Structure and Medical Significance of Streptogramin A

Structure and medical significance of Streptogramin A

Pristinamycin IIA Virginamycin M1 Mikamycin A

1. Table of contents

1. Table of contents 2 2. Introduction 3 3. Overview and general information 3 4. Structural properties 3 5. Synthesis 5 6. Method of action 6 6.1 Translation 6 6.2 Mechanism of action 6 6.3 Resistances 6 7. Utilization as an antibiotic 7 8. Synercid® 8 8.1 Structure 8 8.2 Synthesis 8 9. Importance of antibiotics 9 Literature and internet sources 10

2. Introduction

Antibiotics play an important role in modern medicine. While they are very effective at what they do, their usefulness is very limited by time and extent of use, as bacteria quickly evolve resistances to fight them. It is for this reason that new alternatives need to be found or developed accordingly. The Streptogramins are such an alternative, as they bypass a decent number of bacterial resistance methods. While immunity to Streptogramins themselves can also be observed, it is, in contrast to other representatives of its group, found very infrequently, despite being actively used for several decades.

The paper at hand will primarily focus on explaining the structural details that make Streptogramin A and its chemical successor unique, as well as why and how Streptogramins are used to as an antibiotic.

3. Overview and general information

Streptogramin A is a substance which belongs to the group of A-type Streptogramins - a subgroup of the larger family of Streptogramins. Streptogramins are generally divided into two groups, one group being A-type Streptogramins, the other group being B-type Streptogramins. The Substance Streptogramin A is also known under a lot of different names, including Pristinamycin IIA, Virginamycin M1, Mikamycin A, Ostreogrycin A and Vernamycin A. This assignment will preferably refer to Streptogramin A as such or as Pristinamycin IIA.

The molecular formula for Pristinamycin IIA is C28H35N3O7 and its molecular mass amounts to around 525,602 g/mol.

Figure 1 Chemical Structure of Pristinamycin IIA

4. Structural properties

The Streptogramin A molecule is an unsaturated 23-heterocycle containing an ester group, which makes the molecule a lactone. It contains three different amino acids, namely Glycine, Serine and Proline, and a number of two amid bonds can be found. The Proline group plays an important role regarding the semisynthetic pathway to Pristinamycin IIA’s medically utilized derivate as elaborated in [8.2]. Pristinamycin IIA also contains Isobutyryl-CoA, which originates from a fourth amino acid, Valine, and is the starting point of biosynthesis. Malonyl-CoA extends the carbon chain during biosynthesis and can be found a total of six times.

1 3

Figure 2 Structural elements of Pristinamycin IIA

The molecule is not charged, as it contains no charged groups whatsoever. Overall, 2 Hydrogen bond donors, as well as 8 Hydrogen bond acceptors can be found in the molecule. Despite that, the molecule itself is rather insoluble in water. Salt crystallization is inhibited in comparison to its modern representatives, inasmuch as polarity and charge are less pronounced.

5. Synthesis

Biologically, Streptogramin A is produced by a large genus of bacteria, namely the Streptomyces. Streptomyces synthesise Streptogramin A as well as a B- type Streptogramin derivate (Pristinamycin IA) in combination. The biological precursor to Pristinamycin IIA is Pristinamycin IIB, which, despite its name, is an A- type Streptogramin as well. They differentiate in regards to a carbon double bond inside the Proline ring that PIIB is missing. Figure 3 Light micrograph of Streptomyces

Figure 4 Chemical structure of Pristinamycin IIA Figure 5 Chemical structure of Pristinamycin IIB

As mentioned above, Pristinamycin IIA is Pristinamycin IIB’s successor in biosynthesis, which could be proven via incorporation of radioactive and stable isotopes, as the origin of the 28 carbon atoms could be assigned to the respective positions in Pristinamycin IIB. It was proven that D-proline, as found in Pristinamycin IIB, cannot be derived from dehydroproline and therefore Pristinamycin IIA could not possibly be preceding in the synthesis chain. The oxidation from Pristinamycin IIB to Pristinamycin IIA is catalysed by a two-enzyme system in Streptomyces, one being a monoxygenase and one being FMN reductase. The reaction is shown in a simplified scheme in figure 6.

