The misuse of existing antibiotics in clinical practice and the introduction of antibiotics into the environment in combination with the inherent adaptive nature of microorganisms has led to a major global health crisis with the evolution of antibiotic-resistant organisms. The CDC estimates that more than two million people are sickened every year with antibiotic-resistant infections in the U.S., with at least 23,000 dying as a result [1]. The crisis has been exacerbated by a lack of research and development into the development of novel antibiotics over the past three decades. Recognition of the crisis has led to an increased interest in pursuit of novel antibiotics with initiatives such as CARB-X and the Bill and Linda Gates Foundation along with the NIH promoting research and development in this field. As of June 2018, approximately 42 new antibiotics with the potential to treat serious bacterial infections were in the clinical trial pipeline [2].

In this blog we will review some of the classical targets for antimicrobial therapy, mechanisms of antimicrobial resistance, and clinically-relevant resistant pathogens that are the focus of antimicrobial drug development, and how new technologies may be developed to enrich the antimicrobial drug pipeline.

Development of effective antibiotics requires the new drug to be selective for the killing of the targeted bacteria and safe for the patient. This is most often achieved by identifying molecules and therapeutics that target molecular and biochemical pathways or cellular components that are different between the bacterial (prokaryotic) cells and the host organism.

Most current antibiotic therapies target three main cellular processes- cell wall synthesis, protein synthesis, and nucleic acid synthesis (DNA replication and/or RNA transcription). Mechanisms shared by bacteria for becoming resistant to antimicrobial agents fall into three general categories- direct inactivation or alteration of the drug, modification of the drugs binding or target site, and a change of cell membrane permeability resulting in decreased intracellular drug concentrations. In addition, many pathogenic bacteria have the ability to produce an extracellular biofilm that protects them from exposure to the antimicrobial agent.

Cell wall synthesis and mechanisms of resistance.

The cell wall of bacteria differs greatly in composition and structure to that of mammalian cells and thus has been a well utilized target for antibiotic therapy. Classes of antibiotics targeting the cell wall include the β-lactams (penicillin, methicillin, and cephalosporins), glycopeptides (vancomycin), and the lipopeptides (daptomycin). The most widely used antibiotics are the β-lactams and the glycopeptides are considered a drug of last resort. The main modes of resistance to cell-wall inhibitors are bacterial encoded enzymes that irreversibly modify and inactivate the antibiotic agent. Chief among these are the β-lactamase enzymes, most of which are produced by Gram-negative bacteria, making theses bacteria particularly difficult to treat. A variety of β-lactamase enzymes, including penicillinase, cephalosporinase, broad and extended spectrum β-lactamases, metallo β-lactamase NDM-1, and oxacillin degrading enzymes are found in pathogenic bacteria associated with antibiotic-resistant infections. One particularly troubling aspect of antimicrobial drug resistance is the propensity of the β-lactamase enzymes to be encoded on genetically mobile elements, facilitating the spread of resistance to the most often used and “drug of last resort” antibiotics to different species of bacteria [3].

Bacterial Protein Synthesis

The translational apparatus of bacteria is another distinguishing target for antimicrobial therapy. Antibiotics targeting bacterial translation include the aminoglycosides (streptomycin, kanamycin), tetracyclines, macrolides (erythromycin, clarithromycin) and oxazolidinones. Following the β-lactam antibiotics, the macrolides are the second most prescribe antibiotics. The most common mechanism for resistance to this class of antibiotics is a modification of the antibiotic binding site, predominantly by methylation of the 23S ribosomal RNA by bacterially encoded methylases, such as erm. Erm is usually encoded with transposable elements in pneumococci and dimethylates rRNA at the binding site of erythromycin reducing the ability of erythromycin to bind to the ribosome and inhibit translation. This modification of the 23S rRNA also causes cross-resistance to macrolides, lincosamides, and streptogramin B antibiotics, giving rise to MLSB-resistance phenotype [4]. In addition to the resistance caused by post-transcriptional methylation of the macrolide binding sites in the rRNA target, spontaneous mutation within the ribosomal complex (in 23S rRNA or ribosomal proteins L4 and L22) can occur resulting in alteration of the macrolide binding site. Bacteria may also acquire active efflux, such as the Streptococcal mef genes encoded on genetically mobile elements, which actively transport the antibiotic out of the cell reducing its effectiveness [4].

