Antifungal Drugs & Resistance & New Drugs for Tuberculosis

Fungal Infections and Antifungal Drugs

Fungi as Infectious Agents

• Fungi, including molds and yeasts, are ubiquitous in the environment, thriving in diverse habitats such as soil, air, and water.

• Most fungal species are non-pathogenic, playing crucial roles in ecosystems as decomposers and nutrient recyclers; only a small fraction (approximately 300 out of 100,000) are linked to diseases in animals, including humans.

Common Opportunistic Fungi and Predisposing Conditions

• Candida:

  • Normal flora of the human body but can become pathogenic under certain conditions.

  • Associated with antibiotic therapy (disrupts normal bacterial flora), catheters (provide entry points), diabetes (high glucose levels promote growth), corticosteroid use (immunosuppression), and immunosuppression (including AIDS and genetic conditions). Specifically, Candida albicans is a frequent cause of opportunistic infections. The infections range from superficial mucosal infections to invasive, life-threatening systemic infections, particularly in immunocompromised individuals such as those with HIV/AIDS, transplant recipients, or patients undergoing chemotherapy.

• Aspergillus:

  • Commonly found in soil, decaying vegetation, and indoor air.

  • Linked to leukemia, corticosteroid use, tuberculosis, immunosuppression, and IV drug abuse. Aspergillus fumigatus is the primary culprit. Immunocompromised individuals, such as those with neutropenia or those undergoing hematopoietic stem cell transplantation, are at particularly high risk of developing invasive aspergillosis, which can affect the lungs, sinuses, brain, and other organs.

• Cryptococcus:

  • Primarily Cryptococcus neoformans, found in soil and bird droppings, particularly pigeon droppings.

  • Associated with diabetes, tuberculosis, cancer, corticosteroid use, and immunosuppression. Cryptococcus typically enters the body through inhalation, initially causing a pulmonary infection. In individuals with weakened immune systems, the infection can disseminate to the central nervous system, leading to cryptococcal meningitis, a life-threatening condition. Disseminated cryptococcosis can also affect other organs, such as the skin, bones, and prostate gland.

• Zygomycota Species (Mucor and Rhizopus):

  • Found in soil and decaying organic matter.

  • Linked to diabetes (especially ketoacidosis), cancer, corticosteroid use, IV therapy, and third-degree burns. These fungi can cause severe, rapidly progressing infections known as mucormycosis (formerly zygomycosis). Risk factors for mucormycosis include uncontrolled diabetes mellitus (particularly diabetic ketoacidosis), hematologic malignancies, stem cell transplantation, solid organ transplantation, and deferoxamine therapy. Infections typically begin in the sinuses or lungs and can spread to the brain, eyes, and other tissues.

Candida albicans

• Normal flora of the oral cavity, genitalia, large intestine, or skin in humans but becomes pathogenic when the balance is disrupted.

• Responsible for approximately 70% of nosocomial (hospital-acquired) fungal infections, making it a significant concern in healthcare settings.

• Manifestations include:

  • Thrush: a thick, white, adherent growth on the mucous membranes of the mouth and throat. Common in infants, immunocompromised individuals, and those using corticosteroid inhalers. Untreated thrush can spread to the esophagus and cause difficulty swallowing.

  • Vulvovaginal yeast infection (candidiasis): an inflammatory condition of the female genital region, causing intense itching, burning, ulceration, and discharge. Predisposing factors include antibiotic use, pregnancy, diabetes, and immunosuppression. Recurrent vulvovaginal candidiasis can significantly impact a woman's quality of life.

  • Cutaneous candidiasis: occurs in chronically moist areas of skin (e.g., underarms, groin, skin folds) and in burn patients. Presents as red, itchy patches with satellite lesions. Predisposing factors include obesity, diabetes, and poor hygiene.

• Treatment:

  • Topical antifungals (e.g., clotrimazole, miconazole, nystatin) for superficial infections.

  • Amphotericin B and fluconazole for systemic infections, often requiring intravenous administration and monitoring for side effects.

Candida - Dimorphic Fungus

• Exhibit dimorphism, transitioning between yeast (unicellular) and hyphal (filamentous) forms depending on environmental conditions, nutrient availability, and host factors.