Figure 6 Pristinamycin IIA synthesis from Pristinamycin IA

A total chemically synthesis of Streptogramin A is not known yet. The medically active derivate is produced semi-synthetically as elaborated in [8.2].

6. Method of action

Streptogramins are antibiotics that inhibit biosynthetic protein production of bacteria during translation.

6.1. Required general information: Translation

The translation, the production of polypeptides by using the genetic information of the transcribed mRNA, takes place in the ribosomes. Prokaryotes have 70s ribosomes, which contain a 50s and a 30s subunit. The small 30s subunit binds mRNA and the large 50s subunit binds tRNA. In the ribosomes there is an acceptor site (A-site), a peptidyl site (P-site) and an exit site (E-site). The catalytic site is called the peptidyl transferase center (PTC). The genetic information Figure 7 Translation in form of mRNA is used for the attachment of the fitting tRNA, which is bound to the corresponding amino acid. This unit is called aminoacyl-tRNA. At the A- site tRNA binds with the complementary amino acid. The P-site holds the tRNA, while the polypeptide chain is growing. The binding of the amino acids is catalyzed by an enzyme called peptidyl- transferase.

6.2. Mechanism of action

Streptogramin A and B both bind to the P-site. Only in absence of aminoacyl-tRNA Streptogramin A binds to the PTC. Furthermore, they block substrate attachment to the A and P site and therefore inhibit the process of elongation. Streptogramin A binds through different hydrophobic interactions and Hydrogen bonds. This bond leads to a conformational change of the 50s ribosome subunit. An important observed conformation change is the one of the nucleotide U258. The new conformation has almost the same energy state, which is the reason for the prolonged activity. Furthermore, this change leads to an increased binding affinity of Streptogramin B. Once bound to the P site, Streptogramin A interferes with the positioning of the aminoacyl-tRNA at the A and P site.

6.3. Resistances

There are three types of resistance against streptogramins: enzymatic modification, active efflux and alteration to the target site (figures 8,9,10).

The Vat genes encode acetyltransferases that inactivate the antibiotic. Acetyltransferases catalyze the transfer of an acetyl-group from the acetyl-coA to the secondary alcohol of Streptogramin A. The Vga genes and Lsa gen provide resistance by active Figure 8 Active Efflux transport of Streptogramin A. Resistances have mainly been found in Enterococcus spp. and Staphylococcus spp. The most commonly known resistance to Streptogramin B is encoded by the gene Erm. This gene encodes a methylase, which methylates an adenine residue in the 23 rRNA.

Figure 9 Enzymatic modification Figure 10 Alteration of the target site

Figure 11 contains an overview of most common Streptogramin resistances:

Figure 11 Streptogramin resistances

7. Utilization as an antibiotic

Streptogramin A has been and seldom is used as feed additive for pork, chicken and cows in form of Pristinamycin. This antibiotic contains Pristinamycin IA and Pristinamycin IIA. Optimal efficiency is achieved in the ratio of 30:70. The intention was to increase the growth of cattle and prevent bacterially induced diseases. Amongst other antibiotics, Pristinamycin is currently forbidden in the EU as of 1999 in order to prevent bacteria from developing resistances and rendering it useless as a medical supply. In other countries, i.e. China, it is still in use today.

It serves its main purpose as an antibiotic against bacteria that are gram-positive. These bacteria have a Murein structure in their cell wall, granting extensive immunity against common antibiotics. Streptogramin A plays an important role because of its bacteriostatic properties against multi-resistant bacteria, such as Methicillin-resistant Staphylococcus Aureus (MRSA), vancomycin-resistant Staphylococcus Aureus (VRSA) and vancomycin-resistant Enterococcus Faecium (VREF). An exception to this is Enterococcus faecalis, which shows natural resistance due to its active efflux, as shown in figure 8. Aerobic gram negative bacteria, like Moraxella catarrhalis, or anaerobic bacteria, such as Clostridium perfringens, are also affected, yet are not the main target.