Bacterial Nucleic Acid Synthesis

Bacterial RNA and DNA polymerases are another target for antimicrobial therapeutics that disrupt the ability of the bacteria to replicate its genome or transcribe its genes. Antibiotics targeting bacterial nucleic acid synthesis include the ansamycins (rifamycin), and quinolones (ciprofloxin). Rifamycin works by direct interaction with the bacterial RNA polymerase to prevent the initiation of transcription [5], whereas the quinolones target bacterial gyrase and topoisomerase IV enzymes. These enzymes function by cutting and repairing DNA strands in the bacterial chromosome to relieve torsional stress incurred during DNA replication. The interaction off quinolones with target gyrase and topoisomerase IV enzymes to converts their targets into toxic enzymes that fragment the bacterial chromosome [6]. Resistance to rifamycin can be obtained through a single nucleotide polymorphism in the nucleic acid binding site of the RNA polymerase, decreasing the ability of rifamycin to bind to its target [5]. Similar to rifamycin, the main mechanism of resistance to the quinolones is mutation of their binding site in the targeted gyrase and topoisomerase IV. Resistance to quinolones may also be mediated through genes encoded on plasmids whose products can bind to gyrase and topoisomerase IV decreasing both their ability to bind to DNA and inhibit quinolones from entering cleavage complexes formed by the enzymes. Plasmids may also encode acetyltransferases capable of acetylating and inactivating quinolones, and efflux pumps that actively transport the quinolones out of the cell [6].

Emerging Bacterial Threats

The past couple of decades have witnessed an escalation of emerging bacterial threats as the microbial community becomes resistant to the current standards of microbial therapies (Table 1). Particularly problematic the often multidrug-resistant ESKAPE pathogens -Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, which are the leading global cause of nosocomial infections [7].

(from Perspect Medicin Chem. 2014; 6: 25–64.)

The effective treatment of these and other multidrug-resistant organisms will require the development of novel, potent, and safe antimicrobial agents. Effective strategies include 1) therapeutics that combat the mechanisms of resistant to enhance and extend the effectiveness of antibiotic treatments, such as β-lactamase inhibitors, inhibitors of efflux pumps, and CRSPR-mediated targeting of bacterially-encoded resistance genes; 2) discovery and exploitation of novel bacterial targets; 3) modification of existing classes of antibiotics (new-generations) with greater potency less propensity to spawn resistance; 4) and bacteriophage therapies. New therapeutic entities may be discovered from programmed antimicrobial screening of compounds from natural or synthetic sources.

At Noble Life Sciences we support your efforts in developing new and innovative therapeutics to fight against bacterial resistance and can be your pre-clinical partner in their development. With capabilities in in vitro microbiology including screening against custom drug-resistant bacterial panels and in vivo models of infection with clinically relevant drug resistant bacteria and full-service toxicology, we can be your preclinical drug development “one-stop” partner to take your antimicrobial program from discovery to clinical trial readiness. Contact us today for more information on our antibacterial research services.

References

1. Center for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. (https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf)
2. The PEW Trust Foundation. Sept 2018. Antibiotics Currently in Global Clinical Development (https://www.pewtrusts.org/-/media/assets/2018/09/antibiotics_currently_in_global_clinical_development_sept2018.pdf).
3. S. Santajit and N. Indrawattana. 2016. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Research International 2016. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871955/pdf/BMRI2016-2475067.pdf)
4. Leclercq R.and P. Courvalin. 2002. Resistance to Macrolides and Related Antibiotics in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 46:2727–2734 (https://aac.asm.org/content/aac/46/9/2727.full.pdf)
5. Floss H.G. and T.W. Yu TW. 2005. Rifamycin-mode of action, resistance, and biosynthesis. Chem Rev. 105:621-32.
6. Aldred K. J., Kerns R. J., and N. Osheroff. 2014. Mechanism of quinolone action and resistance. Biochemistry. 53:1565-74. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985860/pdf/bi5000564.pdf)
7. Fair R. J., and Y. Tor. 2014. Antibiotics and Bacterial Resistance in the 21st Century. Perspect Medicin Chem. 6: 25–64. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159373/pdf/pmc-6-2014-025.pdf)