• Yeast form is typically found in the bloodstream, facilitating dissemination throughout the body.

• Hyphal form is usually found in tissues and organs, aiding in escape from macrophages and promoting tissue invasion.

• Present as normal flora in 40-80% of humans, highlighting the importance of immune regulation in preventing pathogenic overgrowth.

Pathogenesis of Invasive Candidiasis

• Candida species are commonly found on mucosal surfaces of healthy individuals (50-70%), coexisting without causing harm under normal circumstances.

• Breaches in intestinal barriers (e.g., post-gastrointestinal surgery, chemotherapy-induced mucositis) can lead to dissemination into the abdominal cavity and bloodstream (candidaemia), allowing the fungus to spread to distant organs.

• Under normal immune conditions, Candida behaves as a commensal organism, kept in check by the host's defense mechanisms.

• Impairment of the immune response (e.g., neutropenia, immunosuppressive therapy) can promote fungal overgrowth in the gut, candidaemia, and subsequent invasive candidiasis in various organs, leading to life-threatening complications.

Invasive Infections Caused by Candida

• High-Risk Factors:

  • Hematologic malignancy (e.g., leukemia, lymphoma)

  • Neutropenia (low neutrophil count)

  • GI surgery (gastrointestinal surgery)

  • Extremes of age (neonates and elderly)

  • Central catheter (provides a direct route into the bloodstream)

  • Hemodialysis

• Exposures:

  • Antibiotics (disrupt normal bacterial flora)

  • Candida colonization (pre-existing Candida presence)

  • ICU stay >3 days (increased risk of exposure and invasive procedures)

• Candida bloodstream infection (BSI) occurs in 5-10/1000 admissions, representing 8%-10% of all BSIs, indicating its significant contribution to hospital-acquired infections.

• Mortality:

  • Death from underlying diseases: 49%

  • Death due to Candida BSI: 12%

  • Survive hospitalization: 39%

• Common species involved:

  • C. albicans (most common species)

  • C. parapsilosis (associated with catheter-related infections)

  • C. glabrata (increasingly common and often resistant to azoles)

Aspergillus fumigatus

• A common airborne soil fungus (hyphal fungus) that releases spores into the air, which can be inhaled by humans.

• Over 600 species exist, but only 8 are involved in human disease; A. fumigatus is the most common cause of infection, especially invasive aspergillosis.

• Serious opportunistic threat to AIDS, leukemia, and transplant patients, who have weakened immune systems that are unable to effectively clear the fungus.

• Infection typically occurs in the lungs, where spores germinate and form fungal balls (aspergillomas); can also colonize sinuses, ear canals, eyelids, and conjunctiva.

• Invasive aspergillosis can lead to necrotic pneumonia and infection of the brain, heart, and other organs, resulting in high mortality rates.

• Treatment: Amphotericin B, voriconazole, and nystatin are used, often in combination with surgical removal of fungal balls.

Cryptococcus neoformans

• A yeast fungus that causes cryptococcosis, a systemic fungal infection that primarily affects individuals with compromised immune systems.

• Common infection in AIDS, cancer, or diabetes patients, who are unable to mount an effective immune response against the fungus.

• Infection of the lungs leads to cough, fever, and lung nodules, often mimicking other respiratory illnesses.

• Dissemination to the meninges and brain can cause severe neurological disturbance and death, resulting in cryptococcal meningitis.

• Systemic infection requires amphotericin B and fluconazole, often administered for extended periods to eradicate the fungus.

Antifungal Drugs

Drug Targets and Examples:

• Plasma Membrane:

  • Polyenes: Amphotericin B, Nystatin

  • Bind to ergosterol, a sterol unique to fungal cell membranes, thereby disrupting the plasma membrane and causing leakage of cytoplasm.

  • Amphotericin B is highly toxic but effective for life-threatening infections; Nystatin is too toxic for systemic use, limited to topical applications for skin and mucosal infections.

  • Azoles: Imidazoles, Triazoles

  • Interfere with ergosterol synthesis by inhibiting the fungal cytochrome P450 enzyme lanosterol 14-alpha demethylase, leading to defective cell membranes.