The bacteriostatic effect becomes bactericidal if Streptogramin A and its Streptogramin B counterpart are used in combination, as in Pristinamycin. Therefore, the subgroups have a synergistic effect and are generally coproduced in nature. This principle is transferred to current antibiotics used to treat human diseases, as they are always co-crystallized type-A and B Streptogramins, an important example being Synercid®.

8. Synercid®

8.1. Structure

Dalfopristin/Quinupristin, also known under its tradename Synercid®, is the only Streptogramin antibiotic currently in use. Synercid® is a combination of Dalfopristin and Quinupristin, which derive from Pristinamycin IIA and Pristinamycin IA respectively. Their synthesis is a semisynthetic pathway, as they are extracted from bacteria and not Figure 12 Synercid® synthetically produced.

Figure 13 Chemical structure of Dalfopristin Figure 14 Chemical structure of Quinupristin

8.2. Synthesis

Dalfopristin, the A-type compound of Synercid®, is a Pristinamycin IIA derivate. Its semisynthetic completion is based on the unsaturated Proline ring of Pristinamycin IIA, which allows for a “Michael-type addition” of Diethylaminoethanethiol.

Figure 15 Dalfopristin synthesis 1 Figure 16 Dalfopristin synthesis 2

As shown in the figures above, Diethylaminoethanethiol replaces the enolate of the classical Michael-type addition and acts as a nucleophile. The addition to the carbon double bond is stereo selective, leading to a thioether. This intermediate state then used to be oxidized via sodium periodate (NaIO4) and Ruthenium(IV)oxide (RuO2). Nowadays, hydrogen peroxide (H2O2) and sodium tungstate (Na2WO4) are used as oxidizers, as they maximize industrial yield and make the procedure more cost-effective. The resulting variant contains a much more ionizable group, so that advantages in salt formation can be observed. This is important, as Quinupristin and Dalfopristin are co- crystallized from acetone solution for Synercid® production. The increased water solubility is advantageous regarding intravenous injection.

9. Importance of antibiotics

Antibiotics play an important role today, and the number of multi-resistant bacteria increases. Due to that, new antibiotics or innovative methods to fight these bacteria need to be found. Creating a new substance needs a lot of time. However, if well-known substances are combined into new drugs, new substances aren’t inevitably necessary. An example for this is Synercid®, as you combine two components to make use of their synergistic effect and increase their respective effectiveness. Isolating new substances directly from bacteria for immediate use is another possibility. Synercid® and other Streptogramins however show a surprising toughness regarding bacterial resistances. This can partly be explained through the likelihood of mutation, as becoming not only immune to a single antibiotic like lone Dalfopristin, but simultaneously developing an answer to Quinupristin, is very unlikely, although not impossible. When used in combination, this likelihood decreases further, as their bactericidal effectiveness increases tremendously. It is mostly due to this that only 2-5% of Staphylococcal isolates examined in France show any resistance to the Streptogramin Pristinamycin, which has been in use for 35 years at that time. Since Streptogramins are such a powerful alternative when other antibiotics fail to achieve their goal, it is important that they are used cautiously and conservatively until alternatives have established.

