2 Terminology Chemotherapeutic agents: Drugs to treat a disease Antimicrobial drugs: Interfere with the growth of microbes within a hostAntibiotic: Substance produced by a microbe that inhibits another microbeSelective toxicity: A drug that kills harmful microbes without damaging the hostChemotherapy drugs that act against a disease: any disease: insulin, antihypertensive drugs and antimicrobial drugs.
3 A Look Back Began in 1909 with Paul Ehrlich Arsenic compound for treatment of syphilisSalvarsan1929 – Fleming discovered penicillin, produced by Penicillium mold.Coined term antibiotic1932 – Gerhard Domagk discovered sulfanilamideFirst to use agent to treat wide array of bacterial infecti0ns.Prontosil: red dyeThe idea of chemotherapy was the brainchild of Paul Ehrlich, a German scientist, who in the 20th century predicted that chemotherapeutic agents could be used to treat diseases caused by microorganisms. Finding a chemotherapeutic agent to kill a pathogenic microorganism wasn’t difficult, but chemotherapeutic agents also harmed and even killed host cells. This proved a major hurdle. A break through in 1928 when Alexander Fleming was growing S aureus in a petri dish. A colony of mold contaminated the dish surrounded the S aureus and prevented it from growing. The mold was penicillium notatum and the active compound Fleming named penicillin.It took roughly tens years from flemings discovery before the first clinical trials were successful. Wasn’t used in humans until 1941
4 Completely synthesized in a lab AntibioticsNaturally produced antimicrobial agentSemi-syntheticsChemically altered antibiotics that are more effective than naturally occurring onesSyntheticsCompletely synthesized in a labMany of the antibiotics in use today are produced from Streptomyces (bacteria that live in soil)Other antibiotics come from the genus Bacillus and from cephalosporium and penicillium both of which are mold
5 Action of Antimicrobials Narrowspectrum: An agent that works against a single Gram negative, Gram positive, or a few organismsBroadspectrum: An agent that is effective against a wide variety of Gram positive and Gram negative organismsBacteriacidal: An agent that kills the organismsBacteriostatic: An agent that temporarily inhibits the growth of the organism long enough for the body’s defense mechanism to take overA major contributing factor to whether an antimicrobial is broadspectrum or narrowspectrum has to do with the ability of the antimicrobial to pass into the cell through the LPS of the gram negative through porins water filled pores and so whether a drug is hydrophilic or hydrophobic will have a big impact on the activity of the drug
8 Inhibitors of Cell Wall Synthesis [INSERT FIGURE 10.3a-b]Recall that the cell wall regulates what exits and enters the cell and thus acts as a barrier. Protects a cell from the effects of osmotic pressure.Major structural component of a bacterial cell wall is its peptidoglycan layer. PTG is a huge macromolecule composed of polysaccharide chain of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) molecules that are cross-linked by short peptide chains extending between NAM subunits.The most common antibacterial agents act by preventing the cross-linkage of NAM subunits. Most prominent among these drugs are beta-lactams such as penicillin and cephalosporins
9 Inhibitors of Cell Wall Synthesis PenicillinSelectively toxicBacteriocidalMechanism of Action (MOA)Competitively bind with transpeptidase (penicillin binding protein)Inhibits cross-linking of peptidylglycanCell wall synthesis is arrested and the bacteria diesLPSPTGCMGram negativeRecall that both gram positive and gram negative bacteria possess peptidoglycan in their cell walls. These are composed of repeating dissaccharide subunits cross-linked with peptide chains. This cross linking is catalyzed by the enzyme transpeptidase. When introduced during WWII, penicillins were the miracle drug and were instantly put to work to treat combat infections. However early penicillins were only effective against Gram positive organismsPenicillin is a competitively inhibits transpeptidase and cell wall synthesis is arrested, the cell dies. Transpeptidase is also called penicillin binding proteinMore effective against gram positive than gram negativeThe enzyme transpeptidase mistakenly accepts the penicillin molecule as a substrate instead of peptidoglycan components. This renders transpeptidase inactive and thus the cell wall cannot be made or repaired.PTGCMGram positive
