PHAR 7: Antimicrobials Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
Which of the following are ‘microorganisms’?
A
- all of them as microorganisms:
- may be acellular, unicellular or multicellular
- may exist individually, as a single cell or in colonies
- are found in each of the three domains of cellular life (Archaea, Bacteria and Eukarya)
3
Q
Compare various characteristics of microorganisms and mammals
A
- Microorganisms have their own distinct characteristics
- Importantly, they all exhibit specific differences from animal cells in terms of their cellular / molecular makeup / behaviour
- as we will see this is important in our ability to develop antimicrobial agents.
- In addition to the above, we might also consider algae as additional microorganisms.
- Algae are only very rarely involved in human infection thus algae are not covered in this eModule (although it should be noted they can produce substances that can be toxic).
4
Q
What are antimicrobials?
What are the different subsets?
A
- We use the term antimicrobial as the broadest term to describe agents that target microorganisms.
- Within that broad grouping, we can define the type of agent depending on the target microorganism, for instance:
- antibacterials, antivirals, antifungals and antiparasitics.
- Although most antibiotics have been discovered by their action against bacteria, they can also target fungi and parasites.
5
Q
Recap bacteria structure
A
- bacteria make up one of the two domains of prokaryotic life (the other being Archaea)
- they are unicellular organisms that do not have a membrane-bound nucleus or membrane-bound organelles (e.g. mitochondria)
- They do contain a bacterial ribosome for synthesis of oligopeptides and proteins, but this differs in its composition from ribosomes in other microorganisms and humans.
6
Q
What are commensal and pathogenic bacteria?
A
- Collectively, bacteria perform a wide variety of biochemical reactions, including a vast array that animals cannot.
- In some cases, bacterial and animal biochemistry work synergistically;
- in this capacity, bacteria are known as commensal (arguably mutualistic).
- In the wrong place, in sufficient numbers, or when in the presence of specific stressors, some bacteria are pathogenic i.e. they cause disease.
- Importantly, it should be noted that the pathogenicity of bacteria is context-dependent; factors that can influence this include compromised host immunity, translocation, etc.
7
Q
A 170 cm tall, 70 kg male has around 30 trillion human cells in their body.
Approximately how many microbial cells do you think are in (or on) them?
A
- 40 trillion.
- Not what you might have imagined or heard? Note that in recent years, the assertion that in the body, human cells are outnumbered by bacterial cells 10:1 has been shown to be somewhat of an overestimate; a revised estimate is much lower (about 40 trillion microbial cells).
8
Q
Describe how bacteria can be described by their shapes
A
- Bacterial cells can typically be described according to their shape including rods (bascilli), spheres (cocci), spirals (spirochaetes), and commas (vibrio), although many others are possible, and aggregations of bacteria can add specificity to this description
9
Q
How can bacteria be described according to the makeup of their cell wall?
A
- Bacteria can also be described according to the makeup of their cell wall, which can differ substantially in composition (in terms of the number of layers of a constituent called peptidoglycan).
- These differences result in radically different responses to several types of antibacterial agents, but also in the degree of staining of the peptidoglycan when exposed to Gram’s stain;
- Gram-positive bacteria take up the stain more readily as they have a thicker peptidoglycan layer.
10
Q
Observe this image showing the divergence of bacteria over evolutionary time
A
- this divergence has resulted in a phenomenal range of environmental niches that prokaryotic life has colonised; there are few places on Earth that are uninhabited by prokaryotes.
- Some of those environmental niches are located on and in the human body.
11
Q
Describe the structure of viruses
A
- viruses are acellular and store their genetic information as RNA or DNA.
- Thus, viruses need to infect a host cell to survive but can do so as they carry the genetic information to produce viral particles or ‘virions’.
- The size of virions can range from small viruses (20 nm, polio virus) to large viruses such as the recently discovered Pandoravirus (1000 nm, Pandoravirus salinus).