Literature and internet sources

Literature:

• Dewick, Paul. Medicinal Natural Products: A Biosynthetic Approach, 2009, John Wiley & Sons Ltd. p450-451 [10.06.18] • Bonfiglio G, Furneri PM. Novel streptogramin antibiotics. Expert Opin Investig Drugs. 2001; 10:185–198. [10.06.18]

Internet sources:

• Harms, Jörg M; Schlünzen, Frank; Fucini, Paola; Bartels Heike; Yonath, Ada: Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin. In: BMC Biology. 2004. https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-2-4 [30.04.18] • Mast, Yvonne; Wohlleben, Wolfgang: Streptogramins – Two are better than one!. In: ScienceDirekt, International Journal of Medical Mircrobiologie Volume 304, Issue 1, Januar 2014, S. 44-50. https://www.sciencedirect.com/science/article/pii/S143842211300132X [24.04.18] • Murchison, Amanda: Quinupristin-dalfopristin: a streptogramin antibiotic. In: ScienceDirekt, Primary Care Update for OB/GYNS Volume 9, Issue 5, Semptember-Oktober 2002, S. 176-177. https://www.sciencedirect.com/science/article/pii/S1068607X02001130 [24.04.18] • In: Pubchem open chemistry database. https://pubchem.ncbi.nlm.nih.gov/compounds/16220095#section=Chemical-Co-Occurrences- in-Literature [24.04.18] • Krey, Angela: Molekularbiologische Resistenzmechanismen und Therapieoptionen bei europäischen Staphylococcus aureus Isolaten. https://docserv.uni- duesseldorf.de/servlets/DerivateServlet/Derivate-2924/924.pdf [12.04.18] • Wikipedia Translation: https://en.wikipedia.org/wiki/Translation_(biology) [27.05.2018] • Tariq A. Mukhtar and Gerard D. Wright; Streptogramins, Oxazolidinones, and Other Inhibitors of Bacterial Protein Synthesis; Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University; Hamilton; https://pubs.acs.org/doi/pdf/10.1021/cr030110z [10.06.18] • Lee Ann Thal Marcus J. Zervos: Occurrence and epidemiology of resistance to virginiamycin and streptogramins; in Journal of Antimicrobial Chemotherapy, Volume 43, Issue 2, 1 February 1999, Pages 171–176; https://academic.oup.com/jac/article/43/2/171/849148 [10.06.18] • Jia et al. 2017. CARD 2017: expansion and model-centric curation of the Comprehensive Antibiotic Resistance Database. Nucleic Acids Research, 45, D566-573.; McArthur & Wright. 2015. Bioinformatics of antimicrobial resistance in the age of molecular epidemiology. Current Opinion in Microbiology; McArthur et al. 2013; https://card.mcmaster.ca/ontology/36592 [10.06.18] • George A. Syrogiannopoulos, Ioanna N. Grivea, Amelia Tait-Kamradt, George D. Katopodis, Nicholas G. Beratis, Joyce Sutcliffe, Peter C. Appelbaum,3 and Todd A. Davies: Identification of an erm(A) Erythromycin Resistance Methylase Gene in Streptococcus pneumoniae Isolated in Greece; Department of Pediatrics, General University Hospital, University of Patras, School of Medicine, Patras, Greece1; Department of Infectious Diseases, Pfizer Global Research and Development, Groton, Connecticut2; and Department of Pathology, The Milton S. Hershey Medical Center, Hershey, Philadelphia, Pennsylvania3; Copyright © 2001, American Society for Microbiology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC90289/ [10.06.18] • In: Pubchem open chemistry database. https://pubchem.ncbi.nlm.nih.gov/compound/5459319#section=Top [27.05.18]