10 Penicillin-Family Antibiotics AmoxicillinAmpicillinKeflexCeclorCefzilVantinAzactamPenicillin G is the original G-man of penicillins usually given IM or IV many organisms have now developed resistance to this form. Pcn V is an oral formAminopenicillins (ampicillin and amoxicillin) is able to penetrate the outer wall of gram negative bacteria and better binding to the transpeptidase. However are still inhibited by penicillinasePenicillinase resistant penicillins: methicillin, nafcillin, and oxacillin are penicillinase resistant drugs that kill S aureus. Use of methicillin in USA DC’d because of nephritis.Antipseudomonal penicillins has expanded gram-negative rod coverage (pseudomonas aeruginosa)cephalosporins there are more than 20 different kinds. New basement makes beta lactam ring much more resistant to beta lactamases (but now cephalosporinases)A new R group side chain allows lots of lab manipulationThree generations division is based on their activity against gram negative and gram positive organismsFirst generation, second generation, third generationCephalosporins2nd, 3rd, and 4th generations more effective against gram-negatives
11 Resistance to Beta-Lactam Antibiotics Bacteria defend themselves from the penicillin family in four waysAlter porinsGm + and Gm- possess beta-lactamase enzymes that cleave beta-lactam ringPenicillinaseAlter structure of transpeptidase so antibiotics can’t bindMethicillin-resistant Staphylococcus aureus (MRSA)Gm + and Gm - may develop the ability to actively pump out beta-lactam before it binds to the transpeptidaseAlter porinsGram-negative bacteria have a LPS layer outside of PTG layer of cell wallPorins are small pores in the outer LPS layer
13 Beta-lactamase Inhibitors These enzymes are inhibitors of beta-lactamaseThey are given in combination with penicillins to create a beta-lactamase resistant combinationClavulanic acidAmoxicillin and clavulanic acid: AugmentinSulbactamAmpicillin and sulbactam: UnasynTazobactamPiperacillin and tazobactam: ZosynA type of beta lactamase is penicillinase so these drugs block the bacteria’s ability to destroy the beta lactam ring of penicillins
14 Inhibitors of Cell Wall Synthesis BacitracinInhibits lipid carrier, bactoprenolPrevents transport of NAM subunits through cell wallEffective as a topical treatment against Gram-positivesStaphylococciStreptococciVancomycinGlycopeptide derived from StreptomycesPrevents insertion of NAMs and NAGs into PTG wallImportant "last line" against antibiotic resistant S. aureusIn addition to the penicillin family other antibiotics are also effective inhibitors of cell wall synthesisBacitracin is a polypeptide antibiotic (name comes from the organism Bacillus was isolated from a wound of a girl named Tracy) MOA is different from pcn acts by inhibiting the linear strands of PTGVancomycin was derived from streptomyces found in the jungles of Borneo, narrow spectrum of activity against gram positive.Vancomycin never became first line treatment for Staphylococcus aureus for several reasons:The drug must be given intravenously, because it is not absorbed orally.β-lactamase-resistant semi-synthetic penicillins such as methicillin (and its successors, nafcillin and cloxacillin) were subsequently developed.Early trials using early impure forms of vancomycin ("Mississippi mud") which were found to be toxic to the ears and to the kidneys; these findings led to vancomycin being relegated to the position of a drug of last resort.