- Structurally, viruses consist of nucleic acid (RNA or DNA) protected by a protein coat called capsid.
- The protein units of the coat are called capsomeres, and the capsid enclosing the nucleic acid is called nucleocapsid.
- Viruses with this structure are called non-enveloped viruses, and viruses that are also protected by a lipid layer are called enveloped viruses.
- Figure 6 shows examples of viruses and the structural differences between enveloped and non-enveloped viruses.
12
Q
Viruses can contain RNA or DNA as genetic material. What is the genetic material of the following viruses?
A
- DNA viruses:
- herpesviruses (chickenpox, shingles, cold sores, glandular fever)
- papillomaviruses (warts)
- adenoviruses (sore throat, conjunctivitis)
- poxviruses (smallpox).
- RNA viruses:
- retroviruses (HIV, HTLV)
- orthomyxoviruses (influenza)
- filoviruses (Ebola virus)
- coronaviruses (MERS, SARS-CoV-2), rhabdoviruses (rabies)
- paramyxoviruses (measles, mumps)
- rubella virus (German measles)
- picornaviruses (colds, meningitis, poliomyelitis)
- arenaviruses (meningitis, Lassa fever)
- hepadnaviruses (serum hepatitis)
- arboviruses (yellow fever).
13
Q
What does viral replication depend on?
A
- it depends on the type of genome, as some viruses have a dsDNA genome while others may have ssDNA, dsRNA, or ssRNA genomes
- replication of DNA and RNA is different
14
Q
For RNA viruses, how many types of RNA genome are possible?
A
- If the viral genome is RNA, there are three possibilities:
- dsRNA
- positive (+) single-strand (+ssRNA)
- negative (−) single-strand RNA (−ssRNA).
15
Q
Describe the herpesvirus life cycle
A
- Binding: envelope viral glycoproteins bind to host cell receptors
- Entry: entry through receptor-mediated endocytosis (2a) or endosome formation (2b).
- Release and Nuclear Transport: viral uncoating and release of nucleocapsid and tegument proteins into the cytoplasm
- Nuclear Entry: nuclear entry via nuclear pores and viral genome circularization.
- Gene Expression: expression of immediately early (IE), early (E) and late (L) viral genes, transport of mRNAs to the cytoplasm, and translation.
- DNA Replication: early viral gene expression and viral DNA replication.
- Packaging: assembly of late proteins into viral capsids and DNA packaging.
- Egress: viral budding through the inner nuclear membrane, transport to the nuclear-associated endoplasmic reticulum and plasma membrane, and particle release
16
Q
Describe the retrovirus life cycle
A
- Binding: viral envelope glycoprotein attachment to the CD4 receptor and co-receptors (CXCR4/CCR5).
- Fusion: entry and fusion of the viral particle.
- Core delivery: transport of the viral core from the cell membrane towards the nucleus.
- Reverse Transcription: initiation of reverse transcription of viral RNA into DNA.
- Import: core import into the nucleus.
- Uncoating and reverse transcription completion: uncoating and reverse transcription completes in the nucleus.
- Integration: viral integrase catalyzes viral genome integration into the host genome (provirus)
- Transcription: Proviral transcription to produce viral RNAs.
- Translation: viral protein production.
- Budding: assembly of viral RNA and proteins to package into virions.
- Release and Maturation: virus release and maturation into an infectious virion.
17
Q
What is Salvarsan?
A
- Salvarsan, an organoarsenic compound was first synthesised in the Ehrlich lab and subsequently found by others to have good antisyphilitic activity (syphilis is caused by Treponema pallidum).
18
Q
What does the term ‘Magic Bullet’ mean?
A
- With respect to antimicrobial therapy, Ehrlich coined the term ‘Magic Bullet’ to describe an ideal therapeutic agent,
- i.e. one that would only affect the target organism selectively.