• In: Wikipedia The Free Encyclopedia. https://en.wikipedia.org/wiki/Streptogramin_A#cite_note-4 [27.05.18] • In: Wikipedia The Free Encyclopedia. https://de.wikipedia.org/wiki/Streptogramine [27.05.18] • https://www.genome.jp/Fig/compound/C00630.gif#, [10.06.18] • https://www.chem.uzh.ch/dam/jcr:4b98357c-5294-4a3a-9cc4-22e402cc9883/10.1.1g.png, [10.06.18] • https://www.spektrum.de/lexika/images/chemie/fff1359_w.jpg, [10.06.18] • https://upload.wikimedia.org/wikipedia/commons/thumb/6/61/D-Proline.svg/2000px-D- Proline.svg.png, [10.06.18] • https://upload.wikimedia.org/wikipedia/commons/thumb/4/46/Glycin_- _Glycine.svg/2000px-Glycin_-_Glycine.svg.png, [10.06.18] • https://upload.wikimedia.org/wikipedia/commons/thumb/7/7c/Malonyl-CoA2.svg/235px- Malonyl-CoA2.svg.png, [10.06.18] • https://upload.wikimedia.org/wikipedia/commons/thumb/b/b9/L-Serin_-_L- Serine.svg/1200px-L-Serin_-_L-Serine.svg.png, [10.06.18] • https://en.wikipedia.org/wiki/Quinupristin/dalfopristin [10.06.18] • https://en.wikipedia.org/wiki/Dalfopristin [10.06.18] • https://en.wikipedia.org/wiki/Hydrogen_peroxide [10.06.18] • https://de.wikipedia.org/wiki/Natriumwolframat [10.06.18] • https://de.wikipedia.org/wiki/Ruthenium(IV)-oxid [10.06.18] • https://de.wikipedia.org/wiki/Natriumperiodat [10.06.18] • https://en.wikipedia.org/wiki/Dalfopristin [10.06.18] • https://www.scbt.com/scbt/product/dalfopristin-as-mesylate-112362-50-2 [10.06.18] • https://en.wikipedia.org/wiki/Pristinamycin [10.06.18] • https://de.wikipedia.org/wiki/Pristinamycin [10.06.18] • https://en.wikipedia.org/wiki/Macrolide [10.06.18] • https://www.arznei-telegramm.de/html/2000_07/0007062_01.html [10.06.18] • https://www.rxlist.com/synercid-drug.htm#medguide [10.06.18] • https://www.sciencedirect.com/science/article/pii/S004040390100973X [10.06.18] • https://pubchem.ncbi.nlm.nih.gov/compound/2-_Diethylamino_ethanethiol#section=Top [10.06.18]

Figures:

• Figure 1: https://upload.wikimedia.org/wikipedia/commons/e/ed/Streptogramin_A.svg [10.06.18] • Figure 2: https://upload.wikimedia.org/wikipedia/commons/e/ed/Streptogramin_A.svg, [10.06.18] • Figure 3: https://de.wikipedia.org/wiki/Streptomycetaceae, [06.06.18] • Figure 4: https://upload.wikimedia.org/wikipedia/commons/e/ed/Streptogramin_A.svg [10.06.18] • Figure 5: https://upload.wikimedia.org/wikipedia/commons/0/02/Pristinamycin_IIB.svg [10.06.18] • Figure 6: https://www.semanticscholar.org/paper/Cloning-and-Analysis-of-Structural-Genes- from-in-of-Blanc-Lagneaux/72423e81fae4134f29e2224a8609508b2f715254/figure/2 [10.06.18] • Figure 7: https://en.wikipedia.org/wiki/Translation_(biology) [10.06.18] • Figure 8,9,10: https://www2.le.ac.uk/projects/vgec/highereducation/topics/microbial- genetics-1/antibiotic-resistance-1 [10.06.18]

• Figure 11: Ellie Hershberger Susan, Donabedian Konstantinos Konstantinou Marcus J. Zervos George M. Eliopoulos: Quinupristin-Dalfopristin Resistance in Gram-Positive Bacteria: Mechanism of Resistance and Epidemiology; published 01.01.2004; https://academic.oup.com/cid/article/38/1/92/356580 [10.06.18] • Figure 12: “Quinupristin-dalfopristin” https://www.idstewardship.com/quinupristin- dalfopristin/, 06.06.2018 [10.06.18] • Figure 13: https://upload.wikimedia.org/wikipedia/commons/e/ef/Dalfopristin_chemical_structure.png [10.06.18] • Figure 14: https://upload.wikimedia.org/wikipedia/commons/7/72/Quinupristin.png [10.06.18] • Figure 15, 16: via chemsketch https://chemsketch.de.softonic.com/ [10.06.18]