15 Inhibitors of Cell Wall Synthesis Members of the genus Mycobacterium have an atypical cell wallContain mycolic acidAcid-fast stainCause leprosy and tuberculosisAntimycobacterium antibioticsIsoniazid (INH)Inhibits mycolic acid synthesisInhibits the enzyme, fatty acid synthaseDrug of choice, generally used in combination with rifampin and ethambutolBecause they don’t have typical bacterial walls (they don’t have PTG but rather incorporate mycolic acid into their walls) mycobacterium are not going to be sensitive to the PCNs instead other antibiotics are used to treat these pathogensBoth Isoziazid and ethambutal inhibit cell wall synthesisEthambutol is not the drug of choice because of its weak activity and generally is used in combo with INH to minimize drug resistance
16 Antifungal Drugs Inhibition of Cell Wall Synthesis Target synthesis of b-glucans and result in an incomplete cell wallEchinocandinsBinds to 1,3 b-glucan synthaseUsed to treat Candida and PneumocystisCaspofungin (Cancidas™)The fungal cell wall is critical for cell viability and pathogenicity. Beyond serving as a protective shell and providing cell morphology, the fungal cell wall is a critical site for exchange and filtration of ions and proteins, as well as metabolism and catabolism of complex nutrients. Because mammalian cells lack a cell wall, it also represents an ideal and specific target for antifungal therapy. Structurally, the fungal cell wall is composed of a complex network of proteins and polycarbohydrates that varies in composition depending on the fungal species. Disruption of this protein/carbohydrate matrix results in a structurally-defective cell wall, rendering the fungal cell sensitive to osmotic lysis.Glucan synthesis inhibitors The glucan synthesis inhibitors are, collectively, agents that are presumed to block fungal cell wall synthesis by inhibiting the enzyme 1,3-beta glucan synthase. Inhibition of this enzyme results in depletion of glucan polymers in the fungal cell, resulting in an abnormally weak cell wall unable to withstand osmotic stress.
17 Disruption of Plasma Membrane Polymyxin BCauses disruption of the plasma membrane by attaching to the phospholipidsEffective against Gram negative bacteriaPseudomonasToxic to human kidneysTopicalCombined with bacitracin and neomycin in over-the-counter preparationCertain antibiotics bring about changes in the permeability of the plasma membrane of bacteriaPolymyxin is produced by bacillus polymyxaIs effective against gram negative bacteria especially Pseudomonas but is also toxic to human kidneys and thus reserved for topical use.
18 Fungal Plasma Membrane Knowledge of fungal cell structure and function is essential for understanding the pharmacology of antifungal agents. Like mammalian cells, fungi are eukaryotes with DNA organized into chromosomes within the cell nucleus and have distinct cytoplasmic organelles including endoplasmic reticulum, Golgi apparatus, mitochondria, and storage vacuoles. This homology to mammalian cells also extends to biosynthetic pathways, where fungi share similar mechanisms for DNA replication and protein synthesis.Fungi and mammalian cells both contain a cell membrane that serves and important role in cell structure, division, and metabolism. Complex lipid particles, called sterols, account for approximately 25% of the weight of the cell membrane. However, the sterol content between mammalian cells and fungal cells is different. Whereas mammalian cell membranes contain primarily cholesterol, ergosterol is the predominant sterol in many pathogenic fungi. This difference in sterol content has been exploited as the target of antifungal drug action by several classes of antifungal agents currently used to treat superficial and invasive fungal infections including the polyenes, azoles, and allylamines.
19 Antifungal Disruption of Plasma Membranes Amphotericin BAttaches to ergosterol and disrupts membrane and causes lysisPolyene antifungals such as amphotericin B act by binding to ergosterol in the fungal cell membrane and create a pores that increase permeability eventually leading to cell death. Amphotericin B may also induce oxidative damage in fungal cells and has been reported to stimulate of host immune cells.