- This approach underpins much of how we try to exploit biological differences between microorganisms that cause disease, and healthy human cells; the more selectively toxic we can make a compound, the greater the opportunity for application without side effects.
- In short, the idea of using chemotherapeutic “Magic Bullets” paved the way for discovering differences in the biochemistry of organisms and designing/ developing therapeutic drugs that exploit them.
- Furthermore, the process by which Ehrlich screened for therapeutic activity across a number of potential candidate compounds - and then selected some for further development - was effectively the forerunner for modern drug development programmes.
19
Q
Looking at this table again, think about how you might target these differences to design chemical or biological agents that might be bacteriostatic or bactericidal
A
- Stop genetic material replication
- Restrict genetic material component availability
- Compromise the structural integrity of the cell
- Prevent synthesis of membranes needed for growth
- Prevent synthesis of cellular proteins
20
Q
Using the table, come up with some ways how therapeutics may target viruses
A
- Inhibition of membrane fusion
- Inhibition of viral uncoating
- Inhibition of viral enzymes (reverse transcriptase, protease, integrase)
- Inhibition of viral nucleic acid synthesis
- Inhibition of viral release (budding)
21
Q
What are the three main classes of antibacterial agents?
A
- Class I: agents target the production of metabolic precursors from substrates such as glucose, and therefore restrict any downstream processes
- Class II: agents target processes involved in the production of small molecules from metabolic precursors
- Class III: agents target processes involved in the production of macromolecules from small molecule substrates
22
Q
Considering bacterial cellular structures, how could we also classify antibacterial agents?
A
- Cell wall synthesis inhibitors
- Bacterial cell membrane disruptors
- Bacterial protein synthesis inhibitors
- Bacterial nucleic acid synthesis and action inhibitors
23
Q
Observe these diagrams of antibacterial agent summaries
A
24
Q
Describe the structure and function of bacterial cell walls
- plus Gram positive and negative
A
- Bacterial cell walls are formed from peptidoglycan (a.k.a. murein), a polymer that encases the cell, in addition to the cell membrane.
- The peptidoglycan polymer itself is made up of N-acetylglucosamine (NAG/NAGA) and N-acetylmuramic acid (NAM/NAMA), bonded in an alternating pattern by beta-(1,4)-glycosidic linkages
- These polymers are cross-linked by short peptide chains that give rise to a mesh-like structure composed of repeating parallel peptidoglycan.
- The bacterial wall serves several purposes, including providing structural rigidity to the cell while remaining porous, and preventing the cell from lysis under the osmotic pressure of the local environment.
- Gram negative bacteria have a single peptidoglycan layer, whereas Gram positive bacteria have multiple layers (up to 40 is common).
- The substantial thickness of the cell wall in Gram positive bacterial cells is responsible for their uptake of Gram’s stain, hence their designation as such.
25
Q
What are beta-lactam drugs?
Describe their structure
A
- beta-lactam drugs inhibit bacterial cell wall synthesis by covalently binding to the DD-transpeptidase enzymes that are responsible for cross-linking the peptides between peptidoglycan chains
- they have a central beta-lactam ring
- The covalent binding of beta-lactams to the enzyme active site inactivates it and therefore prevents cell wall synthesis.
26
Q
What are some of the most commonly-used/studied beta-lactam antibiotics?
A
- penicillins
- cephalosporins
- monobactams
- carbapenems
- clavulanic acid
27
Q
What are the most common of the beta-lactam drugs?
Describe what variety of antibacterial agents there are using its core structure
A
- the pencillins
28
Q
Describe the bacterial cell wall synthesis inhibitors: vancomycin and teicoplanin
A
- Vancomycin, and related compound teichoplanin, prevent the synthesis of the bacterial cell wall by forming strong hydrogen bonds with the peptides that cross-link the peptidoglycan polymer chains,
- and thus prevent the formation of the normal lattice/mesh-like structure.
