gram-negative bacterial infections are their innate ability to resist against many antibiotic therapies. Carbapenems have also been noted as the most effective antimicrobial agents against gram-positive and gram-negative bacteria. They inhibit bacterial cell wall synthesis by binding to and inactivating transpeptidase. This enzyme's function is crucial to the microorganism's survival in that it synthesizes peptidoglycan for cell wall assembly in both gram-positive and gram-negative (peptidoglycan synthesis in the periplasmic space) organisms, making it a bactericidal agent.(4) However, gram-negative bacteria have developed mechanisms in which it can resist against carbapenems as well.
Recent discoveries have demonstrated that a loss of gram-negative outer membrane permeability can be attributed to reduced expression of three porin proteins found in gram-negative bacteria; OmpF, OmpC, and PhoE.(5) Until recently, these three outer membrane proteins were thought to be nonspecific channels that allowed solute into the periplasmic space of a gram-negative species. This was considered to be true until the discovery of an ampicillin binding site within the OmpF porin protein.(6) Other similar β-Lactam antibiotics, such as ertapenem and carbenicillin, have also been shown to have binding sites different from that seen in ampicillin.(4)
The Outer Membrane Porin protein family, commonly abbreviated 'Omp' has similar structural components that are conserved across multiple genera of bacteria. The OmpF porin is a homotrimer that consists of a 16 antiparallel β-barrel (as found in other channel proteins) in each monomer.(4,7) Each monomer is configured in such a way that it forms a separate central pore that works independently in regards to solute permeation. Intracellularly in the periplasmic space, the monomers are held together by α-turn motifs, and have two extracellular loops. Of particular note, one extracellular loop is designated as L3 and has the significant function of folding back on the middle of the barrel and creating a 'constriction site'. This constriction site determines the size of solute able to pass into the periplasmic space.(4) The acidic residues on L3, opposed to the basic amino acids on the adjacent β-barrel wall, provides a strong electric field that is believed to have an effect on the porin's ion selectivity.(4,7)
β-Lactam antibiotics often generate negatively and positively charged side groups when at physiological pH. In combination with the difference in side groups of each antibiotic, the charged state contributes to the different locations in which they bind to OmpF.
Ertapenem, having two negatively charged carboxylic acid side chains and a positively charged amine group, demonstrates some similar as well as different binding interactions when compared with the zwitterion ampicillin and di-anionic carbenicillin. After soaking OmpF crystals in ertapenem solution, the Fobs-Fcalc omit map showed clear density at the extracellular mouth of OmpF, about 17A above the constriction site when using a 1.9A resolution. Rather than binding to spaces in the extracellular pore, such as ampicillin and carbenicillin, ertapenem binds to the extracellular loops on one side of the OmpF porin.(4)
Ertapenem is unique in itself due to its structure and the conformations it can form. It contains a pyrroline ring and what is now commonly referred to as a carbapenem ring. Both groups are attached by a sulfur moiety. The greatest conformation is between the sulfur and the β-lactam due to the presence of rotameric states from 1150-1850 cm-1.(4,8) Using this information; only half of ertapenem was able to be modeled. In its lowest energy conformation state, the ertapenem carboxylic acid group off the benzene ring is negatively charged and forms a hydrogen bound with arginine 168 or R168. This single interaction has also been seen in ampicillin's carboxylate moiety.(4) Ertapenem also takes an orientation that is parallel to the pore axis on the same wall of the β-barrel that carbenicillin does. The difference in binding is most likely due to the arrangement of side chains around the β-lactam ring in ertapenem versus ampicillin and carbenicillin. Ertapenem lacks an amine connector and also has a longer side chain profile on the opposite side of the ring when compared to ampicillin and carbenicillin.
Ertapenem is a beta-lactam anionic molecule that is made up of two rigid planar groups (penem group on one side and phenyl pyrroline rings on the other side) that interact with each other through a sulfur atom bond. Ertapenem is a molecule that belongs to the carbapenem family and has a positive and two negative charges that can interact with substrates. Ertapenem interacts with the OmpF porin (a diffusion porin that has been attributed to antibiotic resistance) of E. coli which consists of large homotrimers of beta barrels that have a 16 strand antiparallel structure. The beta strands on the periplasmic side are connected through beta-turn motifs while the extracellular side has loops that perform a variety of functions (stabilization of the trimer structure as well as the formation of a 'constriction zone' which regulates what size molecules can permeate through the pore). Through crystallization, it was revealed that ertapenem is bound to the OmpF porin in an orientation parallel to the porin near the extracellular loops of the pore. Ertapenem is bound to the extracellular loops through hydrogen bonding (within 3.3 angstroms) of 3 OmpF residues: Gln 203, Arg 167, & Arg 168.(9)
OmpF PorinErtapenem also interacts with other antibiotic resistant bacterial proteins and enzymes. Two ertapenem-binding site interactions that were examined are the β-Lactamase (BlaC) enzyme from Mycobacterium tuberculosis and L, D-Transpeptidase from Enterococcus faecium.