  • Imidazoles: Examples are ketoconazole, miconazole, and clotrimazole, typically used topically due to toxicity.

  • Triazoles: Examples are fluconazole and itraconazole, which have better safety profiles and broader spectrum of activity compared to imidazoles.

  • Allylamines: Naftifine, terbinafine (Lamisil)

  • Inhibit squalene epoxidase, an enzyme in ergosterol synthesis, leading to accumulation of squalene and cell death; administered topically for dermatophyte infections; terbinafine can be taken orally for nail and skin infections.

• Cell Wall:

  • Echinocandins: Caspofungin, Micafungin, Anidulafungin

  • Block the synthesis of β-(1,3)-D-glucan, a polysaccharide component of the cell wall, leading to cell wall weakening and cell death; useful for treating invasive Candida and Aspergillus infections.

• Cell Division:

  • Griseofulvin

  • Used to treat skin and nail infections by concentrating in dead keratinized layers; active only against fungi invading keratinized cells; inhibits fungal mitosis by disrupting the mitotic spindle.

• Nucleic Acid Synthesis:

  • Flucytosine

  • Used for systemic yeast infections; inhibits an enzyme required for nucleic acid synthesis; not effective against most molds; converted to 5-fluorouracil (5-FU) within fungal cells, which inhibits DNA and RNA synthesis.

Ergosterol

• Ergosterol is a sterol found in fungal cell membranes, analogous to cholesterol in mammalian cells, and is crucial for maintaining membrane integrity.

• Azoles (e.g., fluconazole) inhibit Erg11 (lanosterol 14-alpha demethylase), blocking ergosterol production from lanosterol, leading to accumulation of toxic sterol intermediates and cell membrane stress.

• Polyenes (e.g., amphotericin B and nystatin) bind directly to ergosterol, forming pores in the cell membranes, leading to leakage of cellular contents.

Antifungal Azoles

• Function by inhibiting cytochrome P450 enzymes involved in the demethylation of lanosterol to ergosterol, a critical step in fungal cell membrane synthesis.

• This leads to reduced ergosterol concentrations in fungal membranes, resulting in leaky cell membranes and compromised cell integrity.

• Toxicity is related to the affinity of azoles for mammalian cytochrome P450 enzymes, potentially causing drug interactions and side effects.

• Triazoles generally have fewer side effects, better absorption and distribution, and fewer drug interactions compared to older imidazole agents, making them preferred for systemic fungal infections.

• Azoles are synthetic drugs with broad-spectrum fungistatic activity, divided into:

  • Older imidazole agents (clotrimazole, ketoconazole, miconazole) with two nitrogens in the azole nucleus, primarily used topically.

  • Newer triazole compounds (fluconazole, itraconazole, voriconazole) with three nitrogens in the azole nucleus, used for both topical and systemic infections.

Fluconazole

• Approximately 80-90% of an orally administered dose is absorbed, resulting in high serum drug levels and excellent bioavailability.

• The drug penetrates widely into most body tissues, with cerebrospinal fluid levels reaching 60-80% of serum levels, enabling effective treatment for fungal meningitis.

• Fluconazole is effective against most Candida species but has limited activity against Candida krusei and Candida glabrata, which may exhibit resistance.

• A 3-day course of oral fluconazole can effectively treat Candida urinary tract infections, providing a convenient and well-tolerated treatment option.

Azole Resistance Mechanisms

• Resistance to azoles can occur through:

  • Upregulation of efflux pumps (e.g., ABC transporters) that remove the drug from the cell, reducing intracellular drug concentration.

  • Drug target mutations (Erg11) that reduce affinity for the drug, preventing azoles from binding effectively.

  • Overexpression of Erg11, allowing some Erg11 to remain active, compensating for the inhibitory effects of azoles.

  • Loss-of-function mutations in ergosterol biosynthesis (e.g., Erg3), blocking the accumulation of toxic sterol intermediates, reducing the drug's effectiveness.

Polyene Antifungal Drugs

• Amphotericin B and Nystatin

  • Bind to ergosterol in the fungal cell membrane, increasing membrane permeability (leakage of ions and other cellular contents), leading to cell death.