20 Azoles Miconazole/clotrimazole Inhibits lanosterol a-demethylaseEnzyme converts lanosterol to ergosterolDisrupts plasma membraneMiconazole/clotrimazoleTriazoles (fluconazole and itraconazole)Eukaryotes such as fungi, use the same mechanisms to synthesize proteins and nucleic acids as the host the attack, therefore it is more difficult to find a point of selective toxicity in eukaryotes than in prokaryotes. Fungal infections are becoming more prevalent because of immunosuppressed individuals, especially those with AIDsMany antifungal drugs target the sterols in the plasma membrane. The principle sterol is ergosterol (in animals it is cholesterol)Azole antifungals inhibit the fungal cytochrome P A dependent enzyme 14-alpha demethylase, thereby interrupting the synthesis of ergosterol. Inhibition of this critical enzyme in the ergosterol synthesis pathway leads to the depletion of ergosterol in the cell membrane and accumulation of toxic intermediate sterols, causing increased membrane permeability and inhibition of fungal growthAzole antifungals can also inhibit many mammalian cytochrome P450-dependent enzymes involved in hormone synthesis or drug metabolism. Therefore, azole antifungals are particularly susceptible to clinically-significant drug interactions with other medications metabolized through the P450 pathway. Allylamines Allylamines work in a conceptually similar fashion to azole antifungals by inhibiting the synthesis of ergosterol. However, allylamines act at an earlier step in the ergosterol synthesis pathway by inhibiting the enzyme squalene epoxidase. Like the azoles, terbinafine has the potential for drug interactions with other medications metabolized through the mammalian cytochrome P-450 pathway.Inhibits cytochrome P450 14a-demethylase (P45014DM)Enzyme is in the sterol biosynthesis pathway that leads from lanosterol to ergosterolMiconazole/clotrimazoleTriazoles (fluconzole and itraconazoleAllylaminesInhibition of the fungal squalene epoxidaseResults in decrease sterol synthesis, especially ergosterolTerbinafine
21 Inhibitors of Protein Synthesis Recall prokaryotes ribosomes are structurally different from eukaryotes70S ribosome50S unit30S unitAllows creation of antimicrobials that target these structuresHowever mitochondria (eukaryotic cells) also contain 70S ribosomes similar to those of bacteria and therefore some antibiotics that target these structures and can have adverse effects on the cells of the host.Eukaryotes are 80S (consists of a 60S and 40S)
22 Inhibitors of Protein Synthesis ChloramphenicolBroad spectrumBinds 50S subunit, inhibits peptide bond formationSerious toxicitySuppression of bone marrowAminoglycosidesStreptomycin, neomycin, gentamicin, tobramycinBind to 30S and cause misreading of mRNAToxicity can cause hearing impairment and/or kidney damageReacting with the 50S portion of the 70S prokaryote ribosomes chloramphenicol inhibits the formation of peptide bonds in the growing polypeptide chain. Is a broad spectrum antibiotic from Steptomyces but is cheaper to synthesize chemically. Though it is cheap and broad spectrum it causes serious toxicity to the host, suppression of bone marrow, suppresses blood cell formation … aplastic anemia. Used is reserved as a last resort.Aminoglycosides have significant activity against gram negative bacteria
23 Inhibitors of Protein Synthesis TetracyclinesBroad spectrumEffective against Gram + and Gram –, rickettsias, and chlamydiaInterferes with tRNA attachmentForms complexes with calcium and can stain developing teeth and affect strength of developing bones.MacrolidesGram-positivesBinds 50S, prevents translationErythromycinAzithromycinClarithromycinDiscovered in the 1940s, the original TCN was produced by Steptomyces, but newer ones are semi-synthetic. Tetracycline, doxycycline, minocyclineMacrolides: (can’t penetrate wall of gram negatives) erythromycin , azithromycin (Z pac) clarithromycin (Biaxin) penetrate tissues better therefore better coverage
24 Inhibition of Protein Synthesis Antisense nucleic acidsFomiversenCytomegalovirus and eye infectionsPrevents formation of 70S initiation complexOxazolidinones (Zyvox™)Vancomycin and methicillin-resistant Staphylococcus aureusFomivirsen (brand name Vitravene) is an antiviral drug. It is used in the treatment of cytomegalovirus retinitis (CMV) in immunocompromised patients, including those with AIDS.It is an oligonucleotide that blocks translation of viral mRNA by binding to a coding segment of a key CMV gene. It was the first antisense antiviral approved by the FDA.It is 21 nucleotides in length and has the sequence: 5'-GCG TTT GCT CTT CTT CTT GCG-3'Oxazolidinones marketed in US as Zyvox. It works different than other protein synthesis inhibitors because it blocks the ability of the two ribosome subunits 50S and 30S thus no 70S complex can form and no initiation of protein synthesis can occur. Hopeful for antibiotic resistant bacteria but MRSA bacteria have become resistant to this antibiotic as well.