Ser70 is within the active site and is responsible for forming a covalently bound ertapenem acyl-enzyme complex. Hydrophobic interactions are present between Ile117 side chain and the methyl group of the pyrroline ring. Lys73 and Glu166 have hydrogen bonding interactions with the C6 hydroxyethyl group on ertapenem with its carboxyl O2 group. Ser130's hydroxyl group has hydrogen bonding with the nitrogen on the pyrroline ring. Thr 251 has hydrogen bonding between its hydroxyethyl side chain and pyrroline C2 carboxylate group as well as an active site water molecule.(9)
Carbapenems are substrates that acylate the enzyme. Carbapenems are then slowly deacylated once bound and can therefore act as potent inhibitors of BlaC. Like other Class A β-lactamases, BlaC catalyzes the opening of the β-lactam ring via nucleophilic attack by an active site serine residue to generate the acyl-enzyme. This is followed by the hydrolysis of the ester bond to generate an inactive product with an open ring.(10) Ertapenem has both hydrogen bonding and hydrophobic interactions with the active site of L, D-Transpeptidase from Enterococcus faecium.(9)
Ertapenem interacting with transpeptidaseSince the late 1920's, β-Lactam antibiotics have been used to treat various bacterial infections and have become widely used for prophylactic care.(11) Medications such as amoxicillin, ertapenem, ampicillin, and carbenicillin are all medications that fall into this larger category antibiotics, which are irreversible inhibitors of the β-Lactamase enzyme. As early as the 1980's, healthcare practitioners have become aware of growing resistance to our traditional β-Lactam antibiotics in key pathogenic organisms. This is due to modification by inactivating enzymes, expression of multidrug efflux pumps, and reduction of outer membrane permeability (which is least understood).(4,12)
The regular mechanism of action of carbapenem antibiotics (such as ertapenem) is to first enter the periplasmic space via a porin protein, specifically OmpF.(5) OmpF is a specific homotrimer that allows carbapenems to move into the periplasmic space and inhibit cell wall production. If they are not removed into the extracellular space via efflux proteins, they can then enter the periplasmic space and irreversibly inhibit transpeptidases. This is done by crosslinking peptidoglycan within the cell wall structure.(13) Without a cell wall, the microorganism may experience hypotenicity and may rupture. It may also experience loss of form and structure, causing cell apoptosis. In order to work properly, OmpF must first allow a carbapenem to enter. As a result of the discovery of lower rates of transcription caused by mutation, researchers are finding more evidence to support that β-Lactam resistance is linked to decreased expression of the outer membrane channel proteins.
Like many porin proteins, OmpF is comprised of three independent monomer β-barrels that allow larger charged molecules to enter the periplasmic space of the gram-negative bacteria.(6) Ertapenem, a larger, hydrophilic molecule at physiological pH, has two negatively charged carboxylate groups and one positive amine group at physiological pH. It is also attracted to the constriction site on the extracellular surface of OmpF. This may be due to the constriction site's loop domain having a high concentrated negative charge because of the acidic residues. Another reason why this may occur is because the face of the β-barrel has positively charged and basic amino acids. The interaction within the gap forms an electric field that attracts ertapenem's charged groups and allows reversible binding.(4) When in contact with the β-barrel, the negatively charged carboxylate on ertapenem orientate itself parallel with the positively charged β-barrel and basic amine of ertapenem. This conformational change causes the carboxylate groups to face towards the constriction site in the extracellular loops. Though ertapenem is not bound in the β-barrel monomer itself, it exists in a hydrogen bonding state with the amino acid residue arginine 168 (possible hydrogen bonding interactions with Q203 and R167) with a distance of 3.3A between them.