  • Nystatin is too toxic for systemic use and is limited to topical treatment of superficial infections caused by C. albicans, such as oral candidiasis (thrush), mild esophageal candidiasis, and vaginitis.

  • Amphotericin B is used to treat systemic fungal infections but has extensive side effects due to interactions with mammalian cholesterol, necessitating careful monitoring and management. Reserved for severe infections in critically ill or immunocompromised patients.

  • Considered first-line therapy for cryptococcal meningitis and certain Aspergillus and Candida infections, owing to its broad spectrum of activity.

  • Highly effective drug for over fifty years with low drug resistance, making it a valuable option in challenging cases.

Resistance to Polyenes

• Resistance is uncommon and carries significant fitness costs, limiting the fungus's ability to survive and reproduce.

• Susceptibility depends on the ergosterol content in the fungal cell membrane, with higher ergosterol levels generally correlating with increased susceptibility.

• Ergosterol biosynthesis is regulated by 25 known ERG enzymes, each playing a specific role in the pathway.

• Mutations affecting ergosterol biosynthetic genes (ERG1, ERG2, ERG3, ERG5, ERG6, ERG11) decrease Amphotericin B sensitivity in Candida species, leading to reduced drug efficacy.

• These mutations change the sterol content in the membrane, leading to the accumulation of ergosterol intermediates, making the cell less susceptible but also reducing its fitness, highlighting the trade-offs associated with resistance.

Echinocandins

• Caspofungin and Micafungin inhibit β-(1,3)-D-glucan synthase (the catalytic subunit is encoded by FKS1 and FKS2 genes in Saccharomyces cerevisiae) disrupting cell-wall integrity, leading to cell lysis.

• β-glucans are abundant cell wall polysaccharides in fungi, which are important for cell defence and strength, providing structural support.

• Resistance is caused by target mutations (Fks1) that reduce drug-target affinity, making the enzyme less susceptible to inhibition by echinocandins.

Flucytosine

• A fluorinated pyrimidine with the following mechanism of action:

  1. Accumulates in fungal cells via cytosine permease.

  2. Cytosine deaminase converts the drug to 5-fluorouracil (5-FU).

  • Selectivity: Mammalian cells do not accumulate or deaminate flucytosine, minimizing toxicity.

  1. 5-fluorouracil is metabolized to 5-fluorouridylic acid.

  • Incorporation into mRNA leads to misreading and defective proteins, disrupting cellular function.

  • Metabolism to 5-deoxyfluorouridylic acid, a potent inhibitor of thymidylate synthase, blocks DNA synthesis, preventing cell replication.

Flucytosine – Resistance Mechanisms

• Resistance mechanisms include inactivating mutations in:

  • Cytosine permease, reducing drug uptake, preventing the drug from entering the cell.

  • Cytosine deaminase, reducing drug metabolism, preventing conversion to the active form.

  • Uracil phosphoribosyltransferase, preventing incorporation of 5FU into RNA or DNA, reducing its inhibitory effects.

Summary of Antifungal Drug Mechanisms of Action

• Azoles: Inhibit Erg11, disrupting ergosterol biosynthesis and compromising cell membrane integrity.

• Echinocandins: Reduce β-1,3-glucan content in the cell wall by inhibiting Fks1/Fks2, disrupting cell wall synthesis.

• Polyenes: Sequester ergosterol and form pores in the cell membrane, increasing membrane permeability and leading to cell death.

• Flucytosine (5-FC): Inhibits RNA/DNA synthesis, disrupting cell growth and replication.

Summary of Fungal Drug Resistance Mechanisms

• Target mutations: Reduce drug affinity, preventing effective binding and inhibition.

• Overexpression of Erg11: Allows some Erg11 to remain active, compensating for the inhibitory effects of azoles.

• Efflux upregulation: Reduces intracellular drug concentration, limiting drug exposure.

• Loss of function in Erg11 and bypass mechanisms: Rare and incur fitness costs, indicating significant metabolic disruption.

Key Milestones in Antifungal Drug Development

• 1950: Amphotericin B discovered, revolutionizing the treatment of systemic fungal infections.

• 1957: Antifungal activity of 5-flucytosine discovered, expanding the arsenal of antifungal agents.

• 1970: First azole antifungal discovered, paving the way for a new class of antifungals with improved safety and efficacy.