25 Inhibition of Metabolic Pathways Competitive InhibitorsSulfonamides (Sulfa drugs)Structural similar to PABAInhibit folic acid synthesisBroad spectrumSulfanamides were among the first synthetic antimicrobial drugs used to treat microbial disease
26 [INSERT FIGURE 10.6]Sulfanilamides are structurally similar to PABA. PABA is critical for synthesis of nucelotides needed to make DNA and RNA. Many organisms enzymatically convert PABA into dihydrofolic acid and DHF is converted to tetrahydrofolic acid (THF) a form of folic acid that is then used as a coenzyme in the synthesis of purines and pyrimidines. Sulfanilamides are structurally similar to PABA (an analog) and thus blockes the active site and thus no DHF, no THF and nucelotides.Humans do not synthesize THF from PABA instead we use folic acid found in our diet and convert them to THF thus our synthesis of nucleotides is not affected by sulfa drugs.
27 TMP is not a sulfa drug but is added to SMZ…Double whammy!!! Trimethorim also interferes with nucleic acid synthesis. Binds to the enzyme that converts DHF to THF
28 Inhibition of Nucleic Acid Synthesis Compounds can interfere with function of nucleic acids (nucleoside analogs)Nucleoside Analogs can distort shapes of nucleic acid molecules and prevent further replication, transcription, or translationMost often used against viruses; viral DNA polymerases more likely to incorporate and viral nucleic acid synthesis more rapid than that in host cellsAlso effective against rapidly dividing cancer cells
30 Inhibitors of Nucleic Acid Synthesis RifampinBinds to bacterial RNA polymeraseAntituberculosisQuinolones and fluoroquinolonesInhibits DNA gyraseBroader spectrum synthetic versionsCiprofloxacin: anthraxLimited use in childrenA number of antibiotics interfere with the processes of DNA replication and transcription in microorganisms. Some drugs with this mode of action have an extremely limited usefulness because they interfere with mammalian DNA and RNA as well.Quinilones and fluoroquinolones originated from naldixic acid (1960s) had limited activity for UTI but lead to the development of better and broader spectrum antibiotics. Inhibit DNA gyrase an enzyme necessary for correct coiling and uncoiling of repilcation of bacterial DNARifampin binds more readily to bacterial RNA polymerase that prevents the formation of mRNA
31 Antifungal Drugs Inhibition of Microtubules (Mitosis) GriseofulvinProduced by a species of PenicilliumUsed for superficial mycoses of hair and nail (tinea capitis or ringworm)Binds selectively to keratinBlocks microtubules and inhibits mitosisTolnaftateMechanism of action: not knownUsed for athlete's foot
32 Anti-viral Medications No peptidoglycan wallNo ribosomesNo plasma membraneCurrent anti-viral medications attack steps in viral replicationOnly attack actively replicating virusesMost are fake viral nucleotides, derail viral replication
33 Antiviral Drugs Nucleoside and Nucleotide Analogs Acyclovir is best known for treating genital herpes but it is effective against most herpesvirus infectionIt resembles nuceloside 2’deoxyguanosine. The viral enzyme thymidine kinase combines phosphate with such nucelosides to form a nucleotide which are then incorporated into DNAAcyclovir resembles the nucleoside but cannot be incorporated into DNA
34 Antiviral Drugs Enzyme Inhibitors Protease inhibitorsIndinavir:Prevents HIV protease to trim viral proteins down to working sizePrevents capsid formationInhibit attachmentZanamivirInfluenzaInhibit uncoatingAmantadineInterferons prevent spread of viruses to new cellsViral hepatitisReverse transcriptase inhibitors: RNA to DNAHIVThere are a number of antiviral drugs which are enzyme inhibitorsProteases are required for production of infectious viral particlesdrugs developed to kill viruses are targeted at various points in viral reproduction. Such as attachment to host cells, penetration or uncoating, DNA or RNA synthesis or reproductionRTIs inhibit activity of reverse transcriptase, a viral DNA polymerase enzyme that HIV needs to reproduce.