The three primary components of increasing drug resistance in gram-negative species involve expression of porin and efflux proteins. OmpF is a channel protein that allows hydrophilic molecules (such as many β-lactam antibiotics) to enter into the periplasmic space and eventually into the cell itself.(7) These channels are water filled and allow nonspecific entry of smaller ions. These channels also have specific interactions with larger compounds as seen before with ampicillin, ertapenem, and carbenicillin. The biggest hurdle to bacterial resistance are mutations that cause a decreased expression or different phenotype of the outer membrane protein family, OmpF in particular.(4,7) With high mutation rates in bacteria (a study citing three sequence changes in OmpC over the course of a two year trial) and lower production of porin specific channels, the use of traditional β-lactam antibiotics are almost useless in gram-negative species.(5,14)
Carbapenems also have enzymatic bacterial resistance such as chromosomally-encoded inducible beta lactamases. The different classes of enzymes all bear serine in their active centers which cleaves an amide bond on the beta-lactam ring of carbapenems and ultimately inactivates the molecule A subgroup. Class B consists of metallo-beta lactamases (MBLs) which bears zinc and catalyzes the same chemical reaction instead of using one or two divalent cations. The active site of MBLs orients and polarizes the beta-lactam bond to induce nucleophilic attack by zinc-bound water hydroxide, thus aiding to its resistance against the antibiotic. Of the four classes of enzymes contributing to carbapenem resistance, class B is considered the most notable enzyme.(14)
Carbapenems are used as initial inpatient therapy for gram-negative infections because they quickly treat these infections despite the increasing resistance.(15) Per protocol and the hospital policy of Froedtert Hospital, a 35 year old male patient was started on ertapenem upon positive results of gram-negative E. coli cultures. After five days of dosing (1g intravenously over 30 minutes once daily), the patient's symptoms of diarrhea and fever remain unchanged. This resulted in the physician declaring the patient to have carbapenem resistance enterobacteriaceae. The patient will need to be dosed with other antibiotics that are effective against CRE such as Fosfomycin or Tigecyline.(15)
It is possible that our patient has a resistance pathway caused by a decreased expression of OmpF in the outer membrane, therefore causing less ertapenem to move into the periplasmic space.(4) Decreased expression most commonly comes from mutations that bacteria acquire in the environment, typically through a point mutation in the first or second nucleotide in the codon. The bacteria may also mis-repair its own DNA sequence via inappropriate nucleotide excision enzyme check. Specifically for the interaction between ertapenem and OmpF, a mutation in the sequence of arginine 168 could result in the loss of a positive charge needed to interact with a carboxylate on ertapenem. A mutation would lead to less specific binding and would be in the case if the residue became negatively charged or nonpolar. Nonpolar amino acids with hydroxyl groups could potentially interact, but would most likely be too distant from the ertapenem to be able to associate with the theoretical interactions of ertapenem around Q203 and A167. A change in amino acid chain sequence could also result in tighter or similar binding (if arginine were changed to a histidine or lysine) and may result in the inability of ertapenem to interact with the constriction zone/ β-barrel electric field near ertapenem's binding site.
Other possibilities pertaining to the patient case include mutations in the bacterial genome that lead to increased expression of β-lactamase or the efflux proteins the span both cell membranes in gram-negative species. β-lactamase has activity to hydrolyze the β-Lactam ring in ertapenem, therefore causing its inhibition of penicillin binding proteins to decrease.(4) Another growing concern of the β-Lactamase enzyme is its increasing capacity to metabolize newer third generation β-Lactams.(16) Efflux proteins function by pumping out molecules from both the periplasmic space and inner cell. If these structures are mutated, they could become over active. Reduced activity would lead to under pumping these molecules, and may cause greater concentration of molecules within cells. E. coli itself is very prone to point mutations, consequently making it a prime candidate for becoming a multiple-resistant strain.(17)
The correlation between mutations alone and antibiotic resistant bacteria has not yet been determined. It is also unknown what other factors and influences may cause resistance.(18)
Being able to differentiate between resistance pathways can lead to better patient outcomes and decreased duration of hospital visits for patients presenting with any form of multidrug resistant, gram-negative bacteria. Currently, to the authors' knowledge, there are no practical means for a hospital or clinic to determine what resistance pathways these microorganisms have. It is therefore important that staff still rely on protocol when making clinical decisions. Being able to recognize resistance and switching therapies as soon as possible may be imperative for successful treatment outcomes. It is therefore important to identify that different bacterial strains within the hospital practice site and surrounding community to determine the prevalence of certain bacterial strains. This will aid prescribers in choosing an agent that will most effectively target the organism of interest.(19)
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