• 1990: Fluconazole approved, becoming a widely used azole antifungal for various fungal infections.

• 2000: Echinocandin discovered, representing a novel class of antifungals targeting the fungal cell wall.

• 2001: Caspofungin approved, the first echinocandin to be used clinically for invasive fungal infections.

• 2002: Voriconazole approved, a broad-spectrum triazole antifungal with activity against Aspergillus and other molds.

• 2006: Anidulafungin approved, another echinocandin with similar properties to caspofungin and micafungin.

• 2014: Posaconazole approved, a broad-spectrum triazole antifungal with activity against a wide range of molds and yeasts.

Current Antifungal Compounds

• Currently, there are four classes of antifungals available: polyenes, flucytosine, azoles, and echinocandins, each with distinct mechanisms of action and clinical applications.

• Improvements have been made to increase efficacy or reduce toxicity, resulting in more effective and safer treatment options.

• Future generations of these compounds are currently in development, aiming to address emerging resistance and improve patient outcomes.

Summary of Resistance to Antifungal Drugs

• Resistance mechanisms in fungi (mutations – no HGT mechanisms):

  3. Upregulated drug efflux – reducing intracellular drug concentration, limiting drug exposure.

  4. Target mutations reducing drug affinity for the target, impairing drug binding and inhibition.

  5. Mutations reducing the effects of drug-activity-associated toxic products, mitigating drug-induced damage.

• No contribution from horizontal gene transfer, suggesting that resistance primarily arises through spontaneous mutations within the fungal genome.

• Resistance evolution is dependent on mutation rate, drug use (selection pressure), fitness costs of resistance, and rate of fitness compensation, highlighting the complex interplay of factors driving resistance emergence.

Drugs for Tuberculosis

First-Line Antibiotics

• Isoniazid (pro-drug, activated by KatG): Targets cell wall synthesis (mycolic acid), a critical component of the mycobacterial cell wall.

• Pyrazinamide (pro-drug): Target/mechanism unknown, although it is thought to disrupt membrane synthesis and transport functions, essential for cell survival.

• Ethambutol: Targets cell wall synthesis (arabinogalactan synthesis), inhibiting the incorporation of mycolic acid into the cell wall.

• Streptomycin: Targets protein synthesis by binding to 16S rRNA, a component of the bacterial ribosome, disrupting protein production.

• Rifampicin: Targets RNA Polymerase, an enzyme responsible for transcribing DNA into RNA, thereby halting gene expression.

Unusual Cell Wall of Mycobacteria

• The mycobacterial cell wall consists of:

  1. Outer lipids, forming a hydrophobic barrier.

  2. Mycolic acid, a unique long-chain fatty acid.

  3. Polysaccharides (arabinogalactan), linking peptidoglycan to mycolic acid.

  4. Peptidoglycan, providing structural support.

  5. Plasma membrane, enclosing the cytoplasm.

  6. Lipoarabinomannan, a glycolipid with immunomodulatory properties.

  7. Phosphatidylinositol mannoside, a glycolipid involved in cell signaling.

  8. Cell wall skeleton, providing rigidity and structural integrity.

• The presence of mycolic acids confers several characteristics:

  • Increased resistance to chemical damage and dehydration, protecting the bacterium from harsh environmental conditions.

  • Prevention of effective activity of hydrophobic antibiotics, limiting drug penetration.

  • Allows the bacterium to grow readily inside macrophages, effectively hiding it from the host's immune system.

• Mycolate biosynthesis is crucial for survival and pathogenesis of Mycobacterium tuberculosis, making it an attractive drug target.

  • Isoniazid (INH): targets the synthesis of mycolic acid, disrupting cell wall formation.

  • Ethambutol: targets cell wall synthesis, inhibiting the incorporation of mycolic acid into the cell wall.

Anti-mycobacterium Cell Wall Antibiotics

Isoniazid (INH)

• First-line anti-tuberculous medication, widely used in combination therapies.

• Prodrug – activated by KatG catalase (bacterial enzyme), converting it into its active form, isonicotinic acyl-NAD.

• Bacteriocidal (for growing cells), effectively killing actively replicating bacteria.