36 Disk-Diffusion Test Also known as the Kirby-Bauer test Petri plate is “seeded” with test organismFilter paper disks are impregnated with chemotherapeutic agents are place on surfaceZone of inhibitionThe larger the zone the more sensitive the microbe is to the drugThe further the drug diffuses out into the agar the lower it concentrationCompare zone diameter to standards and the organism is reported to be sensitive, intermediate, or resistant
37 E Test More advance diffusion test Can measure minimal inhibitory concentration (MIC)Lowest antibiotic concentration that prevents visible bacterial growth
38 Broth Dilution Test Used to determine MIC Plus minimal bactericidal concentrationWells that show no growth (higher concentration than MIC) are cultured in drug free brothNo growth = bacteriocidalGrowth = basteriostaticHelps to determine if a drug is bactericidal or bacterostatic
39 Minimum Bacterial Concentration (MBC) Test [INSERT FIGURE 10.12]This is similar to MIC test except that this test determines the amount of the drug that will kill the organism rather than just inhibit its growth as the MIC test does. Take a clear MIC tube and check to see what concentration is cidal. Here 8 is bacteriostatic and 16 is cidal.
40 Clinical Considerations in Prescribing Antimicrobial Drugs Routes of AdministrationTopical application of drug if infection is externalOral – simplest; lower drug concentrations; no reliance on health care provider; patients do not always follow prescribing informationIntramuscular – requires needle; concentration never as high as IV administrationIntravenous – requires needle or catheter; drug concentration diminishes as liver and kidneys remove drug from circulationMust know how antimicrobial agent will be distributed to infected tissues
41 Toxicity Safety and Side Effects Disruption of Normal Flora Allergies Exact cause of many adverse reactions poorly understoodDrugs may be toxic to kidneys, liver, or nervesConsiderations needed when prescribing drugs to pregnant womenAllergiesAlthough allergic reactions are rare, they may be life threateningAnaphylactic shockDisruption of Normal Flora
42 Antibiotic Resistance A variety of mutations can lead to antibiotic resistanceMechanisms of antibiotic resistanceEnzymatic destruction of drugPrevention of penetration of drugAlteration of drug's target siteAlter their metabolic activityRapid ejection of the drugMycobacterium tuberculosis produces MfpA protein, which binds to DNA gyrase preventing the binding of fluoroquinolone drugsResistance genes are often on plasmids or transposons that can be transferred between bacteria
43 Resistance to Antimicrobial Drugs [INSERT FIGURE 10.15]
44 Antibiotic Resistance Misuse of antibiotics selects for resistance mutantsMisuse includes:Using outdated, weakened antibioticsUsing antibiotics for the common cold and other inappropriate conditionsUse of antibiotics in animal feedFailure to complete the prescribed regimenUsing someone else's leftover prescription
45 Retarding ResistancePatient should finish entire course of antimicrobialLimit use of antimicrobials to necessary casesUse synergistic antimicrobialsDevelop new variations of existing drugsSecond-generation drugsThird-generation drugsSearch for new antibiotics, semi-synthetics, and syntheticsBacteriocinsDesign drugs complementary to the shape of microbial proteins to inhibit themAntimicrobial peptidesBroad spectrum antibiotics from plants and animalsSqualamine (sharks)Protegrin (pigs)Magainin (frogs)Antisense agentsComplementary DNA or peptide nucleic acids that binds to a pathogen's virulence gene(s) and prevents transcription