- Without cross-linking, the cell wall integrity cannot be achieved, and therefore bacterial cell viability is compromised
29
Q
What type of antibacterial is daptomycin?
Describe its structure and its function
A
- daptomycin is a bacterial cell membrane disruptor
- the molecule is amphipathic;
- it contains both a hydrophobic alkyl chain moiety at one end and a hydrophilic peptide ring at the other which enable it to localise in the bacterial cell membrane.
- When multiple daptomycin molecules aggregate, their collective effect is to substantially distort the cell membrane shape, giving rise to holes.
- Loss of membrane integrity in this way causes depolarisation of the membrane, and consequently, chemical gradients necessary for many synthetic processes cannot be maintained.
- Ultimately, the bacterium dies though an inability to perform critical biochemical functions.
30
Q
What type of antibacterials are polymixins?
Describe their function and structure
A
- Polymixins are peptide antibacterial agents that work in a similar way to daptomycin in that they compromise cell membrane integrity.
- They achieve their selectivity for bacterial cells by binding to lipopolysaccharide (LPS) that is highly abundant in Gram-negative bacteria, although treatment is often accompanied by a range of off-target effects that has limited their use, particularly since the development of agents with fewer associated toxicities.
31
Q
How can processes in bacterial protein synthesis be targeted if humans also synthesise proteins?
What drugs target these?
A
- There are several processes in bacterial protein synthesis that can be targeted by antibacterial agents.
- As in eukaryotes, the synthesis of proteins from amino acid precursors occurs on/in ribosomes, but these are sufficiently different in structure (See Figure 18) to allow drugs to selectively target only bacterial ribosomes.
- bacterial protein synthesis inhibitors can be developed
32
Q
Give some examples of bacterial protein synthesis inhibitors
A
33
Q
Describe why nucleic acid synthesis and action inhibitors are used in antibacterials
A
- Synthesis of DNA and RNA in bacteria is dependent on the availability of folic acid, which in turn is synthesised from the precursor molecule p-aminobenzoic acid (PABA; Figure 19).
- Enzymes involved in catalysing the necessary biotransformation of these precursors include dihydropteroate synthetase and dihydrofolate reductase
- and these can be targeted by competitive inhibitors; sulfonamides are structurally similar to PABA, and trimethoprim is structurally similar to folic acid
- therefore introduction of these compounds will reduce the rate of bacterial DNA/RNA production and are bacteriostatic.
34
Q
Give examples of some nucleic acid synthesis and action inhibitors
A
- DNA gyrase inhibitors
- Bacterial DNA replication requires the action of the enzyme topoisomerase II (bacterial DNA gyrase).
- Inhibition of this process prevents DNA replication and bacterial cell division.
- This can be achieved by quinolones such as ciprofloxacin, norfloxacin, and nalidixic acid.
- RNA synthesis inhibitors
- Preventing the synthesis of RNA also prevent normal bacterial function and growth.
- This is possible using the drug rifampicin (a.k.a. rifampin), which inhibits bacterial DNA-dependent RNA polymerase (Figure 20).
36
Q
What compounds can inhibit nucleic acid biosynthesis in viruses?
A
- Nucleoside analogues, molecules derived from a nucleoside, are used as antiviral agents.
- For instance, the guanosine analogue acyclovir is used to treat herpes virus infections (Figure 21).
- Nucleoside analogues are administered as prodrugs (nucleosides), which are then phosphorylated by kinases to the active triphosphate form (nucleotides).
- Nucleotide analogues (triphosphate) can be incorporated into growing chains during nucleic acid biosynthesis leading to ‘chain termination’.
- Figure 22 shows how the DNA elongation process is affected by nucleotide analogues. Note: these antivirals are also known as DNA polymerase inhibitors.
37
Q
Describe a key antiherpesvirus agent
A
- the nucleoside analogue acyclovir (ACV), which functions by inhibiting nucleic acid biosynthesis via blocking the DNA elongation process (Figure 22).