• Oral administration, well adsorbed, widely distributed, reaching therapeutic concentrations in various tissues and fluids.

• Never used alone to treat active tuberculosis because resistance quickly develops (katG mutations), necessitating combination therapy.

• Inhibits the synthesis of mycolic acid in the mycobacterial cell wall, disrupting its structural integrity.

Ethambutol

• Blocks arabinogalactan synthesis, inhibiting incorporation of mycolic acid into the cell wall, weakening the cell wall structure.

• Bacteriostatic, inhibiting bacterial growth without directly killing the cells.

• Used in combination with isoniazid, pyrazinamide, and rifampicin, complementing their mechanisms of action.

Drugs for TB – Second-Line Drugs

• Used to treat TB that is resistant to first-line therapy: MDR (INH, RIF), XDR (INH, RIF, FQ), representing serious clinical challenges.

• Second-line drugs are used because they are:

  • Less effective (p-aminosalicylic acid), requiring higher doses and longer treatment durations.

  • Have toxic side effects (cycloserine, aminoglycosides), necessitating careful monitoring and management.

  • Unavailable/costly in many countries (fluoroquinolones), limiting access to effective treatment.

• Aminoglycosides: Amikacin, Kanamycin

  • Target protein synthesis (Ribosome, 16S rRNA), inhibiting bacterial protein production.

• Fluoroquinolones: Cipro-, Levo-, Moxifloxacin

  • Target Replication (DNA gyrase / topoisomerase IV), essential enzymes for DNA replication and repair.

• Cycloserine: Targets cell wall synthesis (peptidoglycan precursors), disrupting peptidoglycan formation.

Drugs for TB – Second-Line, Continued

• Ethionamide: Pro-drug, targets cell wall synthesis (mycolic acid, similar to isoniazid), has toxic side effects, limiting its use.

• Capreomycin / Viomycin: Polypeptides, target protein synthesis – toxic side effects, necessitating careful monitoring.

• para-aminosalicylic acid (PAS): Targets folic acid synthesis, an essential metabolic pathway.

Third-Line Drugs

• Third-line because drugs have unproven efficacy, representing experimental or last-resort options.

• Rifabutin: Variant of rifampicin – expensive, for those who do not tolerate rifampicin, offering an alternative for patients with adverse reactions.

• Macrolides: Target protein synthesis, inhibiting bacterial protein production.

• Linezolid: Target protein synthesis, offering a different mechanism of action compared to other antibiotics.

• Bedaquiline (diarylquinoline class):

  • Unique mechanism of action: Targets ATP synthase (energy supply), disrupting cellular energy production.

  • In 2012, bedaquiline received approval by the US FDA for the treatment of MDR-TB, in addition to the current second-line treatment regimen; interim guidance for its use has also been issued by the WHO; multiple side effects (black box warning), necessitating careful risk-benefit assessment.

Recently Approved Drugs

• Bedaquiline (Diarylquinoline): Targets ATP synthase, inhibiting energy metabolism of the cell, leading to cell death.

• Delamanid (Nitroimidazole): Exact target not yet known, inhibits mycolic acid synthesis (keto and methoxy mycolic acids) and cell respiration, disrupting multiple cellular processes.

Drugs in Phase II and Phase III Clinical Trials

• Pretomanid (Nitroimidazole): Exact target not yet known, inhibits cell wall synthesis and causes respiratory poisoning (Phase III), showing promise in clinical trials.

• Delpazolid (Oxazolidinone): Targets the 50S subunit of the ribosome, inhibiting protein synthesis (Phase II), offering a novel approach to combatting TB.

• Sutezolid (Oxazolidinone): Targets the 50S subunit of the ribosome, inhibiting protein synthesis (Phase II), with potential advantages over linezolid.

Fungal, Mycobacterial, and New Drugs Exam Questions:

• Flucytosine is an antifungal drug. Briefly describe what flucytosine targets and how that inhibits fungal growth.

• Azoles and polyenes are two important classes of antifungal drugs that affect the cell membrane. Briefly describe the mode of action of each class.

• Name four first-line anti-tuberculosis antibiotics and their targets.

• What is the target of bedaquiline, a recently approved anti-tuberculosis drug?

• We