- ACV is a synthetic analogue of the nucleoside guanosine (Figure 21).
- ACV is first phosphorylated intracellularly by the viral enzyme thymidine kinase (TK) to ACV-MP, then di- and tri- phosphorylated by cellular kinases to ACV-DP and ACV-TP (Figure 23).
- ACV-TP is the active metabolite that can be incorporated into growing DNA during replication, causing chain termination (Figure 24).
- ACV and other derivatives are used for the treatment of herpes virus infections, including genital herpes, chickenpox, shingles, Epstein-Barr virus infections, and cytomegalovirus infections.
- ACV administration is either topical or systemical, depending on the infection, and due to its high selectivity side effects are uncommon.
39
Q
How does antiviral therapy (ART) suppress HIV disease progression?
A
- HIV-1 requires the host machinery to replicate its genetic material and survive and carries its own viral enzymes, which can be used as therapeutic targets (Figure 7).
- Antiretroviral agents can inhibit different steps in the viral life cycle (Figure25).
- Standard ART uses a combination of at least three antiretroviral drugs to maximally suppress HIV and disease progression
41
Q
Describe the reverse transcriptase inhibitor: Nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs)
A
- First group of anti-HIV drugs, initially with concerning side effects. As we learned in the previous section, nucleoside analogues are prodrugs that are phosphorylated by kinases to the active metabolite.
- NNRTs/NtRTIs include: Abacavir, adefovir, didanosine, dipivoxil, emtricitabine, entecavir, lamivudine, stavudine, telbivudine, tenofovir, zidovudine
- Zidovudine (AZT) and Emtricitabine are examples of nucleoside reverse transcriptase inhibitors (NRTIs), and Tenofovir is a nucleotide reverse transcriptase inhibitor (NtRTI), see Figure 26.
- AZT was the first antiretroviral agent used for the treatment of HIV.
- AZT reduces mother-to-child transmission (MTCT) during pregnancy and then to the newborn.
- It is generally administered orally 2–3 times once/day, with a half-life of around 1 h.
- Most of AZT is metabolised to the inactive glucuronide in the liver, only 20% of the active form being excreted in the urine.
- Tenofovir has limited bioavailability due to poor oral absorption and cell penetration (Figure 26C), thus Gilead developed a prodrug version of tenofovir, tenofovir disoproxil (Figure 26D), in which the two negative charges of the phosphonic acid group are masked thus enhancing oral absorption.
49
Q
What are some ways in which bacteria can reduce the efficacy of antibacterial agents?
A
- Target modification: largely responsible for resistance is the change in the target of the antibacterial agent;
- in general, this means that critical biochemical interactions are no longer effective, and the drug cannot act.
- Immunity or bypass:
- In some cases, the bacterial targets are not sufficiently well targeted or there is redundancy in the system (e.g. an enzyme may be inhibited, but the process is accomplished via another route).
- Reducing the internal dose:
- Efflux pumps can be expressed on the cell membrane to pump out the active agent.
- Chemical inactivation:
- Expression of enzymes or creation of an environment that inactivates a drug, or sequesters it.
61
Q
Which of the following is NOT a mechanism for the development of antimicrobial resistance in bacteria?
A
- Plasmid hybridisation
62
Q
What common structural feature is present in tetracycline antibacterial agents?
A
- Four connected and functionalised hydrocarbon rings
63
Q
Use what you know about antibacterial agent structure to suggest the mechanism of action of the compound shown below:
A
- Covalent binding to DD-transpeptidase (penicillin binding protein)
64
Q
Tenofovir disoproxil is a nucleotide analogue prodrug used in the treatment and prevention of HIV, including PrEP. What is an important feature of this antiretroviral agent?
A
- It was modified to mask the two negative charges of the phosphonic group to enhance oral absorption.
78
Q
Make sure you understand the mechanism of action of these antimicrobials
A
